John Huguenard

All Publications

• Synaptic cell adhesion moleculeCdh6identifies a class of sensory neurons with novel functions in colonic motility. bioRxiv : the preprint server for biology Gomez-Frittelli, J., Devienne, G., Travis, L., Kyloh, M. A., Duan, X., Hibberd, T. J., Spencer, N. J., Huguenard, J. R., Kaltschmidt, J. A. 2024

  Abstract

  Intrinsic sensory neurons are an essential part of the enteric nervous system (ENS) and play a crucial role in gastrointestinal tract motility and digestion. Neuronal subtypes in the ENS have been distinguished by their electrophysiological properties, morphology, and expression of characteristic markers, notably neurotransmitters and neuropeptides. Here we investigated synaptic cell adhesion molecules as novel cell type markers in the ENS. Our work identifies two Type II classic cadherins, Cdh6 and Cdh8, specific to sensory neurons in the mouse colon. We show that Cdh6+ neurons demonstrate all other distinguishing classifications of enteric sensory neurons including marker expression of Calcb and Nmu, Dogiel type II morphology and AH-type electrophysiology and I H current. Optogenetic activation of Cdh6+ sensory neurons in distal colon evokes retrograde colonic motor complexes (CMCs), while pharmacologic blockade of rhythmicity-associated current I H disrupts the spontaneous generation of CMCs. These findings provide the first demonstration of selective activation of a single neurochemical and functional class of enteric neurons, and demonstrate a functional and critical role for sensory neurons in the generation of CMCs.

  View details for DOI 10.1101/2024.08.06.606748

  View details for PubMedID 39149241

• Cross-regional coordination of activity in the human brain during autobiographical self-referential processing. Proceedings of the National Academy of Sciences of the United States of America Stieger, J. R., Pinheiro-Chagas, P., Fang, Y., Li, J., Lusk, Z., Perry, C. M., Girn, M., Contreras, D., Chen, Q., Huguenard, J. R., Spreng, R. N., Edlow, B. L., Wagner, A. D., Buch, V., Parvizi, J. 2024; 121 (32): e2316021121

  Abstract

  For the human brain to operate, populations of neurons across anatomical structures must coordinate their activity within milliseconds. To date, our understanding of such interactions has remained limited. We recorded directly from the hippocampus (HPC), posteromedial cortex (PMC), ventromedial/orbital prefrontal cortex (OFC), and the anterior nuclei of the thalamus (ANT) during two experiments of autobiographical memory processing that are known from decades of neuroimaging work to coactivate these regions. In 31 patients implanted with intracranial electrodes, we found that the presentation of memory retrieval cues elicited a significant increase of low frequency (LF < 6 Hz) activity followed by cross-regional phase coherence of this LF activity before select populations of neurons within each of the four regions increased high-frequency (HF > 70 Hz) activity. The power of HF activity was modulated by memory content, and its onset followed a specific temporal order of ANTHPC/PMCOFC. Further, we probed cross-regional causal effective interactions with repeated electrical pulses and found that HPC stimulations cause the greatest increase in LF-phase coherence across all regions, whereas the stimulation of any region caused the greatest LF-phase coherence between that particular region and ANT. These observations support the role of the ANT in gating, and the HPC in synchronizing, the activity of cortical midline structures when humans retrieve self-relevant memories of their past. Our findings offer a fresh perspective, with high temporal fidelity, about the dynamic signaling and underlying causal connections among distant regions when the brain is actively involved in retrieving self-referential memories from the past.

  View details for DOI 10.1073/pnas.2316021121

  View details for PubMedID 39078679

• Reuniens thalamus recruits recurrent excitation in medial prefrontal cortex. bioRxiv : the preprint server for biology Vantomme, G., Devienne, G., Hull, J. M., Huguenard, J. R. 2024

  Abstract

  Medial prefrontal cortex (mPFC) and hippocampus are critical for memory retrieval, decision making and emotional regulation. While ventral CA1 (vCA1) shows direct and reciprocal connections with mPFC, dorsal CA1 (dCA1) forms indirect pathways to mPFC, notably via the thalamic Reuniens nucleus (Re). Neuroanatomical tracing has documented structural connectivity of this indirect pathway through Re however, its functional operation is largely unexplored. Here we used in vivo and in vitro electrophysiology along with optogenetics to address this question. Whole-cell patch-clamp recordings in acute mouse brain slices revealed both monosynaptic excitatory responses and disynaptic feedforward inhibition for both Re-mPFC and Re-dCA1 pathways. However, we also identified a novel biphasic excitation of mPFC by Re, but not dCA1. These early monosynaptic and late recurrent components are in marked contrast to the primarily feedforward inhibition characteristic of thalamic inputs to neocortex. Local field potential recordings in mPFC brain slices revealed that this biphasic excitation propagates throughout all cortical lamina, with the late excitation specifically enhanced by GABAAR blockade. In vivo Neuropixels recordings in head-fixed awake mice revealed a similar biphasic excitation of mPFC units by Re activation. In summary, Re output produces recurrent feed-forward excitation within mPFC suggesting a potent amplification system in the Re-mPFC network. This may facilitate amplification of dCA1->mPFC signals for which Re acts as the primary conduit, as there is little direct connectivity. In addition, the capacity of mPFC neurons to fire bursts of action potentials in response to Re input suggests that these synapses have a high gain.The interactions between medial prefrontal cortex and hippocampus are crucial for memory formation and retrieval. Yet, it is still poorly understood how the functional connectivity of direct and indirect pathways underlies these functions. This research explores the synaptic connectivity of the indirect pathway through the Reuniens nucleus of the thalamus using electrophysiological recordings and optogenetic manipulations. The study found that Reuniens stimulation recruits recurrent and long-lasting activity in mPFC - a phenomenon not previously recorded. This recurrent activity might create a temporal window ideal for coincidence detection and be an underlying mechanism for memory formation and retrieval.

  View details for DOI 10.1101/2024.05.31.596906

  View details for PubMedID 38854099

  View details for PubMedCentralID PMC11160760

• Antisense oligonucleotide therapeutic approach for Timothy syndrome. Nature Chen, X., Birey, F., Li, M. Y., Revah, O., Levy, R., Thete, M. V., Reis, N., Kaganovsky, K., Onesto, M., Sakai, N., Hudacova, Z., Hao, J., Meng, X., Nishino, S., Huguenard, J., Pașca, S. P. 2024; 628 (8009): 818-825

  Abstract

  Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

  View details for DOI 10.1038/s41586-024-07310-6

  View details for PubMedID 38658687

  View details for PubMedCentralID 1149428

• Atlas of the aging mouse brain reveals white matter as vulnerable foci. Cell Hahn, O., Foltz, A. G., Atkins, M., Kedir, B., Moran-Losada, P., Guldner, I. H., Munson, C., Kern, F., Pálovics, R., Lu, N., Zhang, H., Kaur, A., Hull, J., Huguenard, J. R., Grönke, S., Lehallier, B., Partridge, L., Keller, A., Wyss-Coray, T. 2023

  Abstract

  Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA sequencing of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glial aging was particularly accelerated in white matter compared with cortical regions, whereas specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.

  View details for DOI 10.1016/j.cell.2023.07.027

  View details for PubMedID 37591239

• Adult Gene Therapy for Epilepsy in a Model of Angelman Syndrome: Hope or Hype? Epilepsy currents Huguenard, J. R. 2023; 23 (5): 312-314

  View details for DOI 10.1177/15357597231191885

  View details for PubMedID 37901779

  View details for PubMedCentralID PMC10601042

• Adult Gene Therapy for Epilepsy in a Model of Angelman Syndrome: Hope or Hype? EPILEPSY CURRENTS Huguenard, J. R. 2023

  View details for DOI 10.1177/15357597231191885

  View details for Web of Science ID 001044011300001

• A CMOS-based highly scalable flexible neural electrode interface. Science advances Zhao, E. T., Hull, J. M., Mintz Hemed, N., Uluşan, H., Bartram, J., Zhang, A., Wang, P., Pham, A., Ronchi, S., Huguenard, J. R., Hierlemann, A., Melosh, N. A. 2023; 9 (23): eadf9524

  Abstract

  Perception, thoughts, and actions are encoded by the coordinated activity of large neuronal populations spread over large areas. However, existing electrophysiological devices are limited by their scalability in capturing this cortex-wide activity. Here, we developed an electrode connector based on an ultra-conformable thin-film electrode array that self-assembles onto silicon microelectrode arrays enabling multithousand channel counts at a millimeter scale. The interconnects are formed using microfabricated electrode pads suspended by thin support arms, termed Flex2Chip. Capillary-assisted assembly drives the pads to deform toward the chip surface, and van der Waals forces maintain this deformation, establishing Ohmic contact. Flex2Chip arrays successfully measured extracellular action potentials ex vivo and resolved micrometer scale seizure propagation trajectories in epileptic mice. We find that seizure dynamics in absence epilepsy in the Scn8a+/- model do not have constant propagation trajectories.

  View details for DOI 10.1126/sciadv.adf9524

  View details for PubMedID 37285436

  View details for PubMedCentralID PMC10246892

• Prefrontal PV interneurons facilitate attention and are linked to attentional dysfunction in a mouse model of absence epilepsy. eLife Ferguson, B., Glick, C., Huguenard, J. R. 2023; 12

  Abstract

  Absence seizures are characterized by brief periods of unconsciousness accompanied by lapses in motor function that can occur hundreds of times throughout the day. Outside of these frequent moments of unconsciousness, approximately a third of people living with the disorder experience treatment-resistant attention impairments. Convergent evidence suggests prefrontal cortex (PFC) dysfunction may underlie attention impairments in affected patients. To examine this, we use a combination of slice physiology, fiber photometry, electrocorticography (ECoG), optogenetics, and behavior in the Scn8a+/-mouse model of absence epilepsy. Attention function was measured using a novel visual attention task where a light cue that varied in duration predicted the location of a food reward. In Scn8a+/-mice, we find altered parvalbumin interneuron (PVIN) output in the medial PFC (mPFC) in vitro and PVIN hypoactivity along with reductions in gamma power during cue presentation in vivo. This was associated with poorer attention performance in Scn8a+/-mice that could be rescued by gamma-frequency optogenetic stimulation of PVINs. This highlights cue-related PVIN activity as an important mechanism for attention and suggests PVINs may represent a therapeutic target for cognitive comorbidities in absence epilepsy.

  View details for DOI 10.7554/eLife.78349

  View details for PubMedID 37014118

  View details for PubMedCentralID PMC10072875

• Loss of Rai1 enhances hippocampal excitability and epileptogenesis in mouse models of Smith-Magenis syndrome (vol 119, e2210122119, 2022 ) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, Y., Kowalczyk, M., Fogerson, P., Lee, Y., Haque, M., Wang, D. C., DeNardo, A., Tessier-Lavigne, M., Huguenard, J. R., Adams, E. L., Luo, L., Huang, W. 2022; 119 (52)

  View details for DOI 10.1073/pnas.2219265119

  View details for Web of Science ID 000956548600008

• Loss of Rai1 enhances hippocampal excitability and epileptogenesis in mouse models of Smith-Magenis syndrome. Proceedings of the National Academy of Sciences of the United States of America Chang, Y., Kowalczyk, M., Fogerson, P. M., Lee, Y., Haque, M., Adams, E. L., Wang, D. C., DeNardo, L. A., Tessier-Lavigne, M., Huguenard, J. R., Luo, L., Huang, W. 2022; 119 (43): e2210122119

  Abstract

  Hyperexcitability of brain circuits is a common feature of autism spectrum disorders (ASDs). Genetic deletion of a chromatin-binding protein, retinoic acid induced 1 (RAI1), causes Smith-Magenis syndrome (SMS). SMS is a syndromic ASD associated with intellectual disability, autistic features, maladaptive behaviors, overt seizures, and abnormal electroencephalogram (EEG) patterns. The molecular and neural mechanisms underlying abnormal brain activity in SMS remain unclear. Here we show that panneural Rai1 deletions in mice result in increased seizure susceptibility and prolonged hippocampal seizure duration invivo and increased dentate gyrus population spikes ex vivo. Brain-wide mapping of neuronal activity pinpointed selective cell types within the limbic system, including the hippocampal dentate gyrus granule cells (dGCs) that are hyperactivated by chemoconvulsant administration or sensory experience in Rai1-deficient brains. Deletion of Rai1 from glutamatergic neurons, but not from gamma-aminobutyric acidergic (GABAergic) neurons, was responsible for increased seizure susceptibility. Deleting Rai1 from the Emx1Cre-lineage glutamatergic neurons resulted in abnormal dGC properties, including increased excitatory synaptic transmission and increased intrinsic excitability. Our work uncovers the mechanism of neuronal hyperexcitability in SMS by identifying Rai1 as a negative regulator of dGC intrinsic and synaptic excitability.

  View details for DOI 10.1073/pnas.2210122119

  View details for PubMedID 36256819

• Maturation and circuit integration of transplanted human cortical organoids. Nature Revah, O., Gore, F., Kelley, K. W., Andersen, J., Sakai, N., Chen, X., Li, M. Y., Birey, F., Yang, X., Saw, N. L., Baker, S. W., Amin, N. D., Kulkarni, S., Mudipalli, R., Cui, B., Nishino, S., Grant, G. A., Knowles, J. K., Shamloo, M., Huguenard, J. R., Deisseroth, K., Pașca, S. P. 2022; 610 (7931): 319-326

  Abstract

  Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

  View details for DOI 10.1038/s41586-022-05277-w

  View details for PubMedID 36224417

• Maladaptive myelination promotes generalized epilepsy progression. Nature neuroscience Knowles, J. K., Xu, H., Soane, C., Batra, A., Saucedo, T., Frost, E., Tam, L. T., Fraga, D., Ni, L., Villar, K., Talmi, S., Huguenard, J. R., Monje, M. 2022

  Abstract

  Activity-dependent myelination can fine-tune neural network dynamics. Conversely, aberrant neuronal activity, as occurs in disorders of recurrent seizures (epilepsy), could promote maladaptive myelination, contributing to pathogenesis. In this study, we tested the hypothesis that activity-dependent myelination resulting from absence seizures, which manifest as frequent behavioral arrests with generalized electroencephalography (EEG) spike-wave discharges, promote thalamocortical network hypersynchrony and contribute to epilepsy progression. We found increased oligodendrogenesis and myelination specifically within the seizure network in two models of generalized epilepsy with absence seizures (Wag/Rij rats and Scn8a+/mut mice), evident only after epilepsy onset. Aberrant myelination was prevented by pharmacological seizure inhibition in Wag/Rij rats. Blocking activity-dependent myelination decreased seizure burden over time and reduced ictal synchrony as assessed by EEG coherence. These findings indicate that activity-dependent myelination driven by absence seizures contributes to epilepsy progression; maladaptive myelination may be pathogenic in some forms of epilepsy and other neurological diseases.

  View details for DOI 10.1038/s41593-022-01052-2

  View details for PubMedID 35501379

• Author Correction: Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin. Nature communications Elleman, A. V., Devienne, G., Makinson, C. D., Haynes, A. L., Huguenard, J. R., Du Bois, J. 2022; 13 (1): 2277

  View details for DOI 10.1038/s41467-022-30054-8

  View details for PubMedID 35449160

• NF1 MUTATION DRIVES NEURONAL ACTIVITY-DEPENDENT OPTIC GLIOMA INITIATION Pan, Y., Hysinger, J., Barron, T., Schindler, N., Cobb, O., Guo, X., Yalcin, B., Anastasaki, C., Mulinyawe, S., Ponnuswami, A., Scheaffer, S., Ma, Y., Chang, K., Xia, X., Toonen, J., Lennon, J., Gibson, E., Huguenard, J., Liau, L., Goldberg, J., Monje, M., Gutmann, D. OXFORD UNIV PRESS INC. 2021: 212

  View details for Web of Science ID 000757356200853

• Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin. Nature communications Elleman, A. V., Devienne, G., Makinson, C. D., Haynes, A. L., Huguenard, J. R., Du Bois, J. 2021; 12 (1): 4171

  Abstract

  Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.

  View details for DOI 10.1038/s41467-021-24392-2

  View details for PubMedID 34234116

• NF1 mutation drives neuronalactivity-dependent initiation of optic glioma. Nature Pan, Y., Hysinger, J. D., Barron, T., Schindler, N. F., Cobb, O., Guo, X., Yalcin, B., Anastasaki, C., Mulinyawe, S. B., Ponnuswami, A., Scheaffer, S., Ma, Y., Chang, K., Xia, X., Toonen, J. A., Lennon, J. J., Gibson, E. M., Huguenard, J. R., Liau, L. M., Goldberg, J. L., Monje, M., Gutmann, D. H. 2021

  Abstract

  Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with theneurofibromatosis1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.

  View details for DOI 10.1038/s41586-021-03580-6

  View details for PubMedID 34040258

• Long-term maturation of human cortical organoids matches key early postnatal transitions. Nature neuroscience Gordon, A. n., Yoon, S. J., Tran, S. S., Makinson, C. D., Park, J. Y., Andersen, J. n., Valencia, A. M., Horvath, S. n., Xiao, X. n., Huguenard, J. R., Pașca, S. P., Geschwind, D. H. 2021

  Abstract

  Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.

  View details for DOI 10.1038/s41593-021-00802-y

  View details for PubMedID 33619405

• Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel. Proceedings of the National Academy of Sciences of the United States of America Koster, A. K., Reese, A. L., Kuryshev, Y., Wen, X., McKiernan, K. A., Gray, E. E., Wu, C., Huguenard, J. R., Maduke, M., Du Bois, J. 2020

  Abstract

  CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

  View details for DOI 10.1073/pnas.2009977117

  View details for PubMedID 33277431

• PFC PV Interneurons Facilitate Visual Attention and are Disrupted in a Genetic Model of Absence Epilepsy Ferguson, B., Huguenard, J. SPRINGERNATURE. 2020: 88–89

  View details for Web of Science ID 000596371000178

• Perspective: Is Cortical Hyperexcitability the Only Path to Generalized Absence Epilepsy? EPILEPSY CURRENTS Huguenard, J. R. 2020

  View details for DOI 10.1177/1535759720959325

  View details for Web of Science ID 000581964700001

• Nonlinearities between inhibition and T-type calcium channel activity bidirectionally regulate thalamic oscillations. eLife Lu, A. C., Lee, C. K., Kleiman-Weiner, M., Truong, B., Wang, M., Huguenard, J., Beenhakker, M. P. 2020; 9

  Abstract

  Absence seizures result from 3-5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.

  View details for DOI 10.7554/eLife.59548

  View details for PubMedID 32902384

• Neuronal defects in a human cellular model of 22q11.2 deletion syndrome. Nature medicine Khan, T. A., Revah, O. n., Gordon, A. n., Yoon, S. J., Krawisz, A. K., Goold, C. n., Sun, Y. n., Kim, C. H., Tian, Y. n., Li, M. Y., Schaepe, J. M., Ikeda, K. n., Amin, N. D., Sakai, N. n., Yazawa, M. n., Kushan, L. n., Nishino, S. n., Porteus, M. H., Rapoport, J. L., Bernstein, J. A., O'Hara, R. n., Bearden, C. E., Hallmayer, J. F., Huguenard, J. R., Geschwind, D. H., Dolmetsch, R. E., Paşca, S. P. 2020

  Abstract

  22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

  View details for DOI 10.1038/s41591-020-1043-9

  View details for PubMedID 32989314

• Current Controversy: Spikes, Bursts, and Synchrony in Generalized Absence Epilepsy: Unresolved Questions Regarding Thalamocortical Synchrony in Absence Epilepsy. Epilepsy currents Huguenard, J. 2019; 19 (2): 105–11

  Abstract

  Absence epilepsy is a disorder of thalamocortical networks. Animal models have provided detailed information regarding the core cellular, synaptic, and network features that contribute to the electroencephalogram spike and wave discharge characteristic of typical absence epilepsy. Understanding of seizure networks and dynamics is a critical step toward improving treatments, yet competing conceptual models have evolved to explain seizure initiation and propagation. Recent studies have questioned 2 key model concepts: (1) T-type Ca2+ channel-dependent burst firing in thalamic relay neurons may not be essential for seizure generation, bringing into question the proposed mechanism for the antiepileptic drug ethosuximide in reducing thalamic bursting and (2) widespread synchronized neural activity may not be a core feature of the seizures, indicating that reductions in synchrony would not be a productive therapeutic goal. In this review, I will discuss these current findings, highlight the innovative approaches that have enabled these insights, and provide a unified framework that incorporates these sometimes-conflicting ideas. Finally, I lay out future work that will be necessary to finally resolve the remaining issues.

  View details for PubMedID 30955423

• Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures. Nature neuroscience Marton, R. M., Miura, Y. n., Sloan, S. A., Li, Q. n., Revah, O. n., Levy, R. J., Huguenard, J. R., Pașca, S. P. 2019

  Abstract

  Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

  View details for PubMedID 30692691

• Reliability of human cortical organoid generation NATURE METHODS Yoon, S., Elahi, L. S., Pasca, A. M., Marton, R. M., Gordon, A., Revah, O., Miura, Y., Walczak, E. M., Holdgate, G. M., Fan, H., Huguenard, J. R., Geschwind, D. H., Pasca, S. P. 2019; 16 (1): 75-+

  View details for DOI 10.1038/s41592-018-0255-0

  View details for Web of Science ID 000454162400033

• Reliability of human cortical organoid generation. Nature methods Yoon, S. J., Elahi, L. S., Pașca, A. M., Marton, R. M., Gordon, A. n., Revah, O. n., Miura, Y. n., Walczak, E. M., Holdgate, G. M., Fan, H. C., Huguenard, J. R., Geschwind, D. H., Pașca, S. P. 2019; 16 (1): 75–78

  Abstract

  The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

  View details for PubMedID 30573846

• Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons. Frontiers in molecular neuroscience Ha, H. T., Leal-Ortiz, S., Lalwani, K., Kiyonaka, S., Hamachi, I., Mysore, S. P., Montgomery, J. M., Garner, C. C., Huguenard, J. R., Kim, S. A. 2018; 11: 405

  Abstract

  During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs. Over development, we found a concomitant increase in Shank2 and Shank3 with GluA2 at synapses, implicating these molecules as potential players in AMPAR maturation. Since Shank activation and function require zinc, we next studied whether neuronal activity regulated postsynaptic zinc at glutamatergic synapses. Zinc was found to increase transiently and reversibly with neuronal depolarization at synapses, which could affect Shank and AMPAR localization and activity. Elevated zinc induced multiple functional changes in AMPAR, indicative of a subunit switch. Specifically, zinc lengthened the decay time of AMPAR-mediated synaptic currents and reduced their inward rectification in young hippocampal neurons. Mechanistically, both Shank2 and Shank3 were necessary for the zinc-sensitive enhancement of AMPAR-mediated synaptic transmission and act in concert to promote removal of GluA1 while enhancing recruitment of GluA2 at pre-existing Shank puncta. These findings highlight a cooperative local dynamic regulation of AMPAR subunit switch controlled by zinc signaling through Shank2 and Shank3 to shape the biophysical properties of developing glutamatergic synapses. Given the zinc sensitivity of young neurons and its dependence on Shank2 and Shank3, genetic mutations and/or environmental insults during early development could impair synaptic maturation and circuit formation that underlie ASD etiology.

  View details for DOI 10.3389/fnmol.2018.00405

  View details for PubMedID 30524232

  View details for PubMedCentralID PMC6256285

• Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons FRONTIERS IN MOLECULAR NEUROSCIENCE Ha, H. T., Leal-Ortiz, S., Lalwani, K., Kiyonaka, S., Hamachi, I., Mysore, S. P., Montgomery, J. M., Garner, C. C., Huguenard, J. R., Kim, S. A. 2018; 11

  View details for DOI 10.3389/fnmol.2018.00405

  View details for Web of Science ID 000449665100001

• Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems. Cell Ren, J. n., Friedmann, D. n., Xiong, J. n., Liu, C. D., Ferguson, B. R., Weerakkody, T. n., DeLoach, K. E., Ran, C. n., Pun, A. n., Sun, Y. n., Weissbourd, B. n., Neve, R. L., Huguenard, J. n., Horowitz, M. A., Luo, L. n. 2018

  Abstract

  The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

  View details for PubMedID 30146164

• Absence seizure susceptibility correlates with pre-ictal ß oscillations. Journal of physiology, Paris Sorokin, J. M., Paz, J. T., Huguenard, J. R. 2017

  Abstract

  Absence seizures are generalized, cortico-thalamo-cortical (CTC) high power electroencephalographic (EEG) or electrocorticographic (ECoG) events that initiate and terminate suddenly. ECoG recordings of absence seizures in animal models of genetic absence epilepsy show a sudden spike-wave-discharge (SWD) onset that rapidly emerges from normal ECoG activity. However, given that absence seizures occur most often during periods of drowsiness or quiet wakefulness, we wondered whether SWD onset correlates with pre-ictal changes in network activity. To address this, we analyzed ECoG recordings of both spontaneous and induced SWDs in rats with genetic absence epilepsy. We discovered that the duration and intensity of spontaneous SWDs positively correlate with pre-ictal 20-40Hz (β) spectral power and negatively correlate with 4-7Hz (Ø) power. In addition, the output of thalamocortical neurons decreases within the same pre-ictal window of time. In separate experiments we found that the propensity for SWD induction was correlated with pre-ictal β power. These results argue that CTC networks undergo a pre-seizure state transition, possibly due to a functional reorganization of cortical microcircuits, which leads to the generation of absence seizures.

  View details for DOI 10.1016/j.jphysparis.2017.05.004

  View details for PubMedID 28576554

• Assembly of functionally integrated human forebrain spheroids NATURE Birey, F., Andersen, J., Makinson, C. D., Islam, S., Wei, W., Huber, N., Fan, H. C., Metzler, K. R., Panagiotakos, G., Thom, N., O'Rourke, N. A., Steinmetz, L. M., Bernstein, J. A., Hallmayer, J., Huguenard, J. R., Pasca, S. P. 2017; 545 (7652): 54-?

  Abstract

  The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

  View details for DOI 10.1038/nature22330

  View details for PubMedID 28445465

• Breathing control center neurons that promote arousal in mice SCIENCE Yackle, K., Schwarz, L. A., Kam, K., Sorokin, J. M., Huguenard, J. R., Feldman, J. L., Luo, L., Krasnow, M. A. 2017; 355 (6332): 1411-1415

  Abstract

  Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

  View details for DOI 10.1126/science.aai7984

  View details for Web of Science ID 000397809500040

  View details for PubMedID 28360327

• Regulation of Thalamic and Cortical Network Synchrony by Scn8a. Neuron Makinson, C. D., Tanaka, B. S., Sorokin, J. M., Wong, J. C., Christian, C. A., Goldin, A. L., Escayg, A., Huguenard, J. R. 2017

  Abstract

  Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.

  View details for DOI 10.1016/j.neuron.2017.01.031

  View details for PubMedID 28238546

  View details for PubMedCentralID PMC5393918

• Bidirectional Control of Generalized Epilepsy Networks via Rapid Real-Time Switching of Firing Mode. Neuron Sorokin, J. M., Davidson, T. J., Frechette, E., Abramian, A. M., Deisseroth, K., Huguenard, J. R., Paz, J. T. 2017; 93 (1): 194-210

  Abstract

  Thalamic relay neurons have well-characterized dual firing modes: bursting and tonic spiking. Studies in brain slices have led to a model in which rhythmic synchronized spiking (phasic firing) in a population of relay neurons leads to hyper-synchronous oscillatory cortico-thalamo-cortical rhythms that result in absence seizures. This model suggests that blocking thalamocortical phasic firing would treat absence seizures. However, recent in vivo studies in anesthetized animals have questioned this simple model. Here we resolve this issue by developing a real-time, mode-switching approach to drive thalamocortical neurons into or out of a phasic firing mode in two freely behaving genetic rodent models of absence epilepsy. Toggling between phasic and tonic firing in thalamocortical neurons launched and aborted absence seizures, respectively. Thus, a synchronous thalamocortical phasic firing state is required for absence seizures, and switching to tonic firing rapidly halts absences. This approach should be useful for modulating other networks that have mode-dependent behaviors.

  View details for DOI 10.1016/j.neuron.2016.11.026

  View details for PubMedID 27989462

  View details for PubMedCentralID PMC5268077

• Tapping the Brakes: Cellular and Synaptic Mechanisms that Regulate Thalamic Oscillations NEURON Fogerson, P. M., Huguenard, J. R. 2016; 92 (4): 687-704

  Abstract

  Thalamic oscillators contribute to both normal rhythms associated with sleep and anesthesia and abnormal, hypersynchronous oscillations that manifest behaviorally as absence seizures. In this review, we highlight new findings that refine thalamic contributions to cortical rhythms and suggest that thalamic oscillators may be subject to both local and global control. We describe endogenous thalamic mechanisms that limit network synchrony and discuss how these protective brakes might be restored to prevent absence seizures. Finally, we describe how intrinsic and circuit-level specializations among thalamocortical loops may determine their involvement in widespread oscillations and render subsets of thalamic nuclei especially vulnerable to pathological synchrony.

  View details for DOI 10.1016/j.neuron.2016.10.024

  View details for Web of Science ID 000388572700007

  View details for PubMedID 27883901

  View details for PubMedCentralID PMC5131525

• Catching a wave. eLife Fogerson, P. M., Huguenard, J. R. 2016; 5

  Abstract

  Temporary circuits amplify spontaneous activity in the visual system of neonatal rats.

  View details for DOI 10.7554/eLife.21236

  View details for PubMedID 27725090

  View details for PubMedCentralID PMC5059133

• Two classes of excitatory synaptic responses in rat thalamic reticular neurons. Journal of neurophysiology Deleuze, C., Huguenard, J. R. 2016; 116 (3): 995-1011

  Abstract

  The thalamic reticular nucleus (nRt), composed of GABAergic cells providing inhibition of relay neurons in the dorsal thalamus, receives excitation from the neocortex and thalamus. The two excitatory pathways promoting feedback or feedforward inhibition of thalamocortical neurons contribute to sensory processing and rhythm generation. While synaptic inhibition within the nRt has been carefully characterized, little is known regarding the biophysics of synaptic excitation. To characterize the functional properties of thalamocortical and corticothalamic connections to the nRt, we recorded minimal electrically evoked excitatory postsynaptic currents from nRt cells in vitro. A hierarchical clustering algorithm distinguished two types of events. Type 1 events had larger amplitudes and faster kinetics, largely mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, whereas type 2 responses had more prominent N-methyl-d-aspartate (NMDA) receptor contribution. Type 1 responses showed subnormal axonal propagation and paired pulse depression, consistent with thalamocortical inputs. Furthermore, responses kinetically similar to type 1 events were evoked by glutamate-mediated activation of thalamic neurons. Type 2 responses, in contrast, likely arise from corticothalamic inputs, with larger NMDA conductance and weak Mg(2+)-dependent block, suggesting that NMDA receptors are critical for the cortical excitation of reticular neurons. The long-lasting action of NMDA receptors would promote reticular cell burst firing and produce powerful inhibitory output to relay neurons proposed to be important in triggering epilepsy. This work provides the first complete voltage-clamp analysis of the kinetics and voltage dependence of AMPA and NMDA responses of thalamocortical and corticothalamic synapses in the nRt and will be critical in optimizing biologically realistic neural network models of thalamocortical circuits relevant to sensory processing and thalamocortical oscillations.

  View details for DOI 10.1152/jn.01121.2015

  View details for PubMedID 27281752

  View details for PubMedCentralID PMC5009213

• LSPS/Optogenetics to Improve Synaptic Connectivity Mapping: Unmasking the Role of Basket Cell-Mediated Feedforward Inhibition. eNeuro Brill, J., Mattis, J., Deisseroth, K., Huguenard, J. R. 2016; 3 (4)

  Abstract

  Neocortical pyramidal cells (PYRs) receive synaptic inputs from many types of GABAergic interneurons. Connections between parvalbumin (PV)-positive, fast-spiking interneurons ("PV cells") and PYRs are characterized by perisomatic synapses and high-amplitude, short-latency IPSCs. Here, we present novel methods to study the functional influence of PV cells on layer 5 PYRs using optogenetics combined with laser-scanning photostimulation (LSPS). First, we examined the strength and spatial distribution of PV-to-PYR inputs. To that end, the fast channelrhodopsin variant AAV5-EF1α-DIO-hChR2(E123T)-eYFP (ChETA) was expressed in PV cells in somatosensory cortex of mice using an adeno-associated virus-based viral construct. Focal blue illumination (100-150 µm half-width) was directed through the microscope objective to excite PV cells along a spatial grid covering layers 2-6, while IPSCs were recorded in layer 5 PYRs. The resulting optogenetic input maps showed evoked PV cell inputs originating from an ∼500-μm-diameter area surrounding the recorded PYR. Evoked IPSCs had the short-latency/high-amplitude characteristic of PV cell inputs. Second, we investigated how PV cell activity modulates PYR output in response to synaptic excitation. We expressed halorhodopsin (eNpHR3.0) in PV cells using the same strategy as for ChETA. Yellow illumination hyperpolarized eNpHR3.0-expressing PV cells, effectively preventing action potential generation and thus decreasing the inhibition of downstream targets. Synaptic input maps onto layer 5 PYRs were acquired using standard glutamate-photolysis LSPS either with or without full-field yellow illumination to silence PV cells. The resulting IPSC input maps selectively lacked short-latency perisomatic inputs, while EPSC input maps showed increased connectivity, particularly from upper layers. This indicates that glutamate uncaging LSPS-based excitatory synaptic maps will consistently underestimate connectivity.

  View details for DOI 10.1523/ENEURO.0142-15.2016

  View details for PubMedID 27517089

  View details for PubMedCentralID PMC4976301

• Early postnatal switch in GABAA receptor a-subunits in the reticular thalamic nucleus. Journal of neurophysiology Pangratz-Fuehrer, S., Sieghart, W., Rudolph, U., Parada, I., Huguenard, J. R. 2016; 115 (3): 1183-1195

  Abstract

  The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABAA receptor (GABAAR)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABAARs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABAAR activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABAARs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABAAR subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.

  View details for DOI 10.1152/jn.00905.2015

  View details for PubMedID 26631150

  View details for PubMedCentralID PMC4808120

• Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target BRAIN Hiu, T., Farzampour, Z., Paz, J. T., Wang, E. H., Badgely, C., Olson, A., Micheva, K. D., Wang, G., Lemmens, R., Tran, K. V., Nishiyama, Y., Liang, X., Hamilton, S. A., O'Rourke, N., Smith, S. J., Huguenard, J. R., Bliss, T. M., Steinberg, G. K. 2016; 139: 468-480

  Abstract

  Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem's potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery.

  View details for DOI 10.1093/brain/awv360

  View details for Web of Science ID 000370205100025

  View details for PubMedID 26685158

  View details for PubMedCentralID PMC4805083

• Satb2 Regulates the Differentiation of Both Callosal and Subcerebral Projection Neurons in the Developing Cerebral Cortex. Cerebral cortex Leone, D. P., Heavner, W. E., Ferenczi, E. A., Dobreva, G., Huguenard, J. R., Grosschedl, R., McConnell, S. K. 2015; 25 (10): 3406-3419

  Abstract

  The chromatin-remodeling protein Satb2 plays a role in the generation of distinct subtypes of neocortical pyramidal neurons. Previous studies have shown that Satb2 is required for normal development of callosal projection neurons (CPNs), which fail to extend axons callosally in the absence of Satb2 and instead project subcortically. Here we conditionally delete Satb2 from the developing neocortex and find that neurons in the upper layers adopt some electrophysiological properties characteristic of deep layer neurons, but projections from the superficial layers do not contribute to the aberrant subcortical projections seen in Satb2 mutants. Instead, axons from deep layer CPNs descend subcortically in the absence of Satb2. These data demonstrate distinct developmental roles of Satb2 in regulating the fates of upper and deep layer neurons. Unexpectedly, Satb2 mutant brains also display changes in gene expression by subcerebral projection neurons (SCPNs), accompanied by a failure of corticospinal tract (CST) formation. Altering the timing of Satb2 ablation reveals that SCPNs require an early expression of Satb2 for differentiation and extension of the CST, suggesting that early transient expression of Satb2 in these cells plays an essential role in development. Collectively these data show that Satb2 is required by both CPNs and SCPNs for proper differentiation and axon pathfinding.

  View details for DOI 10.1093/cercor/bhu156

  View details for PubMedID 25037921

• Optogenetics: 10 years after ChR2 in neurons-views from the community NATURE NEUROSCIENCE Adamantidis, A., Arber, S., Bains, J. S., Bamberg, E., Bonci, A., Buzsaki, G., Cardin, J. A., Costa, R. M., Dan, Y., Goda, Y., Graybiel, A. M., Haeusser, M., Hegemann, P., Huguenard, J. R., Insel, T. R., Janak, P. H., Johnston, D., Josselyn, S. A., Koch, C., Kreitzer, A. C., Luescher, C., Malenka, R. C., Miesenboeck, G., Nagel, G., Roska, B., Schnitzer, M. J., Shenoy, K. V., Soltesz, I., Sternson, S. M., Tsien, R. W., Tsien, R. Y., Turrigiano, G. G., Tye, K. M., Wilson, R. I. 2015; 18 (9): 1202–12

  View details for PubMedID 26308981

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature methods Pasca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., Kim, C. H., Park, J., O'Rourke, N. A., Nguyen, K. D., Smith, S. J., Huguenard, J. R., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015; 12 (7): 671-678

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedID 26005811

• Albumin induces excitatory synaptogenesis through astrocytic TGF-beta/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction NEUROBIOLOGY OF DISEASE Weissberg, I., Wood, L., Kamintsky, L., Vazquez, O., Milikovsky, D. Z., Alexander, A., Oppenheim, H., Ardizzone, C., Becker, A., Frigerio, F., Vezzani, A., Buckwalter, M. S., Huguenard, J. R., Friedman, A., Kaufer, D. 2015; 78: 115-125

  Abstract

  Post injury epilepsy (PIE) is a common complication following brain insults, including ischemic and traumatic brain injuries. At present there are no means to identify the patients at-risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures, remains unknown. Here we demonstrate in-vitro and in-vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process, and highlight manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

  View details for DOI 10.1016/j.nbd.2015.02.029

  View details for PubMedID 25836421

• Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ma, Y., Juntti, S. A., Hu, C. K., Huguenard, J. R., Fernald, R. D. 2015; 112 (12): 3805-3810

  Abstract

  Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.

  View details for DOI 10.1073/pnas.1421851112

  View details for Web of Science ID 000351477000061

  View details for PubMedID 25775522

• Microcircuits and their interactions in epilepsy: is the focus out of focus? NATURE NEUROSCIENCE Paz, J. T., Huguenard, J. R. 2015; 18 (3): 351-359

  Abstract

  Epileptic seizures represent dysfunctional neural networks dominated by excessive and/or hypersynchronous activity. Recent progress in the field has outlined two concepts regarding mechanisms of seizure generation, or ictogenesis. First, all seizures, even those associated with what have historically been thought of as 'primary generalized' epilepsies, appear to originate in local microcircuits and then propagate from that initial ictogenic zone. Second, seizures propagate through cerebral networks and engage microcircuits in distal nodes, a process that can be weakened or even interrupted by suppressing activity in such nodes. We describe various microcircuit motifs, with a special emphasis on one that has been broadly implicated in several epilepsies: feed-forward inhibition. Furthermore, we discuss how, in the dynamic network in which seizures propagate, focusing on circuit 'choke points' remote from the initiation site might be as important as that of the initial dysfunction, the seizure 'focus'.

  View details for DOI 10.1038/nn.3950

  View details for Web of Science ID 000350195100012

  View details for PubMedID 25710837

• Seizing upon mechanisms for impaired consciousness. Neuron Farzampour, Z., Huguenard, J. 2015; 85 (3): 453-455

  Abstract

  Transient loss of consciousness associated with focal temporal lobe seizures is a complex phenomenon with life-threatening repercussions. In this issue of Neuron, Motelow et al. (2015) describe decreased cholinergic drive and suppressed subcortical arousal in seizures as a novel mechanism for impaired cortical function.

  View details for DOI 10.1016/j.neuron.2015.01.023

  View details for PubMedID 25654251

• Cholinergic control of gamma power in the midbrain spatial attention network. journal of neuroscience Bryant, A. S., Goddard, C. A., Huguenard, J. R., Knudsen, E. I. 2015; 35 (2): 761-775

  Abstract

  The modulation of gamma power (25-90 Hz) is associated with attention and has been observed across species and brain areas. However, mechanisms that control these modulations are poorly understood. The midbrain spatial attention network in birds generates high-amplitude gamma oscillations in the local field potential that are thought to represent the highest priority location for attention. Here we explore, in midbrain slices from chickens, mechanisms that regulate the power of these oscillations, using high-resolution techniques including intracellular recordings from neurons targeted by calcium imaging. The results identify a specific subtype of neuron, expressing non-α7 nicotinic acetylcholine receptors, that directly drives inhibition in the gamma-generating circuit and switches the network into a primed state capable of producing high-amplitude oscillations. The special properties of this mechanism enable rapid, persistent changes in gamma power. The brain may employ this mechanism wherever rapid modulations of gamma power are critical to information processing.

  View details for DOI 10.1523/JNEUROSCI.4001-14.2015

  View details for PubMedID 25589769

• Optogenetics and Epilepsy: Past, Present and Future EPILEPSY CURRENTS Paz, J. T., Huguenard, J. R. 2015; 15 (1): 34-38

  Abstract

  The holy grail of epilepsy research is to understand the mechanisms underlying seizures so that patients with epilepsy can receive effective treatment or be cured, ideally with no significant side effects. Recent advances in neuroscience give such hope. Optogenetics is a modern neuroscience research tool that allows precise spatiotemporal control of defined cells and circuits and, thus, dissection of critical players and targeting them for responsive treatments. Here we review the state of the art of these approaches and their applications and implications in epilepsy research.

  View details for Web of Science ID 000352153900012

  View details for PubMedID 25678887

• Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D cultures. Nature Methods Pasca, A. M., Sloan, S., Clarke, L. E., Tian, Y., Makinson, C., Huber, N., Kim, C., Park, J., O’Rourke, N., Nguyen, K., Smith, S. J., Huguenard, J., Geschwind, D. H., Barres, B. A., Pasca, S. P. 2015: 671–78

  Abstract

  The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

  View details for DOI 10.1038/nmeth.3415

  View details for PubMedCentralID PMC4489980

• Endozepines. Advances in pharmacology (San Diego, Calif.) Farzampour, Z., Reimer, R. J., Huguenard, J. 2015; 72: 147-164

  Abstract

  Since their introduction in the 1960s, benzodiazepines (BZs) remain one of the most commonly prescribed medications, acting as potent sedatives, hypnotics, anxiolytics, anticonvulsants, and muscle relaxants. The primary neural action of BZs and related compounds is augmentation of inhibitory transmission, which occurs through allosteric modulation of the gamma-aminobutyric acid (GABA)-induced current at the gamma-aminobutyric acid receptor (GABAAR). The discovery of the BZ-binding site on GABAARs encouraged many to speculate that the brain produces its own endogenous ligands to this site (Costa & Guidotti, 1985). The romanticized quest for endozepines, endogenous ligands to the BZ-binding site, has uncovered a variety of ligands that might fulfill this role, including oleamides (Cravatt et al., 1995), nonpeptidic endozepines (Rothstein et al., 1992), and the protein diazepam-binding inhibitor (DBI) (Costa & Guidotti, 1985). Of these ligands, DBI, and affiliated peptide fragments, is the most extensively studied endozepine. The quest for the "brain's Valium" over the decades has been elusive as mainly negative allosteric modulatory effects have been observed (Alfonso, Le Magueresse, Zuccotti, Khodosevich, & Monyer, 2012; Costa & Guidotti, 1985), but recent evidence is accumulating that DBI displays regionally discrete endogenous positive modulation of GABA transmission through activation of the BZ receptor (Christian et al., 2013). Herein, we review the literature on this topic, focusing on identification of the endogenous molecule and its region-specific expression and function.

  View details for DOI 10.1016/bs.apha.2014.10.005

  View details for PubMedID 25600369

• Attentional flexibility in the thalamus: now we're getting SOMwhere. Nature neuroscience Makinson, C. D., Huguenard, J. R. 2014; 18 (1): 2-4

  View details for DOI 10.1038/nn.3902

  View details for PubMedID 25547472

• Frequency-dependent, cell type-divergent signaling in the hippocamposeptal projection. journal of neuroscience Mattis, J., Brill, J., Evans, S., Lerner, T. N., Davidson, T. J., Hyun, M., Ramakrishnan, C., Deisseroth, K., Huguenard, J. R. 2014; 34 (35): 11769-11780

  Abstract

  Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

  View details for DOI 10.1523/JNEUROSCI.5188-13.2014

  View details for PubMedID 25164672

  View details for PubMedCentralID PMC4145178

• Parallel Midbrain Microcircuits Perform Independent Temporal Transformations JOURNAL OF NEUROSCIENCE Goddard, C. A., Huguenard, J., Knudsen, E. 2014; 34 (24): 8130-8138

  Abstract

  The capacity to select the most important information and suppress distracting information is crucial for survival. The midbrain contains a network critical for the selection of the strongest stimulus for gaze and attention. In avians, the optic tectum (OT; called the superior colliculus in mammals) and the GABAergic nucleus isthmi pars magnocellularis (Imc) cooperate in the selection process. In the chicken, OT layer 10, located in intermediate layers, responds to afferent input with gamma periodicity (25-75 Hz), measured at the level of individual neurons and the local field potential. In contrast, Imc neurons, which receive excitatory input from layer 10 neurons, respond with tonic, unusually high discharge rates (>150 spikes/s). In this study, we reveal the source of this high-rate inhibitory activity: layer 10 neurons that project to the Imc possess specialized biophysical properties that enable them to transform afferent drive into high firing rates (∼130 spikes/s), whereas neighboring layer 10 neurons, which project elsewhere, transform afferent drive into lower-frequency, periodic discharge patterns. Thus, the intermediate layers of the OT contain parallel, intercalated microcircuits that generate different temporal patterns of activity linked to the functions of their respective downstream targets.

  View details for DOI 10.1523/JNEUROSCI.4399-13.2014

  View details for Web of Science ID 000338338700008

  View details for PubMedCentralID PMC4051971

• Parallel midbrain microcircuits perform independent temporal transformations. The Journal of neuroscience : the official journal of the Society for Neuroscience Goddard, C. A., Huguenard, J., Knudsen, E. 2014; 34 (24): 8130-8

  Abstract

  The capacity to select the most important information and suppress distracting information is crucial for survival. The midbrain contains a network critical for the selection of the strongest stimulus for gaze and attention. In avians, the optic tectum (OT; called the superior colliculus in mammals) and the GABAergic nucleus isthmi pars magnocellularis (Imc) cooperate in the selection process. In the chicken, OT layer 10, located in intermediate layers, responds to afferent input with gamma periodicity (25-75 Hz), measured at the level of individual neurons and the local field potential. In contrast, Imc neurons, which receive excitatory input from layer 10 neurons, respond with tonic, unusually high discharge rates (>150 spikes/s). In this study, we reveal the source of this high-rate inhibitory activity: layer 10 neurons that project to the Imc possess specialized biophysical properties that enable them to transform afferent drive into high firing rates (∼130 spikes/s), whereas neighboring layer 10 neurons, which project elsewhere, transform afferent drive into lower-frequency, periodic discharge patterns. Thus, the intermediate layers of the OT contain parallel, intercalated microcircuits that generate different temporal patterns of activity linked to the functions of their respective downstream targets.

  View details for DOI 10.1523/JNEUROSCI.4399-13.2014

  View details for PubMedID 24920618

  View details for PubMedCentralID PMC4051971

• A local glutamate-glutamine cycle sustains synaptic excitatory transmitter release. Neuron Tani, H., Dulla, C. G., Farzampour, Z., Taylor-Weiner, A., Huguenard, J. R., Reimer, R. J. 2014; 81 (4): 888-900

  Abstract

  Biochemical studies suggest that excitatory neurons are metabolically coupled with astrocytes to generate glutamate for release. However, the extent to which glutamatergic neurotransmission depends on this process remains controversial because direct electrophysiological evidence is lacking. The distance between cell bodies and axon terminals predicts that glutamine-glutamate cycle is synaptically localized. Hence, we investigated isolated nerve terminals in brain slices by transecting hippocampal Schaffer collaterals and cortical layer I axons. Stimulating with alternating periods of high frequency (20 Hz) and rest (0.2 Hz), we identified an activity-dependent reduction in synaptic efficacy that correlated with reduced glutamate release. This was enhanced by inhibition of astrocytic glutamine synthetase and reversed or prevented by exogenous glutamine. Importantly, this activity dependence was also revealed with an in-vivo-derived natural stimulus both at network and cellular levels. These data provide direct electrophysiological evidence that an astrocyte-dependent glutamate-glutamine cycle is required to maintain active neurotransmission at excitatory terminals.

  View details for DOI 10.1016/j.neuron.2013.12.026

  View details for PubMedID 24559677

• Spatially Reciprocal Inhibition of Inhibition within a Stimulus Selection Network in the Avian Midbrain. PloS one Goddard, C. A., Mysore, S. P., Bryant, A. S., Huguenard, J. R., Knudsen, E. I. 2014; 9 (1)

  View details for DOI 10.1371/journal.pone.0085865

  View details for PubMedID 24465755

• Modulation of Short-Term Plasticity in the Corticothalamic Circuit by Group III Metabotropic Glutamate Receptors JOURNAL OF NEUROSCIENCE Kyuyoung, C. L., Huguenard, J. R. 2014; 34 (2): 675-687

  Abstract

  Recurrent connections in the corticothalamic circuit underlie oscillatory behavior in this network and range from normal sleep rhythms to the abnormal spike-wave discharges seen in absence epilepsy. The propensity of thalamic neurons to fire postinhibitory rebound bursts mediated by low-threshold calcium spikes renders the circuit vulnerable to both increased excitation and increased inhibition, such as excessive excitatory cortical drive to thalamic reticular (RT) neurons or heightened inhibition of thalamocortical relay (TC) neurons by RT. In this context, a protective role may be played by group III metabotropic receptors (mGluRs), which are uniquely located in the presynaptic active zone and typically act as autoreceptors or heteroceptors to depress synaptic release. Here, we report that these receptors regulate short-term plasticity at two loci in the corticothalamic circuit in rats: glutamatergic cortical synapses onto RT neurons and GABAergic synapses onto TC neurons in somatosensory ventrobasal thalamus. The net effect of group III mGluR activation at these synapses is to suppress thalamic oscillations as assayed in vitro. These findings suggest a functional role of these receptors to modulate corticothalamic transmission and protect against prolonged activity in the network.

  View details for DOI 10.1523/JNEUROSCI.1477-13.2014

  View details for Web of Science ID 000329791300032

  View details for PubMedID 24403165

  View details for PubMedCentralID PMC3870944

• Spatially reciprocal inhibition of inhibition within a stimulus selection network in the avian midbrain. PloS one Goddard, C. A., Mysore, S. P., Bryant, A. S., Huguenard, J. R., Knudsen, E. I. 2014; 9 (1)

  Abstract

  Reciprocal inhibition between inhibitory projection neurons has been proposed as the most efficient circuit motif to achieve the flexible selection of one stimulus among competing alternatives. However, whether such a motif exists in networks that mediate selection is unclear. Here, we study the connectivity within the nucleus isthmi pars magnocellularis (Imc), a GABAergic nucleus that mediates competitive selection in the midbrain stimulus selection network. Using laser photostimulation of caged glutamate, we find that feedback inhibitory connectivity is global within the Imc. Unlike typical lateral inhibition in other circuits, intra-Imc inhibition remains functionally powerful over long distances. Anatomically, we observed long-range axonal projections and retrograde somatic labeling from focal injections of bi-directional tracers in the Imc, consistent with spatial reciprocity of intra-Imc inhibition. Together, the data indicate that spatially reciprocal inhibition of inhibition occurs throughout the Imc. Thus, the midbrain selection circuit possesses the most efficient circuit motif possible for fast, reliable, and flexible selection.

  View details for DOI 10.1371/journal.pone.0085865

  View details for PubMedID 24465755

  View details for PubMedCentralID PMC3897538

• Astrocytes potentiate GABAergic transmission in the thalamic reticular nucleus via endozepine signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christian, C. A., Huguenard, J. R. 2013; 110 (50): 20278-20283

  Abstract

  Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition.

  View details for DOI 10.1073/pnas.1318031110

  View details for Web of Science ID 000328061700072

  View details for PubMedID 24262146

  View details for PubMedCentralID PMC3864346

• Sniffer patch laser uncaging response (SPLURgE): an assay of regional differences in allosteric receptor modulation and neurotransmitter clearance JOURNAL OF NEUROPHYSIOLOGY Christian, C. A., Huguenard, J. R. 2013; 110 (7): 1722-1731

  Abstract

  Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-aminobutyric acid (GABA) type-A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the Sniffer Patch Laser Uncaging REsponse (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice, and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators, and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors.

  View details for DOI 10.1152/jn.00319.2013

  View details for Web of Science ID 000325346300023

  View details for PubMedID 23843428

• Endogenous Positive Allosteric Modulation of GABA(A) Receptors by Diazepam binding inhibitor NEURON Christian, C. A., Herbert, A. G., Holt, R. L., Peng, K., Sherwood, K. D., Pangratz-Fuehrer, S., Rudolph, U., Huguenard, J. R. 2013; 78 (6): 1063-1074

  Abstract

  Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABAARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABAARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together, these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking ("endozepine") roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a therapy for epilepsy and other neurological disorders.

  View details for DOI 10.1016/j.neuron.2013.04.026

  View details for Web of Science ID 000321026900012

  View details for PubMedID 23727119

• Reemerging role of cable properties in action potential initiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ma, Y., Huguenard, J. R. 2013; 110 (10): 3715-3716

  View details for DOI 10.1073/pnas.1300520110

  View details for Web of Science ID 000316377400022

  View details for PubMedID 23440200

  View details for PubMedCentralID PMC3593907

• Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury NATURE NEUROSCIENCE Paz, J. T., Davidson, T. J., Frechette, E. S., Delord, B., Parada, I., Peng, K., Deisseroth, K., Huguenard, J. R. 2013; 16 (1): 64-U98

  Abstract

  Cerebrocortical injuries such as stroke are a major source of disability. Maladaptive consequences can result from post-injury local reorganization of cortical circuits. For example, epilepsy is a common sequela of cortical stroke, but the mechanisms responsible for seizures following cortical injuries remain unknown. In addition to local reorganization, long-range, extra-cortical connections might be critical for seizure maintenance. In rats, we found that the thalamus, a structure that is remote from, but connected to, the injured cortex, was required to maintain cortical seizures. Thalamocortical neurons connected to the injured epileptic cortex underwent changes in HCN channel expression and became hyperexcitable. Targeting these neurons with a closed-loop optogenetic strategy revealed that reducing their activity in real-time was sufficient to immediately interrupt electrographic and behavioral seizures. This approach is of therapeutic interest for intractable epilepsy, as it spares cortical function between seizures, in contrast with existing treatments, such as surgical lesioning or drugs.

  View details for DOI 10.1038/nn.3269

  View details for Web of Science ID 000312633900014

  View details for PubMedID 23143518

• Sleep and Epilepsy: A Summary of the 2011 Merritt-Putnam Symposium EPILEPSY CURRENTS DeWolfe, J. L., Malow, B., Huguenard, J., Stickgold, R., Bourgeois, B., Holmes, G. L. 2013; 13 (1): 42-49

  View details for Web of Science ID 000321551400014

  View details for PubMedID 23447740

• Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation NEUROBIOLOGY OF DISEASE Dulla, C. G., Tani, H., Brill, J., Reimer, R. J., Huguenard, J. R. 2013; 49: 232-246

  View details for DOI 10.1016/j.nbd.2012.09.001

  View details for Web of Science ID 000311594600025

• A call for transparent reporting to optimize the predictive value of preclinical research NATURE Landis, S. C., Amara, S. G., Asadullah, K., Austin, C. P., Blumenstein, R., Bradley, E. W., Crystal, R. G., Darnell, R. B., Ferrante, R. J., Fillit, H., Finkelstein, R., Fisher, M., Gendelman, H. E., Golub, R. M., Goudreau, J. L., Gross, R. A., Gubitz, A. K., Hesterlee, S. E., Howells, D. W., Huguenard, J., Kelner, K., Koroshetz, W., Krainc, D., Lazic, S. E., Levine, M. S., Macleod, M. R., McCall, J. M., Moxley, R. T., Narasimhan, K., Noble, L. J., Perrin, S., Porter, J. D., Steward, O., Unger, E., Utz, U., Silberberg, S. D. 2012; 490 (7419): 187-191

  Abstract

  The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

  View details for DOI 10.1038/nature11556

  View details for Web of Science ID 000309733300036

  View details for PubMedID 23060188

  View details for PubMedCentralID PMC3511845

• Mechanism for Hypocretin-mediated sleep-to-wake transitions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Carter, M. E., Brill, J., Bonnavion, P., Huguenard, J. R., Huerta, R., de Lecea, L. 2012; 109 (39): E2635-E2644

  Abstract

  Current models of sleep/wake regulation posit that Hypocretin (Hcrt)-expressing neurons in the lateral hypothalamus promote and stabilize wakefulness by projecting to subcortical arousal centers. However, the critical downstream effectors of Hcrt neurons are unknown. Here we use optogenetic, pharmacological, and computational tools to investigate the functional connectivity between Hcrt neurons and downstream noradrenergic neurons in the locus coeruleus (LC) during nonrapid eye movement (NREM) sleep. We found that photoinhibiting LC neurons during Hcrt stimulation blocked Hcrt-mediated sleep-to-wake transitions. In contrast, when LC neurons were optically stimulated to increase membrane excitability, concomitant photostimulation of Hcrt neurons significantly increased the probability of sleep-to-wake transitions compared with Hcrt stimulation alone. We also built a conductance-based computational model of Hcrt-LC circuitry that recapitulates our behavioral results using LC neurons as the main effectors of Hcrt signaling. These results establish the Hcrt-LC connection as a critical integrator-effector circuit that regulates NREM sleep/wake behavior during the inactive period. This coupling of distinct neuronal systems can be generalized to other hypothalamic integrator nuclei with downstream effector/output populations in the brain.

  View details for DOI 10.1073/pnas.1202526109

  View details for Web of Science ID 000309604500009

  View details for PubMedID 22955882

  View details for PubMedCentralID PMC3465396

• Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation. Neurobiology of disease Dulla, C. G., Tani, H., Brill, J., Reimer, R. J., Huguenard, J. R. 2012; 49C: 232-246

  Abstract

  Cortical malformations can cause intractable epilepsy, but the underlying epileptogenic mechanisms are poorly understood. We used high-speed glutamate biosensor imaging to ask how glutamatergic signaling is altered in cortical malformations induced by neonatal freeze-lesions (FL). In non-lesion neocortical slices from 2 to 8week old rats, evoked glutamate signals were symmetrical in the medio-lateral axis and monotonic, correlating with simple, brief (≈50ms) local field potentials (LFPs). By contrast, in FL cortex glutamate signals were prolonged, increased in amplitude, and polyphasic, which paralleled a prolongation of the LFP. Using glutamate biosensor imaging, we found that glutamate signals propagated throughout large areas of FL cortex and were asymmetric (skewed toward the lesion). Laminar analysis demonstrated a shift in the region of maximal glutamate release toward superficial layers in FL cortex. The ability to remove exogenous glutamate was increased within the FL itself but was decreased in immediately adjacent regions. There were corresponding alterations in astrocyte density, with an increase within the lesion and a decrease in deep cortical layers surrounding the lesion. These findings demonstrate both network connectivity and glutamate metabolism are altered in this cortical malformation model and suggests that the regional ability of astrocytes to remove released glutamate may be inversely related to local excitability.

  View details for DOI 10.1016/j.nbd.2012.09.001

  View details for PubMedID 22982711

  View details for PubMedCentralID PMC3574209

• Enhanced NMDA Receptor-Dependent Thalamic Excitation and Network Oscillations in Stargazer Mice JOURNAL OF NEUROSCIENCE Lacey, C. J., Bryant, A., Brill, J., Huguenard, J. R. 2012; 32 (32): 11067-11081

  Abstract

  Disturbances in corticothalamic circuitry can lead to absence epilepsy. The reticular thalamic nucleus (RTN) plays a pivotal role in that it receives excitation from cortex and thalamus and, when strongly activated, can generate excessive inhibitory output and epileptic thalamocortical oscillations that depend on postinhibitory rebound. Stargazer (stg) mice have prominent absence seizures resulting from a mutant form of the AMPAR auxiliary protein stargazin. Reduced AMPAR excitation in RTN has been demonstrated previously in stg, yet the mechanisms leading from RTN hypoexcitation to epilepsy are unknown and unexpected because thalamic epileptiform oscillatory activity requires AMPARs. We demonstrate hyperexcitability in stg thalamic slices and further characterize the various excitatory inputs to RTN using electrical stimulation and laser scanning photostimulation. Patch-clamp recordings of spontaneous and evoked EPSCs in RTN neurons demonstrate reduced amplitude and increased duration of the AMPAR component with an increased amplitude NMDAR component. Short 200 Hz stimulus trains evoked a gradual approximately threefold increase in NMDAR EPSCs compared with single stimuli in wild-type (WT), indicating progressive NMDAR recruitment, whereas in stg cells, NMDAR responses were nearly maximal with single stimuli. Array tomography revealed lower synaptic, but higher perisynaptic, AMPAR density in stg RTN. Increasing NMDAR activity via reduced [Mg2+]o in WT phenocopied the thalamic hyperexcitability observed in stg, whereas changing [Mg2+]o had no effect on stg slices. These findings suggest that, in stg, a trafficking defect in synaptic AMPARs in RTN cells leads to a compensatory increase in synaptic NMDARs and enhanced thalamic excitability.

  View details for DOI 10.1523/JNEUROSCI.5604-11.2012

  View details for Web of Science ID 000307640000026

  View details for PubMedID 22875939

  View details for PubMedCentralID PMC3430525

• R U OK? The Novel Therapeutic Potential of R Channels in Epilepsy EPILEPSY CURRENTS Paz, J. T., Huguenard, J. R. 2012; 12 (2): 75-76

  View details for Web of Science ID 000303854400012

  View details for PubMedID 22473549

• Influence of a Subtype of Inhibitory Interneuron on Stimulus-Specific Responses in Visual Cortex CEREBRAL CORTEX Mao, R., Schummers, J., Knoblich, U., Lacey, C. J., Van Wart, A., Cobos, I., Kim, C., Huguenard, J. R., Rubenstein, J. L., Sur, M. 2012; 22 (3): 493-508

  Abstract

  Inhibition modulates receptive field properties and integrative responses of neurons in cortical circuits. The contribution of specific interneuron classes to cortical circuits and emergent responses is unknown. Here, we examined neuronal responses in primary visual cortex (V1) of adult Dlx1(-/-) mice, which have a selective reduction in cortical dendrite-targeting interneurons (DTIs) that express calretinin, neuropeptide Y, and somatostatin. The V1 neurons examined in Dlx1(-/-) mice have reduced orientation selectivity and altered firing rates, with elevated late responses, suggesting that local inhibition at dendrites has a specific role in modulating neuronal computations. We did not detect overt changes in the physiological properties of thalamic relay neurons and features of thalamocortical projections, such as retinotopic maps and eye-specific inputs, in the mutant mice, suggesting that the defects are cortical in origin. These experimental results are well explained by a computational model that integrates broad tuning from dendrite-targeting and narrower tuning from soma-targeting interneuron subclasses. Our findings suggest a key role for DTIs in the fine-tuning of stimulus-specific cortical responses.

  View details for DOI 10.1093/cercor/bhr057

  View details for Web of Science ID 000300495800001

  View details for PubMedID 21666125

  View details for PubMedCentralID PMC3278313

• Gamma Oscillations Are Generated Locally in an Attention-Related Midbrain Network NEURON Goddard, C. A., Sridharan, D., Huguenard, J. R., Knudsen, E. I. 2012; 73 (3): 567-580

  Abstract

  Gamma-band (25-140 Hz) oscillations are a hallmark of sensory processing in the forebrain. The optic tectum (OT), a midbrain structure implicated in sensorimotor processing and attention, also exhibits gamma oscillations. However, the origin and mechanisms of these oscillations remain unknown. We discovered that in acute slices of the avian OT, persistent (>100 ms) epochs of large amplitude gamma oscillations can be evoked that closely resemble those recorded in vivo. We found that cholinergic, glutamatergic, and GABAergic mechanisms differentially regulate the structure of the oscillations at various timescales. These persistent oscillations originate in the multisensory layers of the OT and are broadcast to visual layers via the cholinergic nucleus Ipc, providing a potential mechanism for enhancing the processing of visual information within the OT. The finding that the midbrain contains an intrinsic gamma-generating circuit suggests that the OT could use its own oscillatory code to route signals to forebrain networks.

  View details for DOI 10.1016/j.neuron.2011.11.028

  View details for Web of Science ID 000300140600016

  View details for PubMedID 22325207

  View details for PubMedCentralID PMC3291715

• Increased Excitatory Synaptic Input to Granule Cells from Hilar and CA3 Regions in a Rat Model of Temporal Lobe Epilepsy JOURNAL OF NEUROSCIENCE Zhang, W., Huguenard, J. R., Buckmaster, P. S. 2012; 32 (4): 1183-1196

  Abstract

  One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.

  View details for DOI 10.1523/JNEUROSCI.5342-11.2012

  View details for Web of Science ID 000299801100005

  View details for PubMedID 22279204

• Neocortical excitation/inhibition balance in information processing and social dysfunction NATURE Yizhar, O., Fenno, L. E., Prigge, M., Schneider, F., Davidson, T. J., O'Shea, D. J., Sohal, V. S., Goshen, I., Finkelstein, J., Paz, J. T., Stehfest, K., Fudim, R., Ramakrishnan, C., Huguenard, J. R., Hegemann, P., Deisseroth, K. 2011; 477 (7363): 171-178

  Abstract

  Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

  View details for DOI 10.1038/nature10360

  View details for Web of Science ID 000294603900027

  View details for PubMedID 21796121

• A new mode of corticothalamic transmission revealed in the Gria4(-/-) model of absence epilepsy NATURE NEUROSCIENCE Paz, J. T., Bryant, A. S., Peng, K., Fenno, L., Yizhar, O., Frankel, W. N., Deisseroth, K., Huguenard, J. R. 2011; 14 (9): 1167-U225

  Abstract

  Cortico-thalamo-cortical circuits mediate sensation and generate neural network oscillations associated with slow-wave sleep and various epilepsies. Cortical input to sensory thalamus is thought to mainly evoke feed-forward synaptic inhibition of thalamocortical (TC) cells via reticular thalamic nucleus (nRT) neurons, especially during oscillations. This relies on a stronger synaptic strength in the cortico-nRT pathway than in the cortico-TC pathway, allowing the feed-forward inhibition of TC cells to overcome direct cortico-TC excitation. We found a systemic and specific reduction in strength in GluA4-deficient (Gria4(-/-)) mice of one excitatory synapse of the rhythmogenic cortico-thalamo-cortical system, the cortico-nRT projection, and observed that the oscillations could still be initiated by cortical inputs via the cortico-TC-nRT-TC pathway. These results reveal a previously unknown mode of cortico-thalamo-cortical transmission, bypassing direct cortico-nRT excitation, and describe a mechanism for pathological oscillation generation. This mode could be active under other circumstances, representing a previously unknown channel of cortico-thalamo-cortical information processing.

  View details for DOI 10.1038/nn.2896

  View details for Web of Science ID 000294284900017

  View details for PubMedID 21857658

  View details for PubMedCentralID PMC3308017

• Reorganization of Inhibitory Synaptic Circuits in Rodent Chronically Injured Epileptogenic Neocortex CEREBRAL CORTEX Jin, X., Huguenard, J. R., Prince, D. A. 2011; 21 (5): 1094-1104

  Abstract

  Reduced synaptic inhibition is an important factor contributing to posttraumatic epileptogenesis. Axonal sprouting and enhanced excitatory synaptic connectivity onto rodent layer V pyramidal (Pyr) neurons occur in epileptogenic partially isolated (undercut) neocortex. To determine if enhanced excitation also affects inhibitory circuits, we used laser scanning photostimulation of caged glutamate and whole-cell recordings from GAD67-GFP-expressing mouse fast spiking (FS) interneurons and Pyr cells in control and undercut in vitro slices to map excitatory and inhibitory synaptic inputs. Results are 1) the region-normalized excitatory postsynaptic current (EPSC) amplitudes and proportion of uncaging sites from which EPSCs could be evoked (hotspot ratio) "increased" significantly in FS cells of undercut slices; 2) in contrast, these parameters were significantly "decreased" for inhibitory postsynaptic currents (IPSCs) in undercut FS cells; and 3) in rat layer V Pyr neurons, we found significant decreases in IPSCs in undercut versus control Pyr neurons. The decreases were mainly located in layers II and IV, suggesting a reduction in the efficacy of interlaminar synaptic inhibition. Results suggest that there is significant synaptic reorganization in this model of posttraumatic epilepsy, resulting in increased excitatory drive and reduced inhibitory input to FS interneurons that should enhance their inhibitory output and, in part, offset similar alterations in innervation of Pyr cells.

  View details for DOI 10.1093/cercor/bhq181

  View details for Web of Science ID 000289578900012

  View details for PubMedID 20855494

  View details for PubMedCentralID PMC3077430

• Martinotti Cells: Community Organizers NEURON Lee, C. K., Huguenard, J. R. 2011; 69 (6): 1042-1045

  Abstract

  The specificity of connections made by inhibitory interneurons in the neocortex is not well understood. In this issue of Neuron, Fino and Yuste (2011) use an enhanced version of two-photon glutamate uncaging, which preserves inhibitory synaptic transmission, to demonstrate that somatostatin-positive interneurons form densely convergent connections onto pyramidal cells in layer 2/3 of mouse frontal cortex.

  View details for DOI 10.1016/j.neuron.2011.03.003

  View details for PubMedID 21435551

• Enhanced Infragranular and Supragranular Synaptic Input onto Layer 5 Pyramidal Neurons in a Rat Model of Cortical Dysplasia CEREBRAL CORTEX Brill, J., Huguenard, J. R. 2010; 20 (12): 2926-2938

  Abstract

  Cortical dysplasias frequently underlie neurodevelopmental disorders and epilepsy. Rats with a neonatally induced cortical microgyrus [freeze-lesion (FL)], a model of human polymicrogyria, display epileptiform discharges in vitro. We probed excitatory and inhibitory connectivity onto neocortical pyramidal neurons in layers 2/3 and 5 of postnatal day 16-22 rats, approximately 1-2 mm lateral of the lesion, using laser scanning photostimulation (LSPS)/glutamate uncaging. Excitatory input from deep and supragranular layers to layer 5 pyramidal cells was greater in FL cortex, while no significant differences were seen in layer 2/3 cells. The increased input was due to a greater number of LSPS-evoked excitatory postsynaptic currents (EPSCs), without differences in amplitude or kinetics. Inhibitory input was increased in a region-specific manner in pyramidal cells in FL cortex, due to an increased inhibitory postsynaptic current (IPSC) amplitude. Connectivity within layer 5, parts of which are destroyed during lesioning, was more severely affected than connectivity in layer 2/3. Thus, we observed 2 distinct mechanisms of altered synaptic input: 1) increased EPSC frequency suggesting an increased number of excitatory synapses and 2) higher IPSC amplitude, suggesting an increased strength of inhibitory synapses. These increases in both excitatory and inhibitory connectivity may limit the extent of circuit hyperexcitability.

  View details for DOI 10.1093/cercor/bhq040

  View details for Web of Science ID 000284162300015

  View details for PubMedID 20338974

  View details for PubMedCentralID PMC3003588

• Differential effects of Na plus -K plus ATPase blockade on cortical layer V neurons JOURNAL OF PHYSIOLOGY-LONDON Anderson, T. R., Huguenard, J. R., Prince, D. A. 2010; 588 (22): 4401-4414

  Abstract

  Sodium-potassium ATPase ('Na(+)-K(+) ATPase') contributes to the maintenance of the resting membrane potential and the transmembrane gradients for Na(+) and K(+) in neurons. Activation of Na(+)-K(+) ATPase may be important in controlling increases in intracellular sodium during periods of increased neuronal activity. Down-regulation of Na(+)-K(+) ATPase activity is implicated in numerous CNS disorders, including epilepsy. Although Na(+)-K(+) ATPase is present in all neurons, little is known about its activity in different subclasses of neocortical cells. We assessed the physiological properties of Na(+)-K(+) ATPase in fast-spiking (FS) interneurons and pyramidal (PYR) cells to test the hypothesis that Na(+)-K(+) ATPase activity would be relatively greater in neurons that generated high frequency action potentials (the FS cells). Whole-cell patch clamp recordings were made from FS and PYR neurons in layer V of rat sensorimotor cortical slices maintained in vitro using standard techniques. Bath perfusion of Na(+)-K(+) ATPase antagonists (ouabain or dihydro-ouabain) induced either a membrane depolarization in current clamp, or inward current under voltage clamp in both cell types. PYR neurons were divided into two subpopulations based on the amplitude of the voltage or current shift in response to Na(+)-K(+) ATPase blockade. The two PYR cell groups did not differ significantly in electrophysiological properties including resting membrane potential, firing pattern, input resistance and capacitance. Membrane voltage responses of FS cells to Na(+)-K(+) ATPase blockade were intermediate between the two PYR cell groups (P < 0.05). The resting Na(+)-K(+) ATPase current density in FS interneurons, assessed by application of blockers, was 3- to 7-fold larger than in either group of PYR neurons. Na(+)-K(+) ATPase activity was increased either through direct Na(+) loading via the patch pipette or by focal application of glutamate (20 mM puffs). Under these conditions FS interneurons exhibited the largest increase in Na(+)-K(+) ATPase activity. We conclude that resting Na(+)-K(+) ATPase activity and sensitivity to changes in internal Na(+) concentration vary between and within classes of cortical neurons. These differences may have important consequences in pathophysiological disorders associated with down-regulation of Na(+)-K(+) ATPase and hyperexcitability within cortical networks.

  View details for DOI 10.1113/jphysiol.2010.191858

  View details for Web of Science ID 000284276000011

  View details for PubMedID 20819946

  View details for PubMedCentralID PMC3008847

• Astrocytes as Gatekeepers of GABA(B) Receptor Function JOURNAL OF NEUROSCIENCE Beenhakker, M. P., Huguenard, J. R. 2010; 30 (45): 15262-15276

  Abstract

  The long-lasting actions of the inhibitory neurotransmitter GABA result from the activation of metabotropic GABA(B) receptors. Enhanced GABA(B)-mediated IPSCs are critical for the generation of generalized thalamocortical seizures. Here, we demonstrate that GABA(B)-mediated IPSCs recorded in the thalamus are primarily defined by GABA diffusion and activation of distal extrasynaptic receptors potentially up to tens of micrometers from synapses. We also show that this diffusion is differentially regulated by two astrocytic GABA transporters, GAT1 and GAT3, which are localized near and far from synapses, respectively. A biologically constrained model of GABA diffusion and uptake shows how the two GATs differentially modulate amplitude and duration of GABA(B) IPSCs. Specifically, the perisynaptic expression of GAT1 enables it to regulate GABA levels near synapses and selectively modulate peak IPSC amplitude, which is primarily dependent on perisynaptic receptor occupancy. GAT3 expression, however, is broader and includes distal extrasynaptic regions. As such, GAT3 acts as a gatekeeper to prevent diffusion of GABA away from synapses toward extrasynaptic regions that contain a potentially enormous pool of GABA(B) receptors. Targeting this gatekeeper function may provide new pharmacotherapeutic opportunities to prevent the excessive GABA(B) receptor activation that appears necessary for thalamic seizure generation.

  View details for DOI 10.1523/JNEUROSCI.3243-10.2010

  View details for Web of Science ID 000284096300041

  View details for PubMedID 21068331

  View details for PubMedCentralID PMC3056552

• Desynchronization of Neocortical Networks by Asynchronous Release of GABA at Autaptic and Synaptic Contacts from Fast-Spiking Interneurons PLOS BIOLOGY Manseau, F., Marinelli, S., Mendez, P., Schwaller, B., Prince, D. A., Huguenard, J. R., Bacci, A. 2010; 8 (9)

  Abstract

  Networks of specific inhibitory interneurons regulate principal cell firing in several forms of neocortical activity. Fast-spiking (FS) interneurons are potently self-inhibited by GABAergic autaptic transmission, allowing them to precisely control their own firing dynamics and timing. Here we show that in FS interneurons, high-frequency trains of action potentials can generate a delayed and prolonged GABAergic self-inhibition due to sustained asynchronous release at FS-cell autapses. Asynchronous release of GABA is simultaneously recorded in connected pyramidal (P) neurons. Asynchronous and synchronous autaptic release show differential presynaptic Ca(2+) sensitivity, suggesting that they rely on different Ca(2+) sensors and/or involve distinct pools of vesicles. In addition, asynchronous release is modulated by the endogenous Ca(2+) buffer parvalbumin. Functionally, asynchronous release decreases FS-cell spike reliability and reduces the ability of P neurons to integrate incoming stimuli into precise firing. Since each FS cell contacts many P neurons, asynchronous release from a single interneuron may desynchronize a large portion of the local network and disrupt cortical information processing.

  View details for DOI 10.1371/journal.pbio.1000492

  View details for Web of Science ID 000282279200011

  View details for PubMedID 20927409

• Focal Cortical Infarcts Alter Intrinsic Excitability and Synaptic Excitation in the Reticular Thalamic Nucleus JOURNAL OF NEUROSCIENCE Paz, J. T., Christian, C. A., Parada, I., Prince, D. A., Huguenard, J. R. 2010; 30 (15): 5465-5479

  Abstract

  Focal cortical injuries result in death of cortical neurons and their efferents and ultimately in death or damage of thalamocortical relay (TCR) neurons that project to the affected cortical area. Neurons of the inhibitory reticular thalamic nucleus (nRT) receive excitatory inputs from corticothalamic and thalamocortical axons and are thus denervated by such injuries, yet nRT cells generally survive these insults to a greater degree than TCR cells. nRT cells inhibit TCR cells, regulate thalamocortical transmission, and generate cerebral rhythms including those involved in thalamocortical epilepsies. The survival and reorganization of nRT after cortical injury would determine recovery of thalamocortical circuits after injury. However, the physiological properties and connectivity of the survivors remain unknown. To study possible alterations in nRT neurons, we used the rat photothrombosis model of cortical stroke. Using in vitro patch-clamp recordings at various times after the photothrombotic injury, we show that localized strokes in the somatosensory cortex induce long-term reductions in intrinsic excitability and evoked synaptic excitation of nRT cells by the end of the first week after the injury. We find that nRT neurons in injured rats show (1) decreased membrane input resistance, (2) reduced low-threshold calcium burst responses, and (3) weaker evoked excitatory synaptic responses. Such alterations in nRT cellular excitability could lead to loss of nRT-mediated inhibition in relay nuclei, increased output of surviving TCR cells, and enhanced thalamocortical excitation, which may facilitate recovery of thalamic and cortical sensory circuits. In addition, such changes could be maladaptive, leading to injury-induced epilepsy.

  View details for DOI 10.1523/JNEUROSCI.5083-09.2010

  View details for Web of Science ID 000276685100033

  View details for PubMedID 20392967

  View details for PubMedCentralID PMC2861582

• Glutamine Is Required for Persistent Epileptiform Activity in the Disinhibited Neocortical Brain Slice JOURNAL OF NEUROSCIENCE Tani, H., Dulla, C. G., Huguenard, J. R., Reimer, R. J. 2010; 30 (4): 1288-1300

  Abstract

  The neurotransmitter glutamate is recycled through an astrocytic-neuronal glutamate-glutamine cycle in which synaptic glutamate is taken up by astrocytes, metabolized to glutamine, and transferred to neurons for conversion back to glutamate and subsequent release. The extent to which neuronal glutamate release is dependent upon this pathway remains unclear. Here we provide electrophysiological and biochemical evidence that in acutely disinhibited rat neocortical slices, robust release of glutamate during sustained epileptiform activity requires that neurons be provided a continuous source of glutamine. We demonstrate that the uptake of glutamine into neurons for synthesis of glutamate destined for synaptic release is not strongly dependent on the system A transporters, but requires another unidentified glutamine transporter or transporters. Finally, we find that the attenuation of network activity through inhibition of neuronal glutamine transport is associated with reduced frequency and amplitude of spontaneous events detected at the single-cell level. These results indicate that availability of glutamine influences neuronal release of glutamate during periods of intense network activity.

  View details for DOI 10.1523/JNEUROSCI.0106-09.2010

  View details for Web of Science ID 000274050000011

  View details for PubMedID 20107056

  View details for PubMedCentralID PMC2821093

• Maintenance of Thalamic Epileptiform Activity Depends on the Astrocytic Glutamate-Glutamine Cycle JOURNAL OF NEUROPHYSIOLOGY Bryant, A. S., Li, B., Beenhakker, M. P., Huguenard, J. R. 2009; 102 (5): 2880-2888

  Abstract

  The generation of prolonged neuronal activity depends on the maintenance of synaptic neurotransmitter pools. The astrocytic glutamate-glutamine cycle is a major mechanism for recycling the neurotransmitters GABA and glutamate. Here we tested the effect of disrupting the glutamate-glutamine cycle on two types of neuronal activity patterns in the thalamus: sleep-related spindles and epileptiform oscillations. In recording conditions believed to induce glutamine scarcity, epileptiform oscillations showed a progressive reduction in duration that was partially reversible by the application of exogenous glutamine (300 muM). Blocking uptake of glutamine into neurons with alpha-(methylamino) isobutyric acid (5 mM) caused a similar reduction in oscillation duration, as did blocking neuronal GABA synthesis with 3-mercaptoproprionic acid (10 muM). However, comparable manipulations did not affect sleep spindles. Together, these results support a crucial role for the glutamate-glutamine cycle in providing the neurotransmitters necessary for the generation of epileptiform activity and suggest potential therapeutic approaches that selectively reduce seizure activity but maintain normal neuronal activity.

  View details for DOI 10.1152/jn.00476.2009

  View details for Web of Science ID 000271467300028

  View details for PubMedID 19741104

  View details for PubMedCentralID PMC2777838

• Resting Our Cortices by Going DOWN to Sleep NEURON Mignot, E., Huguenard, J. R. 2009; 63 (6): 719-721

  Abstract

  In this issue of Neuron, Vyazovskiy et al. reports on progressive changes in cortical unit activity within extended wakefulness and within extended sleep paralleling changes in EEG slow-wave sleep activity. Sleep debt may be integrated at the level of individual cortical neurons, providing support for the synaptic homeostasis theory.

  View details for DOI 10.1016/j.neuron.2009.09.008

  View details for Web of Science ID 000270569700004

  View details for PubMedID 19778500

• Who let the spikes out? NATURE NEUROSCIENCE Dulla, C. G., Huguenard, J. R. 2009; 12 (8): 959-960

  View details for DOI 10.1038/nn0809-959

  View details for Web of Science ID 000268396300004

  View details for PubMedID 19636347

  View details for PubMedCentralID PMC2907263

• Synergistic Roles of GABA(A) Receptors and SK Channels in Regulating Thalamocortical Oscillations JOURNAL OF NEUROPHYSIOLOGY Kleiman-Weiner, M., Beenhakker, M. P., Segal, W. A., Huguenard, J. R. 2009; 102 (1): 203-213

  Abstract

  Rhythmic oscillations throughout the cortex are observed during physiological and pathological states of the brain. The thalamus generates sleep spindle oscillations and spike-wave discharges characteristic of absence epilepsy. Much has been learned regarding the mechanisms underlying these oscillations from in vitro brain slice preparations. One widely used model to understand the epileptiform oscillations underlying absence epilepsy involves application of bicuculline methiodide (BMI) to brain slices containing the thalamus. BMI is a well-known GABAA receptor blocker that has previously been discovered to also block small-conductance, calcium-activated potassium (SK) channels. Here we report that the robust epileptiform oscillations observed during BMI application rely synergistically on both GABAA receptor and SK channel antagonism. Neither application of picrotoxin, a selective GABAA receptor antagonist, nor application of apamin, a selective SK channel antagonist, alone yielded the highly synchronized, long-lasting oscillations comparable to those observed during BMI application. However, partial blockade of SK channels by subnanomolar concentrations of apamin combined with picrotoxin sufficiently replicated BMI oscillations. We found that, at the cellular level, apamin enhanced the intrinsic excitability of reticular nucleus (RT) neurons but had no effect on relay neurons. This work suggests that regulation of RT excitability by SK channels can influence the excitability of thalamocortical networks and may illuminate possible pharmacological treatments for absence epilepsy. Finally, our results suggest that changes in the intrinsic properties of individual neurons and changes at the circuit level can robustly modulate these oscillations.

  View details for DOI 10.1152/jn.91158.2008

  View details for Web of Science ID 000267446000020

  View details for PubMedID 19386752

  View details for PubMedCentralID PMC2712277

• Neurons that Fire Together Also Conspire Together: Is Normal Sleep Circuitry Hijacked to Generate Epilepsy? NEURON Beenhakker, M. P., Huguenard, J. R. 2009; 62 (5): 612-632

  Abstract

  Brain circuits oscillate during sleep. The same circuits appear to generate pathological oscillations. In this review, we discuss recent advances in our understanding of how epilepsy co-opts normal, sleep-related circuits to generate seizures.

  View details for DOI 10.1016/j.neuron.2009.05.015

  View details for Web of Science ID 000267119500005

  View details for PubMedID 19524522

  View details for PubMedCentralID PMC2748990

• Robust Short-Latency Perisomatic Inhibition onto Neocortical Pyramidal Cells Detected by Laser-Scanning Photostimulation JOURNAL OF NEUROSCIENCE Brill, J., Huguenard, J. R. 2009; 29 (23): 7413-7423

  Abstract

  Inhibitory connectivity onto neocortical pyramidal cells was mapped using LSPS (laser-scanning photostimulation/glutamate uncaging). The average onset latency of IPSCs was shorter than that of EPSCs recorded in the same cells, indicating a specific mechanism for rapid network recruitment of inhibition. The majority of strong inhibitory synaptic inputs originated within 300 mum of the recorded cell's soma, had onset latencies between 4 and 10 ms, and high amplitude [short-latency IPSCs (slIPSCs)]. slIPSCs were GABA(A) receptor- mediated chloride currents that were evoked in an all-or-none manner. We tested whether slIPSCs resulted from somatic depolarization of presynaptic interneurons or from direct excitation of inhibitory presynaptic terminals via kainate receptors. Our evidence supports the former hypothesis: (1) slIPSCs had similar sensitivity to kainate and AMPA receptor blockers as electrically evoked EPSCs. (2) slIPSCs frequently had an notched rising phase suggestive of summated IPSCs resulting from repetitive firing of presynaptic neurons. (3) Latencies and interevent intervals were consistent with spike latencies and interspike intervals in fast-spiking (FS) interneurons. (4) slIPSCs were frequently evoked at spots where the recorded cell was also excited directly, but approximately 15% of spots from which slIPSCs were evoked did not overlap with the recorded neuron's cell body. We propose that slIPSCs from FS interneurons represent a pool of powerful inhibitory signals that can be recruited by local excitation. Because of their magnitude, progressive recruitment, and short latency, slIPSCs are a effective mechanism of regulating excitability in neocortical circuits.

  View details for DOI 10.1523/JNEUROSCI.6098-08.2009

  View details for Web of Science ID 000267130800005

  View details for PubMedID 19515909

  View details for PubMedCentralID PMC2797487

• A gain in GABA(A) receptor synaptic strength in thalamus reduces oscillatory activity and absence seizures PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schofield, C. M., Kleiman-Weiner, M., Rudolph, U., Huguenard, J. R. 2009; 106 (18): 7630-7635

  Abstract

  Neural inhibition within the thalamus is integral in shaping thalamocortical oscillatory activity. Fast, synaptic inhibition is primarily mediated by activation of heteropentameric GABA(A) receptor complexes. Here, we examined the synaptic physiology and network properties of mice lacking GABA(A) receptor alpha3, a subunit that in thalamus is uniquely expressed by inhibitory neurons of the reticular nucleus (nRT). Deletion of this subunit produced a powerful compensatory gain in inhibitory postsynaptic response in nRT neurons. Although, other forms of inhibitory and excitatory synaptic transmission in the circuit were unchanged, evoked thalamic oscillations were strongly dampened in alpha3 knockout mice. Furthermore, pharmacologically induced thalamocortical absence seizures displayed a reduction in length and power in alpha3 knockout mice. These studies highlight the role of GABAergic inhibitory strength within nRT in the maintenance of thalamic oscillations, and demonstrate that inhibitory intra-nRT synapses are a critical control point for regulating higher order thalamocortical network activity.

  View details for DOI 10.1073/pnas.0811326106

  View details for Web of Science ID 000265783600066

  View details for PubMedID 19380748

  View details for PubMedCentralID PMC2678654

• Sequential Changes in AMPA Receptor Targeting in the Developing Neocortical Excitatory Circuit JOURNAL OF NEUROSCIENCE Brill, J., Huguenard, J. R. 2008; 28 (51): 13918-13928

  Abstract

  Many principal neurons undergo an early developmental switch from GluR2-lacking to GluR2-containing synaptic glutamate receptors. We tested the generality and timing of the GluR2 switch in excitatory neurons of rat somatosensory cortex. Previous studies show that the switch occurs between postnatal day 14 (P14) and P16 in layer 5 pyramidal neurons. We show, using sensitivity to intracellular spermine, that a similar switch occurs between P12 and P14 in layer 2/3 pyramidal cells and between P7 and P8 in layer 4 stellate cells. The presence of GluR2-lacking receptors in layer 2/3 pyramidal cells before P12 was confirmed by demonstrating sensitivity to blockade by 1-naphthyl-acetyl-spermine and large single-channel conductances. GluR2 and the postsynaptic protein PSD95 show progressive colocalization in tissue from P10, P14, and P24 rats, mirroring electrophysiological developments. To distinguish whether changes in GluR2 expression or targeting underlie the switch, we characterized dendritic AMPA receptor responses using focal photolysis of caged glutamate. Contrary to synaptic responses, dendritic responses at all ages studied (P6-P40) were characteristic of GluR2-containing receptors. In addition, dendritically and synaptically evoked responses showed a corresponding decrease in NMDA/AMPA ratios in pyramidal cells, suggesting parallel mechanisms that regulate neuronal calcium levels. These data suggest that the GluR2 switch results from changes in AMPA receptor targeting during early postnatal development, and that rather than following the laminar sequence of cortical development, it proceeds sequentially from layer 4 to layer 2/3 and finally to layer 5b.

  View details for DOI 10.1523/JNEUROSCI.3229-08.2008

  View details for Web of Science ID 000261780100020

  View details for PubMedID 19091980

  View details for PubMedCentralID PMC2706010

• The Endocannabinoid 2-Arachidonoylglycerol Is Responsible for the Slow Self-Inhibition in Neocortical Interneurons JOURNAL OF NEUROSCIENCE Marinelli, S., Pacioni, S., Bisogno, T., Di Marzo, V., Prince, D. A., Huguenard, J. R., Bacci, A. 2008; 28 (50): 13532-13541

  Abstract

  In the CNS, endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and 2-arachidonoylglycerol (2-AG). Endocannabinoids are known to inhibit transmitter release from presynaptic terminals; however we have recently demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in rat somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K(+) conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca(2+)](i) such as occurs during sustained neuronal activity. To establish whether 2-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs). Current-clamp recordings from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM251, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons.

  View details for DOI 10.1523/JNEUROSCI.0847-08.2008

  View details for Web of Science ID 000261601800018

  View details for PubMedID 19074027

• Normal sleep homeostasis and lack of epilepsy phenotype in GABA(A) receptor alpha 3 subunit-knockout mice NEUROSCIENCE Winsky-Sommerer, R., Knapman, A., Fedele, D. E., Schofield, C. M., Vyazovskiy, V. V., Rudolph, U., Huguenard, J. R., Fritschy, J., Tobler, I. 2008; 154 (2): 595-605

  Abstract

  Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

  View details for DOI 10.1016/j.neuroscience.2008.03.081

  View details for Web of Science ID 000257172200020

  View details for PubMedID 18485607

• Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4 HUMAN MOLECULAR GENETICS Beyer, B., Deleuze, C., Letts, V. A., Mahaffey, C. L., Boumil, R. M., Lew, T. A., Huguenard, J. R., Frankel, W. N. 2008; 17 (12): 1738-1749

  Abstract

  Absence epilepsy, characterized by spike-wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses-consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures.

  View details for DOI 10.1093/hmg/ddn064

  View details for Web of Science ID 000256275600004

  View details for PubMedID 18316356

  View details for PubMedCentralID PMC2405903

• Imaging of glutamate in brain slices using FRET sensors JOURNAL OF NEUROSCIENCE METHODS Dulla, C., Tani, H., Okumoto, S., Frommer, W. B., Reimer, R. J., Fluguenard, J. R. 2008; 168 (2): 306-319

  Abstract

  The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Forster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.

  View details for DOI 10.1016/j.jneumeth.2007.10.017

  View details for Web of Science ID 000253824400004

  View details for PubMedID 18160134

  View details for PubMedCentralID PMC2267481

• Intrinsic excitability of cholinergic neurons in the rat parabigeminal nucleus JOURNAL OF NEUROPHYSIOLOGY Goddard, C. A., Knudsen, E. I., Huguenard, J. R. 2007; 98 (6): 3486-3493

  Abstract

  Cholinergic neurons in the parabigeminal nucleus of the rat midbrain were studied in an acute slice preparation. Spontaneous, regular action potentials were observed both with cell-attached patch recordings as well as with whole cell current-clamp recordings. The spontaneous activity of parabigeminal nucleus (PBN) neurons was not due to synaptic input as it persisted in the presence of the pan-ionotropic excitatory neurotransmitter receptor blocker, kynurenic acid, and the cholinergic blockers dihydro-beta-erythroidine (DHbetaE) and atropine. This result suggests the existence of intrinsic currents that enable spontaneous activity. In voltage-clamp recordings, I(H) and I(A) currents were observed in most PBN neurons. I(A) had voltage-dependent features that would permit it to contribute to spontaneous firing. In contrast, I(H) was significantly activated at membrane potentials lower than the trough of the spike afterhyperpolarization, suggesting that I(H) does not contribute to spontaneous firing of PBN neurons. Consistent with this interpretation, application of 25 microM ZD-7288, which blocked I(H), did not affect the rate of spontaneous firing in PBN neurons. Counterparts to I(A) and I(H) were observed in current-clamp recordings: I(A) was reflected as a slow voltage ramp observed between action potentials and on release from hyperpolarization, and I(H) was reflected as a depolarizing sag often accompanied by rebound spikes in response to hyperpolarizing current injections. In response to depolarizing current injections, PBN neurons fired at high frequencies, with relatively little accommodation. Ultimately, the spontaneous activity in PBN neurons could be used to modulate cholinergic drive in the superior colliculus in either positive or negative directions.

  View details for DOI 10.1152/jn.00960.2007

  View details for Web of Science ID 000251775700033

  View details for PubMedID 17898138

• GABA affinity shapes IPSCs in thalamic nuclei JOURNAL OF NEUROSCIENCE Schofield, C. M., Huguenard, J. R. 2007; 27 (30): 7954-7962

  Abstract

  Precise neural inhibition in thalamocortical circuits is required for the generation of sleep spindles and suppression of hypersynchrony associated with epileptiform activity. Accordingly, the time course of GABA(A) receptor-mediated IPSC events is an important parameter influencing the strength of inhibitory signaling. In the thalamus, two distinct types of IPSC kinetics are observed: thalamocortical relay neurons in the ventrobasal nucleus (VB) exhibit a fast decaying IPSC, whereas neurons in the adjacent reticular nucleus (RTN) display a long-lasting, slowly decaying IPSC. Here, we used patch-clamp electrophysiology and computational modeling to elucidate the basis for IPSC kinetic heterogeneity in the thalamus. Rapid application of GABA to excised membrane patches revealed that decay kinetics were attributable to intrinsic differences in GABA(A) receptor deactivation. Examination of desensitization and gating properties revealed these to be similar in VB and RTN, with the notable lack of fast and long-lasting desensitized states in both cell types. Computational simulations demonstrate that slow GABA binding and unbinding rates could reproduce the characteristic long-lasting IPSCs in RTN cells. These results indicate that within thalamic circuits, a powerful diversity of inhibitory function can result from simple differences in underlying GABA(A) receptor affinity.

  View details for DOI 10.1523/JNEUROSCI.0377-07.2007

  View details for Web of Science ID 000248484800008

  View details for PubMedID 17652586

• Thalamic synchrony and dynamic regulation of global forebrain oscillations TRENDS IN NEUROSCIENCES Huguenard, J. R., McCormick, D. A. 2007; 30 (7): 350-356

  Abstract

  The circuitry within the thalamus creates an intrinsic oscillatory unit whose function depends critically on reciprocal synaptic connectivity between excitatory thalamocortical relay neurons and inhibitory thalamic reticular neurons along with a robust post-inhibitory rebound mechanism in relay neurons. Feedforward and feedback connections between cortex and thalamus reinforce the thalamic oscillatory activity into larger thalamocortical networks to generate sleep spindles and spike-wave discharge of generalized absence epilepsy. The degree of synchrony within the thalamic network seems to be crucial in determining whether normal (spindle) or pathological (spike-wave) oscillations occur, and recent studies show that regulation of excitability in the reticular nucleus leads to dynamical modulation of the state of the thalamic circuit and provide a basis for explaining how a variety of unrelated genetic alterations might lead to the spike-wave phenotype. In addition, given the central role of the reticular nucleus in generating spike-wave discharge, these studies have suggested specific interventions that would prevent seizures while still allowing normal spindle generation to occur. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).

  View details for DOI 10.1016/j.tins.2007.05.007

  View details for Web of Science ID 000248295100007

  View details for PubMedID 17544519

• PKC and polyamine modulation of GluR2-deficient AMPA receptors in immature neocortical pyramidal neurons of the rat JOURNAL OF PHYSIOLOGY-LONDON Shin, J., Shen, F., Huguenard, J. 2007; 581 (2): 679-691

  Abstract

  AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12-14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function - inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca(2+) with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca(2+)](i) or [PA](i).

  View details for DOI 10.1113/jphysiol.2007.130963

  View details for Web of Science ID 000246756000024

  View details for PubMedID 17379631

  View details for PubMedCentralID PMC2075192

• Giant spontaneous depolarizing potentials in the developing thalamic reticular nucleus JOURNAL OF NEUROPHYSIOLOGY Pangratz-Fuehrer, S., Rudolph, U., Huguenard, J. R. 2007; 97 (3): 2364-2372

  Abstract

  The thalamic reticular nucleus (nRt) provides a major source of inhibition in the thalamo-cortical circuit and is critically involved in the generation of spindle oscillations. Here we describe the properties of thalamic giant depolarizing potentials (tGDPs) that were observed in nRt during early development. tGDPs persisted in presence of ionotropic glutamate antagonists but were completely abolished by GABA(A)R antagonist SR 35591. tGDPs occurred primarily between p3 and p8 (in 30-50% of cells) and occasionally up until p15. tGDPs lasted 0.4-3 s with peak conductances of 2-13 nS and occurred at frequencies between 0.02 and 0.06 Hz. We used mice with a benzodiazepine-insensitive alpha3 subunit [alpha3(H126R)] to probe for the identity of the GABA receptors responsible for tGDP generation. Benzodiazepine enhancement of tGDP amplitude and duration persisted in nRt neurons in alpha3(H126R) mice, indicating that the GABA(A)Rs containing alpha3 are not critical for tGDP generation and suggesting that tGDPs are mediated by GABA(A)Rs containing the alpha5 subunit, which is transiently expressed in nRt neurons in early postnatal development. Furthermore we found that exogenous GABA application depolarized nRt neurons younger than p8, indicating elevated [Cl(-)](i) at this developmental stage. Taken together, these data suggest that in immature nRt, long-lasting depolarizing responses mediated by GABA receptors could trigger Ca(2+) entry and play a role in functional development of the spindle-generating circuitry.

  View details for DOI 10.1152/jn.00646.2006

  View details for Web of Science ID 000244864900048

  View details for PubMedID 17251370

• Gender and age differences in expression of GABA(A) receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model NEUROBIOLOGY OF DISEASE Li, H., Huguenard, J. R., Fisher, R. S. 2007; 25 (3): 623-630

  Abstract

  Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABA(A) receptor subunit gamma2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) [Li, H., Kraus, A., Wu, J., Huguenard, J.R., Fisher, R.S., 2006. Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat. Neuropharmacology 51, 121-128]. In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABA(A) receptor alpha1 and gamma2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABA(A) gamma2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients.

  View details for DOI 10.1016/j.nbd.2006.11.004

  View details for PubMedID 17208003

• Recurrent circuits in layer II of medial entorhinal cortex in a model of temporal lobe epilepsy JOURNAL OF NEUROSCIENCE Kumar, S. S., Jin, X., Buckmaster, P. S., Huguenard, J. R. 2007; 27 (6): 1239-1246

  Abstract

  Patients and laboratory animal models of temporal lobe epilepsy display loss of layer III pyramidal neurons in medial entorhinal cortex and hyperexcitability and hypersynchrony of less vulnerable layer II stellate cells. We sought to test the hypothesis that loss of layer III pyramidal neurons triggers synaptic reorganization and formation of recurrent, excitatory synapses among layer II stellate cells in epileptic pilocarpine-treated rats. Laser-scanning photo-uncaging of glutamate focally activated neurons in layer II while excitatory synaptic responses were recorded in stellate cells. Photostimulation revealed previously unidentified, functional, recurrent, excitatory synapses between layer II stellate cells in control animals. Contrary to the hypothesis, however, control and epileptic rats displayed similar levels of recurrent excitation. Recently, hyperexcitability of layer II stellate cells has been attributed, at least in part, to loss of GABAergic interneurons and inhibitory synaptic input. To evaluate recurrent inhibitory circuits in layer II, we focally photostimulated interneurons while recording inhibitory synaptic responses in stellate cells. IPSCs were evoked more than five times more frequently in slices from control versus epileptic animals. These findings suggest that in this model of temporal lobe epilepsy, reduced recurrent inhibition contributes to layer II stellate cell hyperexcitability and hypersynchrony, but increased recurrent excitation does not.

  View details for DOI 10.1523/JNEUROSCI.3182-06.2007

  View details for Web of Science ID 000244070000003

  View details for PubMedID 17287497

• Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy NEUROBIOLOGY OF DISEASE Tani, H., Bandrowski, A. E., Parada, I., Wynn, M., Huguenard, J. R., Prince, D. A., Reimer, R. J. 2007; 25 (2): 230-238

  Abstract

  Epileptic activity arises from an imbalance in excitatory and inhibitory synaptic transmission. To determine if alterations in the metabolism of glutamate, the primary excitatory neurotransmitter, might contribute to epilepsy we directly and indirectly modified levels of glutamine, an immediate precursor of synaptically released glutamate, in the rat neocortical undercut model of hyperexcitability and epilepsy. We show that slices from injured cortex take up glutamine more readily than control slices, and an increased expression of the system A transporters SNAT1 and SNAT2 likely underlies this difference. We also examined the effect of exogenous glutamine on evoked and spontaneous activity and found that addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex increased the incidence and decreased the refractory period of epileptiform potentials. By contrast, exogenous glutamine increased the amplitude of evoked potentials in normal cortex, but did not induce epileptiform potentials. Addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex greatly increased abnormal spontaneous activity in the form of events resembling spreading depression, again while having no effect on slices from normal cortex. Interestingly, similar spreading depression like events were noted in control slices at supraphysiological levels of glutamine. In the undercut cortex addition of methylaminoisobutyric acid (MeAIB), an inhibitor of the system A glutamine transporters attenuated all physiological effects of added glutamine suggesting that uptake through these transporters is required for the effect of glutamine. Our findings support a role for glutamine transport through SNAT1 and/or SNAT2 in the maintenance of abnormal activity in this in vitro model of epileptogenesis and suggest that system A transport and glutamine metabolism are potential targets for pharmacological intervention in seizures and epilepsy.

  View details for DOI 10.1016/j.nbd.2006.08.025

  View details for Web of Science ID 000243981400002

  View details for PubMedID 17070687

  View details for PubMedCentralID PMC1952182

• NPY signaling through Y-1 receptors modulates thalamic oscillations 8th International NPY Meeting Brill, J., Kwakye, G., Huguenard, J. R. ELSEVIER SCIENCE INC. 2007: 250–56

  Abstract

  Neuropeptide Y is the ligand of a family of G-protein coupled receptors (Y(1) to Y(6)). In the thalamus, exogenous and endogenously released NPY can shorten the duration of thalamic oscillations in brain slices from P13 to P15 rats, an in vitro model of absence seizures. Here, we examine which Y receptors are involved in this modulation. Application of the Y(1) receptor agonist Leu(31)Pro(34)NPY caused a reversible reduction in the duration of thalamic oscillations (-26.6+/-7.8%), while the Y(2) receptor agonist peptideYY((3-36)) and the Y(5) receptor agonist BWX-46 did not exert a significant effect. No Y receptor agonist affected oscillation period. Application of antagonists of Y(1), Y(2) and Y(5) receptors (BIBP3226, BIIE0246 and L152,806, respectively) produced results consistent with those obtained from agonists. BIBP3226 caused a reversible disinhibition, an effect that increases oscillation duration (18.2+/-9.7%) while BIIE0246 and L152,806 had no significant effect. Expression of NPY is limited to neurons in the reticular thalamic nucleus (nRt), but Y(1) receptors are expressed in both nRt and adjacent thalamic relay nuclei. Thus, intra-nRt or nRt to relay nucleus NPY release could cause Y(1) receptor mediated inhibition of thalamic oscillations.

  View details for DOI 10.1016/j.peptides.2006.08.043

  View details for Web of Science ID 000244539800008

  View details for PubMedID 17196708

  View details for PubMedCentralID PMC1945169

• A thalamic sleep tonic. Epilepsy currents Staley, K. J., Huguenard, J. R. 2006; 6 (5): 164-166

  View details for PubMedID 17260047

• Distinct electrical and chemical connectivity maps in the thalamic reticular nucleus: Potential roles in synchronization and sensation JOURNAL OF NEUROSCIENCE Deleuze, C., Huguenard, J. R. 2006; 26 (33): 8633-8645

  Abstract

  GABAergic neurons of the thalamic reticular nucleus (nRt) provide thalamocortical relay neurons with feedback inhibition that influences sensory processing and thalamocortical rhythm generation. Mutual interactions between reticular neurons coordinate oscillatory activities developed within the network during normal sleep and in absence epilepsy, but the chemical versus electrical nature of these connections and their functional influence remain controversial. Here, we investigated the incidence and spatial extent of intra-nRt connectivity in vitro in horizontal and coronal thalamic slices from rat. Laser scanning photostimulation activated presynaptic nRt cells during patch-clamp recordings of postsynaptic neurons. Photolysis of caged glutamate evoked GABAergic IPSCs and/or depolarizing events (spikelets, mediated via electrical coupling) in a large proportion of neurons, thus indicating connectivity with presynaptic cell(s). Synaptic inputs were organized along the major axis of the nucleus in the same orientation as, but commonly exceeding the extent of, dendritic arborization of the postsynaptic neuron. In the anteroposterior (horizontal) plane, chemical connectivity had higher incidence (60% of recorded neurons vs 40% in vertical plane) and longer spatial extent, whereas in the dorsoventral (vertical) plane, electrical coupling dominated (47% incidence vs 37% in horizontal plane) and was more widely distributed. These data demonstrate that both electrical and chemical synapses are prominent within nRt and suggest different roles for the two types of connections. We thus propose that, along the vertical plane, electrical connectivity will promote coordinated rhythmic activity of sleep and/or thalamocortical epilepsy, whereas along the horizontal plane, chemical connectivity will oppose widespread thalamocortical synchronization and modulate sensory throughput.

  View details for Web of Science ID 000239817700024

  View details for PubMedID 16914689

• Electrophysiological classification of somatostatin-positive interneurons in mouse sensorimotor cortex JOURNAL OF NEUROPHYSIOLOGY Halabisky, B., Shen, F., Huguenard, J. R., Prince, D. A. 2006; 96 (2): 834-845

  Abstract

  Classification of inhibitory interneurons is critical in determining their role in normal information processing and pathophysiological conditions such as epilepsy. Classification schemes have relied on morphological, physiological, biochemical, and molecular criteria; and clear correlations have been demonstrated between firing patterns and cellular markers such as neuropeptides and calcium-binding proteins. This molecular diversity has allowed generation of transgenic mouse strains in which GFP expression is linked to the expression of one of these markers and presumably a single subtype of neuron. In the GIN mouse (EGFP-expressing Inhibitory Neurons), a subpopulation of somatostatin-containing interneurons in the hippocampus and neocortex is labeled with enhanced green fluorescent protein (EGFP). To optimize the use of the GIN mouse, it is critical to know whether the population of somatostatin-EGFP-expressing interneurons is homogeneous. We performed unsupervised cluster analysis on 46 EGFP-expressing interneurons, based on data obtained from whole cell patch-clamp recordings. Cells were classified according to a number of electrophysiological variables related to spontaneous excitatory postsynaptic currents (sEPSCs), firing behavior, and intrinsic membrane properties. EGFP-expressing interneurons were heterogeneous and at least four subgroups could be distinguished. In addition, multiple discriminant analysis was applied to data collected during whole cell recordings to develop an algorithm for predicting the group membership of newly encountered EGFP-expressing interneurons. Our data are consistent with a heterogeneous population of neurons based on electrophysiological properties and indicate that EGFP expression in the GIN mouse is not restricted to a single class of somatostatin-positive interneuron.

  View details for DOI 10.1152/jn.01079.2005

  View details for Web of Science ID 000238974700033

  View details for PubMedID 16707715

• Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat NEUROPHARMACOLOGY Li, H., Kraus, A., Wu, J., Huguenard, J. R., Fisher, R. S. 2006; 51 (1): 121-128

  Abstract

  Neonatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 has been shown to increase occurrence of spike-waves in EEG recordings and decrease benzodiazepines sensitivity of GABA(A) receptor-mediated responses in neurons from the thalamic reticular nuclei (nRt, Wu et al., 2004). The present experiments were designed to investigate the changes in the gamma2 and alpha1 subunits of the GABA(A) receptor in CSI model rats as possible mechanisms of these changes. Western blot, immunohistochemistry and real-time PCR techniques were performed to measure the levels of GABA(A) receptor gamma2 and alpha1 subunit transcripts and protein in the nRt and ventrobasal (VB) relay nuclei of thalamus and in somatosensory cortex. In CSI model animals, Western blot results showed that gamma2 subunit expression significantly decreased in thalamus (control, n=6: 0.17+/-0.02 relative to actin vs. CSI model, n=6: 0.11+/-0.01, P<0.05) but neither in cortex nor in hippocampal tissues. Conversely, alpha1 subunit expression decreased in CSI model somatosensory cortex, but not in nRt and VB. The present results demonstrate that neonatal block of cholesterol synthesis produces region- and subunit-specific decreases in GABA(A) receptor subunits in thalamus and cortex. Selective reductions in GABA(A) receptor subunits in thalamus may play a role in pathophysiology of absence epilepsy.

  View details for DOI 10.1016/j.neuropharm.2006.03.003

  View details for PubMedID 16678865

• Chronic valproic acid treatment triggers increased neuropeptide Y expression and signaling in rat nucleus reticularis thalami JOURNAL OF NEUROSCIENCE Brill, J., Lee, M., Zhao, S., Fernald, R. D., Huguenard, J. R. 2006; 26 (25): 6813-6822

  Abstract

  Valproate (VPA) can suppress absence and other seizures, but its precise mechanisms of action are not completely understood. We investigated whether VPA influences the expression of neuropeptide Y (NPY), an endogenous anticonvulsant. Chronic VPA administration to young rats (300-600 mg.kg(-1).d(-1) in divided doses over 4 d) resulted in a 30-50% increase in NPY mRNA and protein expression in the nucleus reticularis thalami (nRt) and hippocampus, but not in the neocortex, as shown by real-time PCR, radioimmunoassay, and immunohistochemistry. No increased expression was observed after a single acute dose of VPA. Chronic treatment with the pharmacologically inactive VPA analog octanoic acid did not elicit changes in NPY expression. No significant expression changes could be shown for the mRNAs of the Y1 receptor or of the neuropeptides somatostatin, vasoactive intestinal polypeptide, and choleocystokinin. Fewer synchronous spontaneous epileptiform oscillations were recorded in thalamic slices from VPA-treated animals, and oscillation duration as well as the period of spontaneous and evoked oscillations were decreased. Application of the Y1 receptor inhibitor N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-amide (BIBP3226) enhanced thalamic oscillations, indicating that NPY is released during those oscillations and acts to downregulate oscillatory strength. Chronic VPA treatment significantly potentiated the effect of BIBP3226 on oscillation duration but not on oscillation period. These results demonstrate a novel mechanism for the antiepileptic actions of chronic VPA therapy.

  View details for DOI 10.1523/JNEUROSCI.5320-05.2006

  View details for Web of Science ID 000238473600018

  View details for PubMedID 16793888

• Enhanced excitatory synaptic connectivity in layer V pyramidal neurons of chronically injured epileptogenic neocortex in rats JOURNAL OF NEUROSCIENCE Jin, X. M., Prince, D. A., Huguenard, J. R. 2006; 26 (18): 4891-4900

  Abstract

  Formation of new recurrent excitatory circuits after brain injuries has been hypothesized as a major factor contributing to epileptogenesis. Increases in total axonal length and the density of synaptic boutons are present in layer V pyramidal neurons of chronic partial isolations of rat neocortex, a model of posttraumatic epileptogenesis. To explore the functional consequences of these changes, we used laser-scanning photostimulation combined with whole-cell patch-clamp recording from neurons in layer V of somatosensory cortex to map changes in excitatory synaptic connectivity after injury. Coronal slices were submerged in artificial CSF (23 degrees C) containing 100 microM caged glutamate, APV (2-amino-5-phosphonovaleric acid), and high divalent cation concentration to block polysynaptic responses. Focal uncaging of glutamate, accomplished by switching a pulsed UV laser to give a 200-400 micros light stimulus, evoked single- or multiple-component composite EPSCs. In neurons of the partially isolated cortex, there were significant increases in the fraction of uncaging sites from which EPSCs could be evoked ("hot spots") and a decrease in the mean amplitude of individual elements in the composite EPSC. When plotted along the cortical depth, the changes in EPSCs took place mainly between 150 and 200 microm above and below the somata, suggesting a specific enhancement of recurrent excitatory connectivity among layer V pyramidal neurons of the undercut neocortex. These changes may shift the balance within cortical circuits toward increased synaptic excitation and contribute to epileptogenesis.

  View details for DOI 10.1523/JNEUROSCI.4361-05.2006

  View details for Web of Science ID 000237271700020

  View details for PubMedID 16672663

• Intrinsic and synaptic dynamics interact to generate emergent patterns of rhythmic bursting in thalamocortical neurons JOURNAL OF NEUROSCIENCE Sohal, V. S., Pangratz-Fuehrer, S., Rudolph, U., Huguenard, J. R. 2006; 26 (16): 4247-4255

  Abstract

  Rhythmic inhibition entrains the firing of excitatory neurons during oscillations throughout the brain. Previous work has suggested that the strength and duration of inhibitory input determines the synchrony and period, respectively, of these oscillations. In particular, sleep spindles result from a cycle of events including rhythmic inhibition and rebound bursts in thalamocortical (TC) neurons, and slowing and strengthening this inhibitory input may transform spindles into spike-wave discharges characteristic of absence epilepsy. Here, we used dynamic clamp to inject TC neurons with spindle-like trains of IPSCs and studied how modest changes in the amplitude and/or duration of these IPSCs affected the responses of the TC neurons. Contrary to our expectations, we found that prolonging IPSCs accelerates postinhibitory rebound (PIR) in TC neurons, and that increasing either the amplitude or duration of IPSCs desynchronizes PIR activity in a population of TC cells. Tonic injection of hyperpolarizing or depolarizing current dramatically alters the timing and synchrony of PIR. These results demonstrate that rhythmic PIR activity is an emergent property of interactions between intrinsic and synaptic currents, not just a passive reflection of incoming synaptic inhibition.

  View details for DOI 10.1523/JNEUROSCI.3812-05.2006

  View details for Web of Science ID 000236912100011

  View details for PubMedID 16624945

• Fast IPSCs in rat thalamic reticular nucleus require the GABA(A) receptor beta(1) subunit JOURNAL OF PHYSIOLOGY-LONDON Huntsman, M. M., Huguenard, J. R. 2006; 572 (2): 459-475

  Abstract

  Synchrony within the thalamocortical system is regulated in part by intranuclear synaptic inhibition within the reticular nucleus (RTN). Inhibitory postsynaptic currents (IPSCs) in RTN neurons are largely characterized by slow decay kinetics that result in powerful and prolonged suppression of spikes. Here we show that some individual RTN neurons are characterized by highly variable mixtures of fast, slow and mixed IPSCs. Heterogeneity arose largely through differences in the contribution of an initial decay component (tau(D) approximately 10 ms) which was insensitive to loreclezole, suggesting involvement of the GABA(A) receptor beta(1) subunit. Single-cell RT-PCR revealed the presence of beta(1) subunit mRNA only in those neurons whose IPSCs were dominated by a rapid and prominent initial decay phase. These data show that brief, beta(1)-dependent, loreclezole-insensitive IPSCs are present in a subpopulation of RTN neurons, and suggest that striking differences in IPSC heterogeneity within single neurons can result from of the presence or absence of a single GABA(A) receptor subunit.

  View details for DOI 10.1113/jphysiol.2006.106617

  View details for Web of Science ID 000237225300014

  View details for PubMedID 16469775

  View details for PubMedCentralID PMC1779681

• Barrel cortex microcircuits: Thalamocortical feedforward inhibition in spiny stellate cells is mediated by a small number of fast-spiking interneurons JOURNAL OF NEUROSCIENCE Sun, Q. Q., Huguenard, J. R., Prince, D. A. 2006; 26 (4): 1219-1230

  Abstract

  Inhibitory and excitatory neurons located in rodent barrel cortex are known to form functional circuits mediating vibrissal sensation. Excitatory neurons located in a single barrel greatly outnumber interneurons, and form extensive reciprocal excitatory synaptic contacts. Inhibitory and excitatory networks must interact to shape information ascending to cortex. The details of these interactions, however, have not been completely explored. Using paired intracellular recordings, we studied the properties of synaptic connections between spiny neurons (i.e., spiny stellate and pyramidal cells) and interneurons, as well as integration of thalamocortical (TC) input, in layer IV barrels of rat thalamocortical slices. Results show the following: (1) the strength of unitary excitatory connections of spiny neurons is similar among different targets; (2) although inhibition from regular-spiking nonpyramidal interneurons to spiny neurons is comparable in strength to excitatory connections, inhibition mediated by fast-spiking (FS) interneurons is 10 times more powerful; (3) TC EPSPs elicit reliable and precisely timed action potentials in FS neurons; and (4) a small number of FS neurons mediate thalamocortical feedforward inhibition in each spiny neuron and can powerfully shunt TC-mediated excitation. The ready activation of FS cells by TC inputs, coupled with powerful feedforward inhibition from these neurons, would profoundly influence sensory processing and constrain runaway excitation in vivo.

  View details for DOI 10.1523/JNEUROSCI.4727-04.2006

  View details for Web of Science ID 000234896200020

  View details for PubMedID 16436609

• Enhancement of spike-timing precision by autaptic transmission in neocortical inhibitory interneurons NEURON Bacci, A., Huguenard, J. R. 2006; 49 (1): 119-130

  Abstract

  In vivo studies suggest that precise firing of neurons is important for correct sensory representation. Principal neocortical neurons fire imprecisely when repeatedly activated by fixed sensory stimuli or current depolarizations. Here we show that in contrast to pyramidal neurons, firing in neocortical GABAergic fast-spiking (FS) interneurons is quite precise. FS interneurons are self-innervated by powerful GABAergic autaptic connections reliably activated after each spike, suggesting that autapses strongly regulate FS-cell spike timing. Indeed, blockade of autaptic transmission degraded temporal precision in multiple ways. Under these conditions, realistic dynamic-clamp hyperpolarizing autapses restored precision of spike timing, even in the presence of synaptic noise. Furthermore, firing precision was increased in pyramidal neurons by artificial GABAergic autaptic conductances, suggesting that tightly coupled synaptic feedback inhibition regulates spike timing in principal cells. Thus, well-timed inhibition, whether autaptic or synaptic, facilitates precise spike timing and promotes synchronized cortical network oscillations relevant to several behaviors.

  View details for DOI 10.1016/j.neuron.2005.12.014

  View details for Web of Science ID 000236699300014

  View details for PubMedID 16387644

• Inhibitory coupling specifically generates emergent gamma oscillations in diverse cell types PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sohal, V. S., Huguenard, J. R. 2005; 102 (51): 18638-18643

  Abstract

  Networks of inhibitory neurons regulate synchrony during many physiological and pathological oscillations. To explore how these effects depend on cellular, network, and synaptic factors, we developed and validated a semisynthetic inhibitory network that approximates simultaneous activity in multiple neurons by using consecutive responses from single cells. We recorded from three types of neurons, each of which forms interconnected networks in vivo, but has unique intrinsic properties. In all three cell types, fast inhibitory coupling generated emergent gamma oscillations. By contrast, inhibitory coupling desynchronized slower, spindle-frequency responses specifically in thalamic reticular neurons. The emergent gamma-frequency synchronization was also specific to tonic input and did not occur during responses to phasic inputs. These results illustrate how particular features of inhibitory networks (e.g., cell or input type) contribute to their synchronizing or desynchronizing functions. They also demonstrate phenomena (emergent gamma oscillations) that occur robustly in multiple cell types and may thus be a generic feature of inhibitory networks throughout the brain.

  View details for DOI 10.1073/pnas.0509291102

  View details for Web of Science ID 000234174300070

  View details for PubMedID 16339306

  View details for PubMedCentralID PMC1317969

• REORGANIZATION OF BARREL CIRCUITS LEADS TO THALAMICALLY-EVOKED CORTICAL EPILEPTIFORM ACTIVITY. Thalamus & related systems Sun, Q., Huguenard, J. R., Prince, D. A. 2005; 3 (4): 261-273

  Abstract

  We studied circuit activities in layer IV of rat somatosensory barrel cortex containing microgyri induced by neonatal freeze lesions. Structural abnormalities in GABAergic interneurons are present in the epileptogenic paramicrogyral area (PMG) and we therefore tested the hypothesis that decreased postsynaptic inhibition within barrel microcircuits occurs in the PMG and contributes to epileptogenesis when thalamocortical afferents are activated. In thalamocortical (TC) slices from naïve animals, single electrical stimuli within the thalamic ventrobasal (VB) nucleus evoked transient cortical multi-unit activity lasting 65±42 ms. Similar stimuli in TC slices from lesioned barrel cortex elicited prolonged 850 ±100 ms paroxysmal discharges that originated in the PMG and propagated laterally over several mm. Paroxysmal discharges were shortened in duration by ~70 % when APV was applied, and were totally abolished by CNQX. The cortical paroxysmal discharges did not evoke thalamic oscillations. Whole cell patch clamp recordings showed that there was a shift in the balance of TC evoked responses in the PMG that favored excitation over inhibition. Dual whole-cell recordings in layer IV of the PMG indicated that there was selective loss of inhibition from fast-spiking interneurons to spiny neurons in the barrel circuits that likely contributed to unconstrained cortical recurrent excitation with generation and spread of paroxysmal discharges.

  View details for PubMedID 18185849

• Modulation of neocortical interneurons: extrinsic influences and exercises in self-control TRENDS IN NEUROSCIENCES Bacci, A., Huguenard, J. R., Prince, D. A. 2005; 28 (11): 602-610

  Abstract

  Neocortical GABAergic interneurons are a highly heterogeneous cell population that forms complex functional networks and has key roles in information processing within the cerebral cortex. Mechanisms that control the output of these cells are therefore crucial in regulating excitability within the neocortex during normal and pathophysiological activities. In addition to subtype-specific modulation of GABAergic cells by neurotransmitters released by afferents from subcortical nuclei, interneurons belonging to different classes are controlled by distinct self-modulatory mechanisms, each unique and powerful. In this article, we review the diverse responses of neocortical interneurons to extrinsic and intrinsic neuromodulators. We discuss how specificity of responses might differentially influence inhibition in somatodendritic compartments of pyramidal neurons and affect the balance of activities in neocortical circuits.

  View details for DOI 10.1016/j.tins.2005.08.007

  View details for Web of Science ID 000233213700006

  View details for PubMedID 16139371

• Polyamines modulate AMPA receptor-dependent synaptic responses in immature layer V pyramidal neurons JOURNAL OF NEUROPHYSIOLOGY Shin, J., Shen, F., Huguenard, J. R. 2005; 93 (5): 2634-2643

  Abstract

  Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) mediate the majority of fast excitation in the CNS. Receptors lacking GluR2 exhibit inward rectification and paired-pulse facilitation (PPF) due to polyamine (PA)-dependent block and unblock, respectively. In this study, we tested whether rectification and PPF in immature, but not mature, pyramidal neurons depend not only on the absence of functional GluR2 but also on the level of endogenous PAs. Whole cell recordings were obtained from layer V pyramidal neurons of P12-P14 or P16-P20 rats in the presence or absence of spermine in the pipette (50 microM). Isolated minimal excitatory synaptic responses were obtained, and paired (20 Hz) stimuli were used to investigate the rectification index (RI) and paired-pulse ratio (PPR). Spermine and its synthetic enzyme, ornithine decarboxylase (ODC), expression was examined using immunostaining and Western blot, respectively. At the immature stage (

  View details for DOI 10.1152/jn.01054.2004

  View details for Web of Science ID 000228575200025

  View details for PubMedID 15574796

• Impaired Cl- extrusion in layer V pyramidal neurons of chronically injured epileptogenic neocortex JOURNAL OF NEUROPHYSIOLOGY Jin, X. M., Huguenard, J. R., Prince, D. A. 2005; 93 (4): 2117-2126

  Abstract

  In the mature brain, the K(+)/Cl- cotransporter KCC2 is important in maintaining low [Cl-]i, resulting in hyperpolarizing GABA responses. Decreases in KCC2 after neuronal injuries result in increases in [Cl-]i and enhanced neuronal excitability due to depolarizing GABA responses. We used the gramicidin perforated-patch technique to measure E(Cl) ( approximately E(GABA)) in layer V pyramidal neurons in slices of partially isolated sensorimotor cortex of adult rats to explore the potential functional consequence of KCC2 downregulation in chronically injured cortex. E(GABA) was measured by recording currents evoked with brief GABA puffs at various membrane potentials. There was no significant difference in E(Cl) between neurons in control and undercut animals (-71.2 +/- 2.6 and -71.8 +/- 2.8 mV, respectively). However, when loaded with Cl- by applying muscimol puffs at 0.2 Hz for 60 s, neurons in the undercut cortex had a significantly shorter time constant for the positive shift in E(Cl) during the Cl- loading phase (4.3 +/- 0.5 s for control and 2.2 +/- 0.4 s for undercut, P < 0.01). The positive shift in E(Cl) 3 s after the beginning of Cl- loading was also significantly larger in the undercut group than in the control, indicating that neurons in undercut cortex were less effective in maintaining low [Cl-]i during repetitive activation of GABA(A) receptors. Application of furosemide eliminated the difference between the control and undercut groups for both of these measures of [Cl-]i regulation. The results suggest an impairment in Cl- extrusion resulting from decreased KCC2 expression that may reduce the strength of GABAergic inhibition and contribute to epileptogenesis.

  View details for DOI 10.1152/jn.00728.2004

  View details for Web of Science ID 000227701600029

  View details for PubMedID 15774713

• Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids NATURE Bacci, A., Huguenard, J. R., Prince, D. A. 2004; 431 (7006): 312-316

  Abstract

  Neocortical GABA-containing interneurons form complex functional networks responsible for feedforward and feedback inhibition and for the generation of cortical oscillations associated with several behavioural functions. We previously reported that fast-spiking (FS), but not low-threshold-spiking (LTS), neocortical interneurons from rats generate a fast and precise self-inhibition mediated by inhibitory autaptic transmission. Here we show that LTS cells possess a different form of self-inhibition. LTS, but not FS, interneurons undergo a prominent hyperpolarization mediated by an increased K+-channel conductance. This self-induced inhibition lasts for many minutes, is dependent on an increase in intracellular [Ca2+] and is blocked by the cannabinoid receptor antagonist AM251, indicating that it is mediated by the autocrine release of endogenous cannabinoids. Endocannabinoid-mediated slow self-inhibition represents a powerful and long-lasting mechanism that alters the intrinsic excitability of LTS neurons, which selectively target the major site of excitatory connections onto pyramidal neurons; that is, their dendrites. Thus, modulation of LTS networks after their sustained firing will lead to long-lasting changes of glutamate-mediated synaptic strength in pyramidal neurons, with consequences during normal and pathophysiological cortical network activities.

  View details for DOI 10.1038/nature02913

  View details for Web of Science ID 000223864000042

  View details for PubMedID 15372034

• T-Channel Defects in Patients with Childhood Absence Epilepsy. Epilepsy currents / American Epilepsy Society Huguenard, J. R. 2004; 4 (1): 7-8

  View details for DOI 10.1111/j.1535-7597.2004.04102.x

  View details for PubMedID 15346133

  View details for PubMedCentralID PMC324574

• Pathway-specific differences in subunit composition of synaptic NMDA receptors on pyramidal neurons in neocortex JOURNAL OF NEUROSCIENCE Kumar, S. S., Huguenard, J. R. 2003; 23 (31): 10074-10083

  Abstract

  Heterogeneity of synaptic inputs onto neocortical layer 5 pyramidal neurons could result from differences in the underlying receptors, yet previous work has shown that functional attributes of AMPA receptors are uniform among synaptic connections onto these neurons. To determine whether NMDA receptors (NMDARs) would be similarly uniform, we compared in the same pyramidal neurons pharmacologically isolated NMDAR-mediated EPSCs evoked by stimulation of two anatomically distinguishable pathways, callosal or intracortical. Based on differences in voltage dependence, decay kinetics, apparent Mg2+sensitivity, and subunit-specific (NR2A, NR2B, and NR2C/D) pharmacology, we found NMDARs at these inputs to be distinct. Furthermore, NMDARs activated by the intracortical pathway were more efficient at integrating EPSPs and bringing the neuron closer to the spike-firing threshold than the callosal pathway. These results suggest that pyramidal neurons encode information differentially depending on the origin of their neocortical inputs and that NMDAR-dependent synaptic plasticity may be pathway specific.

  View details for Web of Science ID 000186474200014

  View details for PubMedID 14602822

• Target-specific neuropeptide Y-ergic synaptic inhibition and its network consequences within the mammalian thalamus JOURNAL OF NEUROSCIENCE Sun, Q. Q., Baraban, S. C., Prince, D. A., Huguenard, J. R. 2003; 23 (29): 9639-9649

  Abstract

  Neuropeptides are commonly colocalized with classical neurotransmitters, yet there is little evidence for peptidergic neurotransmission in the mammalian CNS. We performed whole-cell patch-clamp recording from rodent thalamic brain slices and repetitively stimulated corticothalamic fibers to strongly activate NPY-containing GABAergic reticular thalamic (RT) neurons. This resulted in long-lasting (approximately 10 sec) feedforward slow IPSPs (sIPSPs) in RT cells, which were mimicked and blocked by NPY1 (Y1) receptor agonists and antagonists, respectively, and were present in wild-type mice but absent in NPY-/- mice. NPYergic sIPSPs were mediated via G-proteins and G-protein-activated, inwardly rectifying potassium channels, as evidenced by sensitivity to GDP-beta-S and 0.1 mm Ba2+. In rat RT neurons, NPYergic sIPSPs were also present but were surprisingly absent in the major synaptic targets of RT, thalamic relay neurons, where instead robust GABA(B) IPSPs occurred. In vitro oscillatory network responses in rat thalamus were suppressed and augmented by Y1 agonists and antagonists, respectively. These findings provide evidence for segregation of postsynaptic actions between two targets of RT cells and support a role for endogenously released NPY within RT in the regulation of oscillatory thalamic responses relevant to sleep and epilepsy.

  View details for Web of Science ID 000186107100018

  View details for PubMedID 14573544

• Major differences in inhibitory synaptic transmission onto two neocortical interneuron subclasses JOURNAL OF NEUROSCIENCE Bacci, A., Rudolph, U., Huguenard, J. R., Prince, D. A. 2003; 23 (29): 9664-9674

  Abstract

  Locally projecting GABAergic interneurons are the major providers of inhibition in the neocortex and play a crucial role in several brain functions. Neocortical interneurons are connected via electrical and chemical synapses that may be crucial in modulating complex network oscillations. We investigated the properties of spontaneous and evoked IPSCs in two morphologically and physiologically identified interneuron subtypes, the fast-spiking (FS) and low threshold-spiking (LTS) cells in layer V of rodent sensorimotor cortex. We found that IPSCs recorded in FS cells were several orders of magnitude more frequent, larger in amplitude, and had faster kinetics than IPSCs recorded in LTS cells. GABA(A) receptor alpha- and beta-subunit selective modulators, zolpidem and loreclezole, had different effects on IPSCs in FS and LTS interneurons, suggesting differential expression of GABA(A) receptor subunit subtypes. These pharmacological data indicated that the alpha1 subunit subtype is poorly expressed by LTS cells but makes a large contribution to GABA(A) receptors on FS cells. This was confirmed by experiments performed in genetically modified mice in which the alpha1 subunit had been made insensitive to benzodiazepine-like agonists. These results suggest that differences in IPSC waveform are likely attributable to distinctive expression of GABA(A) receptor subunits in FS and LTS cells. The particular properties of GABAergic input on different interneuronal subtypes might have important consequences for generation and pacing of cortical rhythms underlying several brain functions. Moreover, selective pharmacological manipulation of distinct inhibitory circuits might allow regulation of pyramidal cell activities under specific physiological and pathophysiological conditions.

  View details for Web of Science ID 000186107100020

  View details for PubMedID 14573546

• Inhibitory interconnections control burst pattern and emergent network synchrony in reticular thalamus JOURNAL OF NEUROSCIENCE Sohal, V. S., Huguenard, J. R. 2003; 23 (26): 8978-8988

  Abstract

  Inhibitory connections between neurons of the thalamic reticular (RE) nucleus are thought to help prevent spike-wave discharge (SWD), characteristic of generalized absence epilepsy, in thalamic and thalamocortical circuits. Indeed, oscillations in thalamic slices resemble SWD when intra-RE inhibition is blocked and are suppressed when intra-RE inhibition is enhanced. To elucidate the cellular mechanisms underlying these network changes, we recorded from RE cells during oscillations in thalamic slices and either blocked intra-RE inhibition with picrotoxin or enhanced it with clonazepam. We found that intra-RE inhibition limits the number and synchrony, but not the duration, of RE cell bursts. We then performed simulations that demonstrate how inhibition can shift network activity into a desynchronized mode simply by vetoing occasional RE cell bursts. In contrast, when intra-RE inhibition is blocked, RE cells burst synchronously, enabling even short RE cell bursts to promote epileptogenesis in two ways: first, by activating GABA(B) receptors, and second, through the GABA(B) receptor-independent emergence of network synchrony.

  View details for Web of Science ID 000185666600020

  View details for PubMedID 14523100

• Dynamic GABA(A) receptor subtype-specific modulation of the synchrony and duration of thalamic oscillations JOURNAL OF NEUROSCIENCE Sohal, V. S., Keist, R., Rudolph, U., Huguenard, J. R. 2003; 23 (9): 3649-3657

  Abstract

  Networks of interconnected inhibitory neurons, such as the thalamic reticular nucleus (TRN), often regulate neural oscillations. Thalamic circuits generate sleep spindles and may contribute to some forms of generalized absence epilepsy, yet the exact role of inhibitory connections within the TRN remains controversial. Here, by using mutant mice in which the thalamic effects of the anti-absence drug clonazepam (CZP) are restricted to either relay or reticular nuclei, we show that the enhancement of intra-TRN inhibition is both necessary and sufficient for CZP to suppress evoked oscillations in thalamic slices. Extracellular and intracellular recordings show that CZP specifically suppresses spikes that occur during bursts of synchronous firing, and this suppression grows over the course of an oscillation, ultimately shortening that oscillation. These results not only identify a particular anatomical and molecular target for anti-absence drug design, but also elucidate a specific dynamic mechanism by which inhibitory networks control neural oscillations.

  View details for Web of Science ID 000182700100012

  View details for PubMedID 12736336

• Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide activate hyperpolarization activated cationic current and depolarize thalamocortical neurons in vitro JOURNAL OF NEUROSCIENCE Sun, Q. Q., Prince, D. A., Huguenard, J. R. 2003; 23 (7): 2751-2758

  Abstract

  Ascending pathways mediated by monoamine neurotransmitters regulate the firing mode of thalamocortical neurons and modulate the state of brain activity. We hypothesized that specific neuropeptides might have similar actions. The effects of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) were tested on thalamocortical neurons using whole-cell patch-clamp techniques applied to visualized neurons in rat brain slices. VIP (2 microm) and PACAP (100 nm) reversibly depolarized thalamocortical neurons (7.8 +/- 0.6 mV; n = 16), reduced the membrane resistance by 33 +/- 3%, and could convert the firing mode from bursting to tonic. These effects on resting membrane potential and membrane resistance persisted in the presence of TTX. Morphologically diverse thalamocortical neurons located in widespread regions of thalamus were all depolarized by VIP and PACAP38. In voltage-clamp mode, we found that VIP and PACAP38 reversibly activated a hyperpolarization-activated cationic current (I(H)) in thalamocortical neurons and altered voltage- and time-dependent activation properties of the current. The effects of VIP on membrane conductance were abolished by the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN)-specific antagonist ZD7288, showing that HCN channels are the major target of VIP modulation. The effects of VIP and PACAP38 on HCN channels were mediated by PAC(1) receptors and cAMP. The actions of PACAP-related peptides on thalamocortical neurons suggest an additional and novel endogenous neurophysiological pathway that may influence both normal and pathophysiological thalamocortical rhythm generation and have important behavioral effects on sensory processing and sleep-wake cycles.

  View details for Web of Science ID 000182107200028

  View details for PubMedID 12684461

• Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking delta subunit JOURNAL OF NEUROPHYSIOLOGY Porcello, D. M., Huntsman, M. M., Mihalek, R. M., Homanics, G. E., Huguenard, J. R. 2003; 89 (3): 1378-1386

  Abstract

  Robust GABA-mediated inhibitory postsynaptic currents (IPSCs) in neurons of the thalamic relay (TC) nuclei are important in sustaining oscillatory activity within thalamic and thalamocortical circuits. The biophysical properties and pharmacological sensitivities of these IPSCs both depend on the subunit combination of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors. Recombinant GABA(A) receptors containing the delta subunit (heavily expressed in TC nuclei) have been shown to exhibit slowed desensitization rates and high affinity for GABA in heterologous expression systems. We tested whether the GABA(A)-mediated synaptic inhibition in TC neurons would be affected by loss of the delta subunit. Spontaneous and evoked IPSCs were recorded from neurons in the ventral basal complex (VB) of the thalamus from brain slices of wild-type (delta(+/+)) and homozygous delta subunit deficient mice (delta(-/-)). Spontaneous IPSCs (sIPSCs) from delta(-/-) mice had no significant differences in amplitude, duration, or frequency compared with their delta(+/+) counterparts. However, baseline noise (63% of control) and the relative contribution of the slow component to overall decay (79% of control) were significantly lower in delta(-/-) VB recordings. Evoked IPSCs (eIPSCs) in delta(-/-) neurons showed no difference in peak amplitude, but had an accelerated slow decay component (40- vs. 55-ms time constant). We further tested whether neurosteroid modulation of GABA(A) receptors was dependent on the presence of the delta subunit, as previously reported in recombinant systems. Pregnenolone sulfate (PS) significantly reduced eIPSC peak amplitude (-30%) and increased duration in delta(-/-), but not in delta(+/+) mice. sIPSCs were not affected in any neurons, delta(-/-) or delta(+/+). In contrast, 3-alpha,5-alpha-tetrahydrodeoxycorticosterone (THDOC) increased the durations of eIPSCs and sIPSCs in both delta(-/-) and delta(+/+) VB neurons. Our findings show that although the delta subunit confers a striking PS insensitivity to eIPSCs in VB neurons, it plays only a minor role in the synaptic inhibition of VB neurons. This suggests delta subunit containing GABA(A) receptors may be functionally limited to an extrasynaptic locus in VB neurons.

  View details for DOI 10.1152/jn.0899.2002

  View details for Web of Science ID 000181426400021

  View details for PubMedID 12626617

• Neurotransmitter Supply and Demand in Epilepsy. Epilepsy currents / American Epilepsy Society Huguenard, J. 2003; 3 (2): 61-63

  View details for DOI 10.1046/j.1535-7597.2003.03210.x

  View details for PubMedID 15309087

  View details for PubMedCentralID PMC321172

• Baseline glutamate levels affect group I and II mGluRs in layer V pyramidal neurons of rat sensorimotor cortex JOURNAL OF NEUROPHYSIOLOGY Bandrowski, A. E., Huguenard, J. R., Prince, D. A. 2003; 89 (3): 1308-1316

  Abstract

  Possible functional roles for glutamate that is detectable at low concentrations in the extracellular space of intact brain and brain slices have not been explored. To determine whether this endogenous glutamate acts on metabotropic glutamate receptors (mGluRs), we obtained whole cell recordings from layer V pyramidal neurons of rat sensorimotor cortical slices. Blockade of mGluRs with (+)-alpha-amino-4-carboxy-alpha-methyl-benzeacetic acid (MCPG, a general mGluR antagonist) increased the mean amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), an effect attributable to a selective increase in the occurrence of large amplitude sEPSCs. 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495, a group II antagonist) increased, but R(-)-1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA) and (RS)-hexyl-HIBO (group I antagonists) decreased sEPSC amplitude, and (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III antagonist) did not change it. The change in sEPSCs elicited by MCPG, AIDA, and LY341495 was absent in tetrodotoxin, suggesting that it was action potential-dependent. The increase in sEPSCs persisted in GABA receptor antagonists, indicating that it was not due to effects on inhibitory interneurons. AIDA and (S)-3,5-dihydroxyphenylglycine (DHPG, a group I agonist) elicited positive and negative shifts in holding current, respectively. LY341495 and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, a group II agonist) elicited negative and positive shifts in holding current, respectively. The AIDA and LY341495 elicited currents persisted in TTX. Finally, in current clamp, LY341495 depolarized cells by approximately 2 mV and increased the number of action potentials to a given depolarizing current pulse. Thus ambient levels of glutamate tonically activate mGluRs and regulate cortical excitability.

  View details for DOI 10.1152/jn.00644.2002

  View details for Web of Science ID 000181426400014

  View details for PubMedID 12626613

• Functional autaptic neurotransmission in fast-spiking interneurons: A novel form of feedback inhibition in the neocortex JOURNAL OF NEUROSCIENCE Bacci, A., Huguenard, J. R., Prince, D. A. 2003; 23 (3): 859-866

  Abstract

  Autapses are synapses made by a neuron onto itself. Although morphological evidence for existence of autapses has been reported in several brain areas, it is not known whether such self-innervation in the neocortex is functional and robust. Here we report that GABAergic autaptic activity is present in fast-spiking, but not in low-threshold spiking, interneurons of layer V in neocortical slices. Recordings made with the perforated-patch technique, in which physiological intracellular chloride homeostasis was unperturbed, demonstrated that autaptic activity has significant inhibitory effects on repetitive firing and increased the current threshold for evoking action potentials. These results show that autapses are not rudimentary nonfunctional structures, but rather they provide a novel and powerful form of feedback inhibitory synaptic transmission in one class of cortical interneurons.

  View details for Web of Science ID 000180865400016

  View details for PubMedID 12574414

• Actions of U-92032, a T-type Ca2+ channel antagonist, support a functional linkage between I-T and slow intrathalamic rhythms JOURNAL OF NEUROPHYSIOLOGY Porcello, D. M., Smith, S. D., Huguenard, J. R. 2003; 89 (1): 177-185

  Abstract

  Thalamic relay neurons express high levels of T-type Ca(2+) channels, which support the generation of robust burst discharges. This intrinsically mediated form of phasic spike firing is thought to be critical in the generation of slow (3-4 Hz) synchronous oscillatory activity of absence epilepsy. Recordings made from brain slices or whole animals have shown that slow synchronous absence-like activity can be abolished when Ca(2+)-dependent burst firing in relay neurons is interrupted by the pharmacological or genetic inactivation of T-channels. Because succinimide drugs act as incomplete and nonspecific antagonists, we tested whether the novel T-channel antagonist U-92032 could provide stronger support for a role of T-channels in slow oscillatory activity. Ca(2+)-dependent rebound (LTS) bursts were recorded using whole cell current clamp in relay cells of the ventral basal complex (VB) from thalamic slices of adult rats. We used LTS kinetics to measure the availability of T-channels in VB cells after TTX. U-92032 (1 and 10 microM) reduced the maximum rate of depolarization of the isolated LTS by 51% and 90%, respectively, compared with the 35% reduction due to 2 mM methylphenylsuccinimide (MPS), the active metabolite of the antiabsence drug methsuximide. U-92032 (1 and 10 microM) also suppressed evoked, slow oscillations in thalamic slices with a time course similar for observed intracellular effects. Unlike MPS, we observed no substantial effects of short-term U-92032 applications (< or =2 h) on the generation of action potentials in VB cells. Our findings show U-92032 is a more potent, effective, and specific T-channel antagonist than previously studied succinimide antiabsence drugs and that it dramatically reduces epileptiform synchronous activity. This suggests that U-92032 or other specific T-channel antagonists may provide effective drug treatments for absence epilepsy.

  View details for DOI 10.1152/jn.00667.2002

  View details for PubMedID 12522170

• Differential modulation of synaptic transmission by neuropeptide Y in rat neocortical neurons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bacci, A., Huguenard, J. R., Prince, D. A. 2002; 99 (26): 17125-17130

  Abstract

  Neuropeptide Y (NPY) is widely expressed throughout the nervous system and is known to reduce excitatory (but also inhibitory) synaptic transmission in many CNS areas, leading to the proposal that it is an endogenous antiepileptic agent. In the neocortex, where NPY is present in gamma-aminobutyric acid (GABA)ergic interneurons, its effects on inhibitory and excitatory synaptic activities have not been completely explored. Here we report that NPY application elicits a long-lasting decrease in evoked excitatory postsynaptic current amplitude and a delayed, long-lasting increase in the amplitude of evoked monosynaptic inhibitory postsynaptic current (IPSC) in layer V pyramidal neurons of rat neocortex. The novel, late, NPY-mediated increase of inhibitory synaptic transmission is caused by modulation of Ca2+-dependent GABA release onto pyramidal neurons, as it was accompanied by an increase in Ca2+-dependent miniature IPSC frequency. NPY decreased evoked monosynaptic IPSCs in GABAergic interneurons, indicating that this neuropeptide has differential effects on different neuronal subtypes in the neocortex. Each of these NPY actions would decrease excitability in cortical circuits, a result that has important implications for both physiological neocortical operations as well as pathophysiological epileptiform activities.

  View details for Web of Science ID 000180101600110

  View details for PubMedID 12482942

• Synaptic inhibition of pyramidal cells evoked by different interneuronal subtypes in layer V of rat visual cortex JOURNAL OF NEUROPHYSIOLOGY Xiang, Z. X., Huguenard, J. R., Prince, D. A. 2002; 88 (2): 740-750

  Abstract

  Properties of GABA(A) receptor-mediated unitary inhibitory postsynaptic currents (uIPSCs) in pyramidal (P) cells, evoked by fast spiking (FS) and low-threshold spike (LTS) subtypes of interneurons in layer V of rat visual cortex slices were examined using dual whole cell recordings. uIPSCs evoked by FS cells were larger and faster rising than those evoked by LTS cells, consistent with the known primary projections of FS and LTS cell axons to perisomatic and distal dendritic areas of layer V pyramidal cells, respectively, and the resulting electrotonic attenuation for LTS-P synaptic events. Unexpectedly, the decay time constants for LTS-P and FS-P uIPSCs were not significantly different. Modeling results were consistent with differences in the underlying GABA(A) receptor-mediated conductance at LTS-P and FS-P synapses. Paired-pulse depression (PPD), present at both synapses, was associated with an increase in failure rate and a decrease in coefficient of variation, indicating that presynaptic mechanisms were involved. Furthermore, the second and first uIPSC amplitudes during PPD were not inversely correlated, suggesting that PPD at both synapses is independent of previous release and might not result from depletion of the releasable pool of synaptic vesicles. Short, 20-Hz trains of action potentials in presynaptic interneurons evoked trains of uIPSCs with exponentially decreasing amplitudes at both FS-P and LTS-P synapses. FS-P uIPSC amplitudes declined more slowly than those of LTS-P uIPSCs. Thus FS and LTS cells, with their differences in firing properties, synaptic connectivity with layer V P cells, and short-term synaptic dynamics, might play distinct roles in regulating the input-output relationship of the P cells.

  View details for DOI 10.1152/jn.00635.2001

  View details for Web of Science ID 000177276100018

  View details for PubMedID 12163526

• Somatostatin inhibits thalamic network oscillations in vitro: Actions on the GABAergic neurons of the reticular nucleus JOURNAL OF NEUROSCIENCE Sun, Q. Q., Huguenard, J. R., Prince, D. A. 2002; 22 (13): 5374-5386

  Abstract

  We examined the effects of somatostatin (SST) on neurons in the thalamic reticular nucleus (RT) using whole-cell patch-clamp techniques applied to visualized neurons in rat thalamic slices. SST, acting via sst(5) receptors and pertussis toxin-sensitive G-proteins, activated an inwardly rectifying K(+) (GIRK) current in 20 of 28 recorded cells to increase input conductance 15 +/- 3% above control and inhibited N-type Ca(2+) currents in 17 of 24 neurons via voltage-dependent mechanisms. SST reversibly depressed evoked EPSCs (eEPSCs) to 37 +/- 8% of control without altering their kinetics. SST-mediated inhibition of eEPSCs showed short-term relief from block during 25 Hz stimulus trains. SST also reduced the frequency (33 +/- 8%) but not the amplitude of miniature EPSCs (mEPSCs). These data indicate that SST mediates presynaptic inhibition of glutamate release onto RT neurons. In current-clamp recordings, SST preferentially inhibited burst discharges mediated by near-threshold corticothalamic EPSPs and intracellularly applied depolarizing currents. SST had powerful effects on in vitro intrathalamic rhythms, which included a shortening of the duration and a reduction in spike count within each oscillatory event. Furthermore, there was a paradoxical increase in the synchrony of epileptiform oscillations, likely mediated by a suppression of the responses to weak synaptic inputs in RT. We conclude that SST suppresses discharges in RT neurons, via presynaptic inhibition of glutamate release and postsynaptic activation of GIRK channels, leading to the dampening of both spindle-like and epileptiform thalamic network oscillations. SST may act as an important endogenous regulator of physiological and pathological thalamocortical network activities.

  View details for Web of Science ID 000176599100017

  View details for PubMedID 12097489

• Reciprocal inhibition controls the oscillatory state in thalamic networks 10th Computational Neuroscience Meeting (CSN 01) Sohal, V. S., Huguenard, J. R. ELSEVIER SCIENCE BV. 2002: 653–659

  View details for Web of Science ID 000176839200095

• A developmental switch of AMPA receptor subunits in neocortical pyramidal neurons JOURNAL OF NEUROSCIENCE Kumar, S. S., Bacci, A., Kharazia, V., Huguenard, J. R. 2002; 22 (8): 3005-3015

  Abstract

  AMPA receptors mediate most of the fast excitatory neurotransmission in the brain, and those lacking the glutamate receptor 2 (GluR2) subunit are Ca(2+)-permeable and expressed in cortical structures primarily by inhibitory interneurons. Here we report that synaptic AMPA receptors of excitatory layer 5 pyramidal neurons in the rat neocortex are deficient in GluR2 in early development, approximately before postnatal day 16, as evidenced by their inwardly rectifying current-voltage relationship, blockade of AMPA receptor-mediated EPSCs by external and internal polyamines, permeability to Ca(2+), and GluR2 immunoreactivity. Overall, these results indicate that neocortical pyramidal neurons undergo a developmental switch in the Ca(2+) permeability of their AMPA receptors through an alteration of their subunit composition. This has important implications for plasticity and neurotoxicity.

  View details for Web of Science ID 000174841300004

  View details for PubMedID 11943803

• Resilient RTN fast spiking in Kv3.1 null mice suggests redundancy in the action potential repolarization mechanism JOURNAL OF NEUROPHYSIOLOGY Porcello, D. M., Ho, C. S., Joho, R. H., Huguenard, J. R. 2002; 87 (3): 1303-1310

  Abstract

  Fast spiking (FS), GABAergic neurons of the reticular thalamic nucleus (RTN) are capable of firing high-frequency trains of brief action potentials, with little adaptation. Studies in recombinant systems have shown that high-voltage-activated K(+) channels containing the Kv3.1 and/or Kv3.2 subunits display biophysical properties that may contribute to the FS phenotype. Given that RTN expresses high levels of Kv3.1, with little or no Kv3.2, we tested whether this subunit was required for the fast action potential repolarization mechanism essential to the FS phenotype. Single- and multiple-action potentials were recorded using whole-cell current clamp in RTN neurons from brain slices of wild-type and Kv3.1-deficient mice. At 23 degrees C, action potentials recorded from homozygous Kv3.1 deficient mice (Kv3.1(-/-)) compared with their wild-type (Kv3.1(+/+)) counterparts had reduced amplitudes (-6%) and fast after-hyperpolarizations (-16%). At 34 degrees C, action potentials in Kv3.1(-/-) mice had increased duration (21%) due to a reduced rate of repolarization (-30%) when compared with wild-type controls. Action potential trains in Kv3.1(-/-) were associated with a significantly greater spike decrement and broadening and a diminished firing frequency versus injected current relationship (F/I) at 34 degrees C. There was no change in either spike count or maximum instantaneous frequency during low-threshold Ca(2+) bursts in Kv3.1(-/-) RTN neurons at either temperature tested. Our findings show that Kv3.1 is not solely responsible for fast spikes or high-frequency firing in RTN neurons. This suggests genetic redundancy in the system, possibly in the form of other Kv3 members, which may suffice to maintain the FS phenotype in RTN neurons in the absence of Kv3.1.

  View details for DOI 10.1152/jn.00556.2001

  View details for Web of Science ID 000174735700014

  View details for PubMedID 11877504

• Block of T -Type Ca(2+) Channels Is an Important Action of Succinimide Antiabsence Drugs. Epilepsy currents Huguenard, J. R. 2002; 2 (2): 49-52

  Abstract

  The role of calcium channel blockade in the antiepileptic action of ethosuximide is controversial, especially regarding the potency and efficacy of block. However, recent evidence obtained from transgenic animals and heterologous expression systems supports a major role of T-type calcium channels in both the generation of absence seizures and the action of ethosuximide in human absence epilepsy.

  View details for PubMedID 15309165

• Sodium channels: Grit, determination, and persistence NEURON Huguenard, J. R. 2002; 33 (4): 492-494

  Abstract

  Persistent sodium channel activity modulates neuronal gain in a neurotransmitter-dependent fashion. Previous studies have suggested that persistent and spike-related sodium channel activities are mediated by separate species. In this issue of Neuron, Taddese and Bean (2002) show that a single channel population is sufficient to explain both gating behaviors. A simple allosteric model is provided that can explain the results.

  View details for Web of Science ID 000173911100003

  View details for PubMedID 11856523

• Prolactin-releasing peptide (PrRP) promotes awakening and suppresses absence seizures NEUROSCIENCE Lin, S. H., Arai, A. C., Espana, R. A., Berridge, C. W., Leslie, F. M., Huguenard, J. R., Vergnes, M., Civelli, O. 2002; 114 (1): 229-238

  Abstract

  Prolactin releasing peptide (PrRP) is a recently identified neuropeptide that stimulates prolactin release from pituitary cells. The presence of its receptor outside the hypothalamic-pituitary axis suggests that it may have other functions. We present here evidence that PrRP can modulate the activity of the reticular thalamic nucleus, a brain region with prominent PrRP receptor expression that is critical for sleep regulation and the formation of non-convulsive absence seizures. Intracerebroventricular injection of PrRP (1-10 nmol) into sleeping animals significantly suppresses sleep oscillations and promotes rapid and prolonged awakening. Higher concentrations of PrRP (10-100 nmol) similarly suppress spike wave discharges seen during absence seizures in genetic absence epilepsy rats from Strasbourg, an animal model for this disorder. In concordance with these findings, PrRP suppressed evoked oscillatory burst activity in reticular thalamic slices in vitro. These results indicate that PrRP modulates reticular thalamic function and that activation of its receptor provides a new target for therapies directed at sleep disorders and absence seizures.

  View details for Web of Science ID 000178011200021

  View details for PubMedID 12207968

• Properties of excitatory synaptic connections mediated by the corpus callosum in the developing rat neocortex JOURNAL OF NEUROPHYSIOLOGY Kumar, S. S., Huguenard, J. R. 2001; 86 (6): 2973-2985

  Abstract

  Despite the major role of excitatory cortico-cortical connections in mediating neocortical activities, little is known about these synapses at the cellular level. Here we have characterized the synaptic properties of long-range excitatory-to-excitatory contacts between visually identified layer V pyramidal neurons of agranular frontal cortex in callosally connected neocortical slices from postnatal day 13 to 21 (P13-21) rats. Midline stimulation of the corpus callosum with a minimal stimulation paradigm evoked inward excitatory postsynaptic currents (EPSCs) with an averaged peak amplitude of 56.5 +/- 5 pA under conditions of whole cell voltage clamp at -70 mV. EPSCs had fixed latencies from stimulus onset and could follow stimulus trains (1-20 Hz) without changes in kinetic properties. Bath application of 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) abolished these responses completely, indicating that they were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs). Evoked responses were isolated in picrotoxin to yield purely excitatory PSCs, and a low concentration of NBQX (0.1 microM) was used to partially block AMPARs and prevent epileptiform activity in the tissue. Depolarization of the recorded pyramidal neurons revealed a late, slowly decaying component that reversed at approximately 0 mV and was blocked by D-2-amino-5-phosphonovaleric acid. Thus AMPA and N-methyl-D-aspartate receptors (NMDARs) coexist at callosal synapses and are likely to be activated monosynaptically. The peak amplitudes and decay time constants for EPSCs evoked using minimal stimulation (+/-40 mV) were similar to spontaneously occurring sEPSCs. Typical conductances associated with AMPA and NMDAR-mediated components, deduced from their respective current-voltage (I-V) relationships, were 525 +/- 168 and 966 +/- 281 pS, respectively. AMPAR-mediated responses showed age-dependent changes in the rectification properties of their I-V relationships. While I-Vs from animals >P15 were linear, those in the younger (

  View details for Web of Science ID 000172460500029

  View details for PubMedID 11731554

• Kinetic and pharmacological properties of GABA(A) receptors in single thalamic neurons and GABA(A) subunit expression JOURNAL OF NEUROPHYSIOLOGY Browne, S. H., Kang, J., Akk, G., Chiang, L. W., Schulman, H., Huguenard, J. R., Prince, D. A. 2001; 86 (5): 2312-2322

  Abstract

  Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.

  View details for Web of Science ID 000172012800017

  View details for PubMedID 11698521

• GABA(B) and NMDA receptors contribute to spindle-like oscillations in rat thalamus in vitro JOURNAL OF NEUROPHYSIOLOGY Jacobsen, R. B., Ulrich, D., Huguenard, J. R. 2001; 86 (3): 1365-1375

  Abstract

  Thalamic slice preparations, in which intrathalamic connectivity between the reticular nucleus and relay nuclei is maintained, are capable of sustaining rhythmic burst firing activity in rodents and ferret. These in vitro oscillations occur spontaneously in the ferret and have frequencies (6-10 Hz) within the range of sleep spindles observed in vivo. In the rat, mainly lower frequency (2-4 Hz) oscillations, evoked under conditions of low bath [Mg(2+)] and/or GABA(A) receptor blockade, have been described. Here we show that faster rhythms in the range of 4-9 Hz can be evoked in rat thalamic slices by electrical stimulation of the internal capsule and also occur spontaneously. When bath [Mg(2+)] was 2 mM, these spindle-like oscillations were most common in a brief developmental time window, peaking at postnatal day 12 (P12). The oscillations were almost completely blocked by the GABA(A) receptor antagonist picrotoxin, and, in some cases, the frequency of oscillations was increased by the GABA(B) receptor antagonist CGP-35348. The selective blockade of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the antagonists 2-amino-5-phosphonovaleric acid or 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), respectively, significantly shortened oscillations but did not completely block them. A combination of the two drugs was necessary to abolish oscillatory activity. The barbituate pentobarbital, which enhances GABA(A)R responses, initially slowed and synchronized oscillations before completely blocking them. When bath [Mg(2+)] was reduced from 2 to 0.65 mM, evoked oscillations became more robust and were often accompanied by spontaneously arising oscillations. Under these conditions, GABA(A) receptor blockade no longer inhibited oscillations, but instead converted them into the slow, synchronous rhythms that have been observed in other studies. The effects of GABA(B) or NMDA receptor blockade were more pronounced in 0.65 mM than in 2 mM external [Mg(2+)]. Thus spindle-like oscillations occur in rat thalamic slices in vitro, and we find that, in addition to the previously demonstrated contributions of GABA(A) and AMPA receptors to these oscillations, NMDA and GABA(B) receptors are also involved. The strong influence of external [Mg(2+)] on GABAergic pharmacology and a contribution of NMDA receptors during oscillations suggest a link between the excitability of NMDA receptors and the activation of GABA(B)R-mediated inhibitory postsynaptic currents.

  View details for Web of Science ID 000171024400028

  View details for PubMedID 11535683

• Thalamocortical Circuits and Excitability. Epilepsy currents / American Epilepsy Society Huguenard, J. 2001; 1 (1): 13

  View details for DOI 10.1046/j.1535-7597.2001.00008.x

  View details for PubMedID 15309032

  View details for PubMedCentralID PMC320682

• It takes T to tango NEURON Sohal, V. S., Huguenard, J. R. 2001; 31 (1): 3-4

  Abstract

  Of three recently cloned T-type voltage-gated calcium channels, alpha(1g) is most likely responsible for burst firing in thalamic relay cells. These neurons burst during various thalamocortical oscillations including absence seizures. In this issue of Neuron, Kim et al. inactivated alpha(1g), and resultant mice were deficient in relay cell bursting and resistant to GABA(B) receptor-dependent absence seizures, suggesting roles for alpha(1g) and relay cell bursting in absences.

  View details for Web of Science ID 000170101100002

  View details for PubMedID 11498042

• The role of H-current in regulating strength and frequency of thalamic network oscillations. Thalamus & related systems Yue, B. W., Huguenard, J. R. 2001; 1 (2): 95-103

  Abstract

  Intrathalamic oscillations related to sleep and epilepsy depend on interactions between synaptic mechanisms and intrinsic membrane excitability. One intrinsic conductance implicated in the genesis of thalamic oscillations is the H current - a cationic current activated by membrane hyperpolarization. Activation of H current promotes rebound excitation of thalamic relay neurons and can thus enhance recurrent network activity.We examined the effects of H current modulation on bicuculline-enhanced network oscillations (2-4 Hz) in rat thalamic slices. The adrenergic agonist norepinephrine, a known regulator of H current, caused an alteration of the internal structure of the oscillations - they were enhanced and accelerated as the interval between bursts was shortened. The acceleration was blocked by the β-adrenergic antagonist propranolol. The β agonist isoproterenol mimicked the effect of norepinephrine on oscillation frequency and truncated the responses suggesting that a β-adrenergic upregulation of H current modifies the internal structure (frequency) of thalamic oscillations. Consistent with this, we found that H channel blockade by Cs(+) or ZD7288 could decelerate the oscillations and produce more robust (longer lasting) responses. High concentrations of either Cs(+) or ZD7288 blocked the oscillations.These results indicate that a critical amount of H current is necessary for optimal intrathalamic oscillations in the delta frequency range. Up- or downregulation of H current can not only alter the oscillation frequency but also retard or promote the development of thalamic synchronous oscillations. This conclusion has important implications regarding the development of epilepsy in thalamocortical circuits.

  View details for DOI 10.1016/S1472-9288(01)00009-7

  View details for PubMedID 18239728

  View details for PubMedCentralID PMC2222919

• Clonazepam suppresses oscillations in rat thalamic slices 9th Annual Computational Neuroscience Meeting (CNS*00) Sohal, V. S., Huguenard, J. R. ELSEVIER SCIENCE BV. 2001: 907–913

  View details for Web of Science ID 000169129200121

• Differential regulation of GABA release and neuronal excitability mediated by neuropeptide Y-1 and Y-2 receptors in rat thalamic neurons JOURNAL OF PHYSIOLOGY-LONDON Sun, Q. Q., Akk, G., Huguenard, J. R., Prince, D. A. 2001; 531 (1): 81-94

  Abstract

  1. Neuropeptide Y (NPY) produced inhibitory effects on neurons of the thalamic reticular nucleus (RT; n = 18) and adjacent ventral basal complex (VB; n = 22), which included hyperpolarization (approximately 4 mV), a reduction in rebound and regular spikes and an increased membrane conductance. These effects were mediated predominantly via NPY1 receptor activation of G-protein-activated, inwardly rectifying K+ (GIRK) channels. 2. NPY reduced the frequency of spontaneous GABAA receptor-mediated inhibitory postsynaptic currents (sIPSCs) in RT (by 60 +/- 7 %, n = 14) and VB neurons (by 25 +/- 11 %, n = 16), but had no effect on the kinetic properties of sIPSCs. After removal of the RT nucleus, the inhibitory effects of NPY on sIPSCs in VB neurons remained (29 +/- 7 %, n = 5). The synaptic effects were mediated via NPY2 receptors. 3. NPY inhibited the frequency of miniature IPSCs (mIPSCs) in RT and VB neurons (by 63 +/- 7 %, n = 5, and 37 +/- 8 %, n = 10, respectively) in the presence of tetrodotoxin (TTX) (1 microM) but not TTX (1 microM) and Cd2+ (200 microM). 4. NPY inhibited evoked IPSCs in both RT (by 18 +/- 3 %, n = 6) and VB (by 5 +/- 4 %, n = 6) neurons without change in short-term synaptic plasticity. 5. We conclude that NPY1 and NPY2 receptors are functionally segregated in the thalamus: NPY1 receptors are predominantly expressed at the somata and dendrites and directly reduce the excitability of neurons in both the RT and VB nuclei by activating GIRK channels. NPY2 receptors are located at recurrent (RT) and feed-forward GABAergic terminals (VB) and downregulate GABA release via inhibition of Ca2+ influx from voltage-gated Ca2+ channels.

  View details for Web of Science ID 000167409800007

  View details for PubMedID 11179393

• Neuropeptide Y receptors differentially modulate G-protein-activated inwardly rectifying K+ channels and high-voltage-activated Ca2+ channels in rat thalamic neurons JOURNAL OF PHYSIOLOGY-LONDON Sun, Q. Q., Huguenard, J. R., Prince, D. A. 2001; 531 (1): 67-79

  Abstract

  1. Using whole-cell patch-clamp recordings, infrared videomicroscopy and fast focal solution exchange methods, the actions of neuropeptide Y (NPY) were examined in thalamic slices of postnatal (10-16 days) rats. 2. NPY activated a K+-selective current in neurons of the thalamic reticular nucleus (RT; 20/29 neurons) and ventral basal complex (VB; 19/25 neurons). The currents in both nuclei had activation and deactivation kinetics that were very similar to those of GABAB receptor-induced currents, were totally blocked by 0.1 mM Ba2+ and showed voltage-dependent relaxation. These properties indicate that the NPY-sensitive K+ current is mediated by G-protein-activated, inwardly rectifying K+ (GIRK) channels. 3. In RT neurons, NPY application reversibly reduced high-voltage-activated (HVA) currents to 33 +/- 5 % (n = 40) of the control level but did not affect the T-type currents. Inhibition of Ca2+ currents was voltage independent and was largely mediated by effects on N- and P/Q-type channels. 4. NPY activation of GIRK channels was mediated via NPY1 receptors, whereas inhibition of N- and P/Q-type Ca2+ channels was mediated by NPY2 receptors. 5. These results show that neuropeptide Y activates K+ channels and simultaneously inhibits HVA Ca2+ channels via different receptor subtypes.

  View details for Web of Science ID 000167409800006

  View details for PubMedID 11179392

• Nonlinear thermodynamic models of voltage-dependent currents JOURNAL OF COMPUTATIONAL NEUROSCIENCE Destexhe, A., Huguenard, J. R. 2000; 9 (3): 259-270

  Abstract

  Hodgkin and Huxley provided the first quantitative description of voltage-dependent currents and adjusted their model to experimental data using empirical functions of voltage. A physically plausible formalism was proposed later by assuming that transition rates depend exponentially on a free-energy barrier, by analogy with the theory of reaction rates. It was also assumed that the free energy depends linearly on voltage. This thermodynamic formalism can accurately describe many processes, but the resulting time constants can be arbitrarily fast, which may also lead to aberrant behavior. We considered here a physically plausible solution to this problem by including nonlinear effects of the electrical field on the free energy. We show that including effects such as mechanical constraints, inherent to the structure of the ion channel protein, leads to more accurate thermodynamic models. These models can account for voltage-dependent transitions that are rate-limited in a given voltage range, without invoking additional states. We illustrate their applicability to fit experimental data by considering the case of the T-type calcium current in thalamic neurons.

  View details for Web of Science ID 000165637400003

  View details for PubMedID 11139042

• Reliability of axonal propagation: The spike doesn't stop here PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huguenard, J. R. 2000; 97 (17): 9349-9350

  View details for Web of Science ID 000088840500001

  View details for PubMedID 10944204

• Circuit mechanisms of spike-wave discharge: Are there similar underpinnings for centrotemporal spikes? Workshop on Benign Focal Epilepsies of Childhood: Genetically Determined Pathophysiology Huguenard, J. R. WILEY-BLACKWELL PUBLISHING, INC. 2000: 1076–77

  View details for Web of Science ID 000088661000034

  View details for PubMedID 10961648

• Reciprocal inhibitory connections produce desynchronizing phase lags during intrathalamic oscillations 8th Annual Computational Neurosciences Meeting (CNS 99) Sohal, V. S., Huntsman, M. M., Huguenard, J. R. ELSEVIER SCIENCE BV. 2000: 509–516

  View details for Web of Science ID 000087897800071

• Reciprocal inhibitory connections regulate the spatiotemporal properties of intrathalamic oscillations JOURNAL OF NEUROSCIENCE Sohal, V. S., Huntsman, M. M., Huguenard, J. R. 2000; 20 (5): 1735-1745

  Abstract

  Mice with an inactivated GABA(A) receptor beta(3) subunit gene have features of Angelman syndrome, including absence-like seizures. This suggests the occurrence of abnormal hypersynchrony in the thalamocortical system. Within the thalamus, the efficacy of inhibitory synapses between thalamic reticular (RE) neurons is selectively compromised, and thalamic oscillations in vitro are prolonged and lack spatial phase gradients (). Here we used computational models to examine how intra-RE inhibition regulates intrathalamic oscillations. A major effect is an abbreviation of network responses, which is caused by long-lasting intra-RE inhibition that shunts recurrent excitatory input. In addition, differential activation of RE cells desynchronizes network activity. Near the slice center, where many cells are initially activated, there is a resultant high level of intra-RE inhibition. This leads to RE cell burst truncation in the central region and a gradient in the timing of thalamocortical cell activity similar to that observed in vitro. Although RE cell burst durations were shortened by this mechanism, there was very little effect on the times at which RE cells began to burst. The above results depended on widespread stimuli that activated RE cells in regions larger than the diameter of intra-RE connections. By contrast, more focal stimuli could elicit oscillations that lasted several cycles and remained confined to a small region. These results suggest that intra-RE inhibition restricts intrathalamic activity to particular spatiotemporal patterns to allow focal recurrent activity that may be relevant for normal thalamocortical function while preventing widespread synchronization as occurs in seizures.

  View details for Web of Science ID 000085462600011

  View details for PubMedID 10684875

• Voltage-gated potassium channels activated during action potentials in layer V neocortical pyramidal neurons JOURNAL OF NEUROPHYSIOLOGY Kang, J., Huguenard, J. R., Prince, D. A. 2000; 83 (1): 70-80

  Abstract

  To investigate voltage-gated potassium channels underlying action potentials (APs), we simultaneously recorded neuronal APs and single K(+) channel activities, using dual patch-clamp recordings (1 whole cell and 1 cell-attached patch) in single-layer V neocortical pyramidal neurons of rat brain slices. A fast voltage-gated K(+) channel with a conductance of 37 pS (K(f)) opened briefly during AP repolarization. Activation of K(f) channels also was triggered by patch depolarization and did not require Ca(2+) influx. Activation threshold was about -20 mV and inactivation was voltage dependent. Mean duration of channel activities after single APs was 6.1 +/- 0.6 ms (mean +/- SD) at resting membrane potential (-64 mV), 6.7 +/- 0.7 ms at -54 mV, and 62 +/- 15 ms at -24 mV. The activation and inactivation properties suggest that K(f) channels function mainly in AP repolarization but not in regulation of firing. K(f) channels were sensitive to a low concentration of tetraethylammonium (TEA, 1 mM) but not to charybdotoxin (ChTX, 100 nM). Activities of A-type channels (K(A)) also were observed during AP repolarization. K(A) channels were activated by depolarization with a threshold near -45 mV, suggesting that K(A) channels function in both repolarization and timing of APs. Inactivation was voltage dependent with decay time constants of 32 +/- 6 ms at -64 mV (rest), 112 +/- 28 ms at -54 mV, and 367 +/- 34 ms at -24 mV. K(A) channels were localized in clusters and were characterized by steady-state inactivation, multiple subconductance states (36 and 19 pS), and inhibition by 5 mM 4-aminopyridine (4-AP) but not by 1 mM TEA. A delayed rectifier K(+) channel (K(dr)) with a unique conductance of 17 pS was recorded from cell-attached patches with TEA/4-AP-filled pipettes. K(dr) channels were activated by depolarization with a threshold near -25 mV and showed delayed long-lasting activation. K(dr) channels were not activated by single action potentials. Large conductance Ca(2+)-activated K(+) (BK) channels were not triggered by neuronal action potentials in normal slices and only opened as neuronal responses deteriorated (e.g., smaller or absent spikes) and in a spike-independent manner. This study provides direct evidence for different roles of various K(+) channels during action potentials in layer V neocortical pyramidal neurons. K(f) and K(A) channels contribute to AP repolarization, while K(A) channels also regulate repetitive firing. K(dr) channels also may function in regulating repetitive firing, whereas BK channels appear to be activated only in pathological conditions.

  View details for Web of Science ID 000084777800008

  View details for PubMedID 10634854

• Nucleus-specific differences in GABA(A)-receptor-mediated inhibition are enhanced during thalamic development JOURNAL OF NEUROPHYSIOLOGY Huntsman, M. M., Huguenard, J. R. 2000; 83 (1): 350-358

  Abstract

  Inhibitory postsynaptic currents (IPSCs) mediated by GABA(A) receptors are much slower in neurons of the thalamic reticular nucleus (RTN) versus those in the ventrobasal complex (VB) of young rats. Here we confirm and extend those findings regarding GABA(A) response heterogeneity especially in relation to development. Whole cell patch-clamp recordings were used to investigate GABA(A) spontaneous and electrically evoked IPSCs (sIPSCs/eIPSCs) in RTN and VB cells of different aged rats. Consistent with earlier findings, sIPSC duration at P8-12 was considerably longer in RTN (weighted decay time constant: tau(D,W) = 56.2 +/- 4.9 ms; mean +/- SE) than in VB (tau(D,W) = 15.8 +/- 1.0 ms) neurons. Decay kinetics in RTN neurons did not differ at P21-30 (45.5 +/- 4.7 ms) or P42-60 (51.6 +/- 10.6 ms). In contrast, VB sIPSCs were significantly faster at both P21-30 (tau(D,W) = 10.8 +/- 0.9 ms) and P42-60 (tau(D,W) = 9.2 +/- 0.4 ms) compared with P8-12 animals. IPSCs displayed differential outward rectification and temperature dependence, providing further support for nucleus-specific responses. tau(D,W) increased with membrane depolarization but with a net larger effect in VB. By contrast, tau(D,W) was always smaller at higher temperatures but with relatively greater difference observed in RTN. Thus nuclear differences in GABA(A) IPSCs are not only maintained, but enhanced in the mature rodent under physiological conditions. These findings support our hypothesis that unique GABA(A) receptors mediate slowly decaying RTN IPSCs that are a critical and enduring feature of the thalamic circuit. This promotes powerful intranuclear inhibition and likely prevents epileptiform thalamocortical hypersynchrony.

  View details for Web of Science ID 000084777800032

  View details for PubMedID 10634878

• Long-range connections synchronize rather than spread intrathalamic oscillatory activity: Computational modeling and in vitro electrophysiology NEUROCOMPUTING Sohal, V. S., Huguenard, J. R. 1999; 26-7: 525-531

  View details for Web of Science ID 000081462700070

• Reciprocal inhibitory connections and network synchrony in the mammalian thalamus SCIENCE Huntsman, M. M., Porcello, D. M., Homanics, G. E., Delorey, T. M., Huguenard, J. R. 1999; 283 (5401): 541-543

  Abstract

  Neuronal rhythmic activities within thalamocortical circuits range from partially synchronous oscillations during normal sleep to hypersynchrony associated with absence epilepsy. It has been proposed that recurrent inhibition within the thalamic reticular nucleus serves to reduce synchrony and thus prevents seizures. Inhibition and synchrony in slices from mice devoid of the gamma-aminobutyric acid type-A (GABAA) receptor beta3 subunit were examined, because in rodent thalamus, beta3 is largely restricted to reticular nucleus. In beta3 knockout mice, GABAA-mediated inhibition was nearly abolished in reticular nucleus, but was unaffected in relay cells. In addition, oscillatory synchrony was dramatically intensified. Thus, recurrent inhibitory connections within reticular nucleus act as "desynchronizers."

  View details for Web of Science ID 000078203300038

  View details for PubMedID 9915702

• Neuronal circuitry of thalamocortical epilepsy and mechanisms of antiabsence drug action. Advances in neurology Huguenard, J. R. 1999; 79: 991-999

  Abstract

  Powerful mechanisms exist within the thalamus that lead to the promotion of synchronous and phasic 3 Hz neuronal activity. These mechanisms include robust burst-firing capability of thalamic neurons, recurrent excitatory and inhibitory synaptic connectivity, and long-lasting and powerful inhibitory synaptic responses arising from activity in thalamic reticular neurons and mediated by gamma-aminobutyric acid (GABA) receptors. The 3 Hz thalamic synchronization appears to arise from a perturbation of a physiologic, higher frequency spindle oscillation. Two currently available antiabsence medications interact with this circuitry with the net result of decreased synchronization, largely through reduction in inhibitory output from the thalamic reticular nucleus. Ethosuximide blocks T-type calcium channels and thus reduces the ability of thalamic neurons to fire bursts of spikes, thereby reducing inhibitory (and excitatory) output within the circuit. By contrast, clonazepam enhances recurrent inhibitory strength within the reticular nucleus. This results in a decreased ability of neighboring inhibitory neurons to fire synchronously and produce the powerful inhibitory synaptic responses that are required for network synchronization.

  View details for PubMedID 10514881

• Low-voltage-activated (T-type) calcium-channel genes identified TRENDS IN NEUROSCIENCES Huguenard, J. R. 1998; 21 (11): 451-452

  View details for Web of Science ID 000076828500001

  View details for PubMedID 9829683

• Long-range connections synchronize rather than spread intrathalamic oscillations: Computational modeling and in vitro electrophysiology JOURNAL OF NEUROPHYSIOLOGY Sohal, V. S., Huguenard, J. R. 1998; 80 (4): 1736-1751

  Abstract

  A thalamic network model was developed based on recent data regarding heterogeneous thalamic reticular (RE) cell axonal arborizations that indicate at least two projection patterns, short-range cluster projections and long-range diffuse projections. The model was constrained based on expected convergence and the biophysical properties of RE and thalamocortical (TC) cells and their synapses. The model reproduced in vitro synchronous slow (3-Hz) oscillatory activity and the known effects of T-channel blockade and cholecystokinin (CCK) application on this activity. Whereas previous models used the speed at which approximately 3-Hz oscillations propagate in vitro to infer the spatial extent of intrathalamic projections, we found that, so long as the gamma-aminobutyric acid-B synaptic conductance was adjusted appropriately, a network with only short-range projections and another network with both short- and long-range projections could both produce physiologically realistic propagation speeds. Although the approximately 3-Hz oscillations propagated at similar speeds in both networks, phase differences between oscillatory activity at different locations in the network were much smaller in the network containing both short- and long-range projections. We measured phase differences in vitro and found that they were similar to those that arise in the network containing both short- and long-range projections but are inconsistent with the much larger phase differences that occur in the network containing only short-range projections. These results suggest that, although they extend much further than do short-range cluster projections, long-range diffuse projections do not spread activity over greater distances or increase the speed at which intrathalamic oscillations propagate. Instead, diffuse projections may function to synchronize activity and minimize phase shifts across thalamic networks. One prediction of this hypothesis is that, immediately after a collision between propagating oscillations, phase gradients should vary smoothly across the thalamic slice. The model also predicts that phase shifts between oscillatory activity at different points along a thalamic slice should be unaffected by T-channel blockers and decreased by suppression of synaptic transmission or application of CCK.

  View details for Web of Science ID 000076487400011

  View details for PubMedID 9772235

• Cholinergic switching within neocortical inhibitory networks SCIENCE Xiang, Z. X., Huguenard, J. R., Prince, D. A. 1998; 281 (5379): 985-988

  Abstract

  Differential actions of acetylcholine on the excitability of two subtypes of interneurons in layer V of the rat visual cortex were examined. Acetylcholine excited low-threshold spike (LTS) cells through nicotinic receptors, whereas it elicited hyperpolarization in fast spiking (FS) cells through muscarinic receptors. Axons of LTS cells were mainly distributed vertically to upper layers, and those of FS cells were primarily confined to layer V. Thus, cortical cholinergic activation may reduce some forms of intralaminar inhibition, promote intracolumnar inhibition, and change the direction of information flow within cortical circuits.

  View details for Web of Science ID 000075412700046

  View details for PubMedID 9703513

• Anatomical and physiological considerations in thalamic rhythm generation JOURNAL OF SLEEP RESEARCH Huguenard, J. R. 1998; 7: 24-29

  Abstract

  The thalamus, known as the pacemaker for spindle rhythms in sleep, has several enabling features that promote such pacemaking. These include a circuitry that interconnects large groups of excitatory and inhibitory neurons, all of which are essentially capable of firing high-frequency 'bursts' of discharges. Bursts in thalamic reticular neurons produce powerful inhibition in thalamic relay neurons, which leads to rebound excitation. The timing properties of the inhibition regulate the network activity by controlling rebound burst latency. Anatomical features within thalamus such as convergence and divergence determine the spread and synchronization of pacemaking activity. The anatomical basis of divergence, i.e. the degree of axonal arborization of elements within the thalamic circuit, can be functionally modified in a dynamic fashion by biochemical pathways that regulate the properties of synaptic release. These data suggest that it will be possible to therapeutically regulate the thalamus to modify not only the propensity to sleep but also forms of epilepsy that rely on similar thalamic circuitry.

  View details for Web of Science ID 000074916400005

  View details for PubMedID 9682190

• Dendritic low-threshold calcium currents in thalamic relay cells JOURNAL OF NEUROSCIENCE Destexhe, A., Neubig, M., Ulrich, D., Huguenard, J. 1998; 18 (10): 3574-3588

  Abstract

  The low-threshold calcium current (IT) underlies burst generation in thalamocortical (TC) relay cells and plays a central role in the genesis of synchronized oscillations by thalamic circuits. Here we have combined in vitro recordings and computational modeling techniques to investigate the consequences of dendritically located IT in TC cells. Simulations of a reconstructed TC cell were compared with the recordings obtained in the same cell to constrain the values of its passive parameters. T-current densities in soma and proximal dendrites were then estimated by matching the model to voltage-clamp recordings obtained in dissociated TC cells, which lack most of the dendrites. The distal dendritic T-current density was constrained by recordings in intact TC cells, which show 5-14 times larger peak T-current amplitudes compared with dissociated cells. Comparison of the model with the recordings of the same cell constrained further the T-current density in dendrites, which had to be 4.5-7.6 times higher than in the soma to reproduce all experimental results. Similar conclusions were reached using a simplified three-compartment model. Functionally, the model shows that the same amount of T-channels can lead to different bursting behaviors if they are exclusively somatic or distributed throughout the dendrites. In conclusion, this combination of models and experiments shows that dendritic T-currents are necessary to reproduce low-threshold calcium electrogenesis in TC cells. Dendritic T-current may also have significant functional consequences, such as an efficient modulation of thalamic burst discharges by corticothalamic feedback.

  View details for Web of Science ID 000073484300010

  View details for PubMedID 9570789

• Localization of CCK receptors in thalamic reticular neurons: A modeling study JOURNAL OF NEUROPHYSIOLOGY Sohal, V. S., Cox, C. L., Huguenard, J. R. 1998; 79 (5): 2820-2824

  Abstract

  In an earlier experimental study, intracellular recording suggested that cholecystokinin (CCK) suppresses a K+ conductance in thalamic reticular (RE) neurons, yet the reversal potential of the CCK response, revealed using voltage clamp, was hyperpolarized significantly relative to the K+ equilibrium potential. Here, biophysical models of RE neurons were developed and used to test whether suppression of the K+ conductance, gK, can account for the CCK response observed in vitro and also to determine the likely site of CCK receptors on RE neurons. Suppression of gK in model RE neurons can reproduce the relatively hyperpolarized reversal potential of CCK responses found using voltage clamp if the voltage clamp becomes less effective at hyperpolarized potentials. Three factors would reduce voltage-clamp effectiveness in this model: the nonnegligible series resistance of the voltage-clamp electrode, a hyperpolarization-activated mixed cation current (Ih) in RE neurons, and the dendritic location of CCK-sensitive K+ channels. Although suppression of gK in the dendritic compartments of model RE neurons simulates both the magnitude and reversal potential of the CCK response, suppression of gK in just the somatic compartment of model RE neurons fails to do so. Thus the model predicts that CCK should effectively suppress K+ conductance RE neuron dendrites and thereby regulate burst firing in RE neurons. This may explain the potent effects of CCK on intrathalamic oscillations in vitro.

  View details for Web of Science ID 000073768400049

  View details for PubMedID 9582249

• Adrenergic modulation of GABA(A) receptor-mediated inhibition in rat sensorimotor cortex JOURNAL OF NEUROPHYSIOLOGY Bennett, B. D., Huguenard, J. R., Prince, D. A. 1998; 79 (2): 937-946

  Abstract

  The effect of adrenoceptor activation on pharmacologically isolated monosynaptic inhibitory postsynaptic currents (IPSCs) detected in layer V pyramidal neurons was examined by using whole cell voltage-clamp in a slice preparation of rat sensorimotor cortex. Epinephrine (EPI; 10 muM) reversibly altered the amplitude of evoked IPSCs (eIPSCs) in slices from postnatal day 9-12 (P9-12) and P15-18 rats. The effects of EPI were heterogeneous in both age groups, and in individual cases an enhancement, a depression or no effect of eIPSCs was observed, although depression was observed more commonly in the younger age group. The effects of EPI on eIPSC amplitude were likely mediated through presynaptic mechanisms because they occurred in the absence of any alteration in the current produced by direct application of gamma-aminobutyric acid (GABA), or in input resistance. EPI always elicited an increase in the frequency of spontaneous IPSCs (sIPSCs) irrespective of whether or not it induced any change in the amplitude of eIPSCs in the same neuron. The increase in sIPSC frequency was blocked by phentolamine (10 muM) but not by propranolol (10 muM), supporting the conclusion that EPI-mediated effects on sIPSC frequency result from activation of alpha-adrenoceptors located on presynaptic inhibitory interneurons. In a subpopulation of neurons (3/9) from P15-18 rats, EPI increased both the amplitude and frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) and under conditions where postsynaptic EPI effects were blocked, suggesting activation of adrenoceptors on presynaptic terminals in these cells. Results of these experiments are consistent with an action of EPI at adrenoceptors located on presynaptic GABAergic interneurons. Adrenergic activation thus has multiple and complex influences on excitability in cortical circuits, some of which are a consequence of interactions that regulate the strength of GABAergic inhibition.

  View details for Web of Science ID 000072115600038

  View details for PubMedID 9463454

• GABA(A) receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex JOURNAL OF PHYSIOLOGY-LONDON Xiang, Z. X., Huguenard, J. R., Prince, D. A. 1998; 506 (3): 715-730

  Abstract

  1. We compared gamma-aminobutyric acid (GABA)-mediated responses of identified pyramidal cells and fast spiking interneurons in layer V of visual cortical slices from young rats (P11-14). 2. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was similar in pyramidal cells and interneurons (1.7 vs. 1.9 Hz). For events with 10-90% rise times less than 0.9 ms, no significant differences were found in mean amplitude (61 vs. 65 pA), mean rise time (0.58 vs. 0.61 ms), or the first time constant of decay (tau 1, 6.4 vs. 6.5 ms) between pyramidal cells and interneurons. The second decay time constant (tau 2) was significantly longer in interneurons than in pyramidal cells (49 vs. 22 ms). The difference in sIPSC decay kinetics between two cell types also existed in adult rats (P36-42), suggesting the kinetic difference in not due to differential development of GABAA receptors in these cell types. 3. The decay kinetics of monosynaptic evoked IPSCs were also longer in interneurons. As in the case of sIPSCs, the difference was accounted for by the second decay time constant. tau 1 and tau 2 were, respectively, 13 and 64 ms for interneurons and 12 and 47 ms for pyramidal cells. 4. Cell-attached patch recordings revealed that the mean open time for single Cl- channels in response to 2 microM GABA was significantly longer in interneurons than pyramidal cells (5.0 vs. 2.8 ms). The chord conductance of these channels in interneurons (12 pS) was significantly smaller than in pyramidal cells (15 pS). Single channel currents reversed polarity when the pipette potential was approximately -10 mV for both cell types. 5. These results show that there is a functional diversity of GABAA receptors in electrophysiologically and morphologically identified cortical pyramidal cells and interneurons. This diversity might derive from the different molecular composition of the receptors in these two cell types.

  View details for Web of Science ID 000072026800011

  View details for PubMedID 9503333

• Dendritic calcium currents in thalamic relay cells 6th Annual Computational Neuroscience Conference Neubig, M., Ulrich, D., Huguenard, J., Destexhe, A. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1998: 233–238

  View details for Web of Science ID 000075569500039

• GABA(A) receptor-mediated Cl- currents in rat thalamic reticular and relay neurons JOURNAL OF NEUROPHYSIOLOGY ZHANG, S. L., Huguenard, J. R., Prince, D. A. 1997; 78 (5): 2280-2286

  Abstract

  GABAA receptor-mediated Cl- currents in rat thalamic reticular and relay neurons. J. Neurophysiol. 78: 2280-2286, 1997. Spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs) and responses to exogenously applied gamma-aminobutyric acid (GABA), mediated by GABA type A (GABAA) receptors, were recorded in inhibitory neurons of nucleus reticularis thalami (nRt) and their target relay cells in ventrobasal (VB) nuclei by using patch clamp techniques in rat thalamic slices. The decay of sIPSCs in both nRt and VB neurons was best fitted with two exponential components. The decay time constants of sIPSCs in nRt neurons were much slower (tau1 = 38 ms; tau2 = 186 ms) than those previously reported in a variety of preparations and two to three times slower than those in VB neurons (tau1 = 17 ms; tau2 = 39 ms). GABAA receptor-mediated Cl- currents directly evoked by local GABA application also had a much slower decay time constant in nRt (225 ms) than in VB neurons (115 ms). Slow decay of GABA responses enhances the efficacy of recurrent intranuclear inhibition in nRt. The results suggest a functional diversity of GABAA receptors that may relate to the known heterogeneity of GABAA receptor subunits in these two thalamic nuclei.

  View details for Web of Science ID A1997YG93900005

  View details for PubMedID 9356381

• GAB(A)-receptor-mediated rebound burst firing and burst shunting in thalamus JOURNAL OF NEUROPHYSIOLOGY Ulrich, D., Huguenard, J. R. 1997; 78 (3): 1748-1751

  Abstract

  The role of gamma-aminobutyric acid-A (GABA(A))-receptor-mediated inhibitory postsynaptic potentials (IPSPs) in 1) generating rebound burst firing and 2) burst inhibition in thalamocortical (TC) relay cells and inhibitory neurons of nucleus reticularis thalami (nRt) was investigated. Experimental data from previous studies were used to generate artificial synaptic responses in neurons via a computer-driven dynamic clamp. On average, in nRt neurons trains of six or more 10-nS GABA(A) IPSPs generated rebound bursts of action potentials with a mean delay of 605 +/- 32 (SE) ms. In contrast, 10 IPSPs were required for rebound bursts in relay cells, and these occurred with a significantly shorter delay of 327 +/- 35 ms. Ca2+-dependent burst responses could be shunted by single IPSPs. Half-maximal burst inhibition was obtained in nRt cells when IPSP conductance was 1.5 times the whole cell input conductance. Burst shunting in TC cells was less effective and required a synaptic- to input-conductance ratio of 3. The relative time window of IPSP burst shunting was broader in nRt (approximately 20 ms) than TC cells (approximately 10 ms). We conclude that in nRt cells GABA(A)-dependent rebound burst responses would occur with a latency that is incompatible with pacemaking of fast (>3-Hz) thalamic rhythm generation such as spindles, yet burst inhibition is powerful. Therefore a likely role for reciprocal intra-nRt connectivity is to mediate lateral inhibition between nRt cells.

  View details for Web of Science ID A1997XY50300051

  View details for PubMedID 9310462

• Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cox, C. L., Huguenard, J. R., Prince, D. A. 1997; 94 (16): 8854-8859

  Abstract

  Detailed information regarding the contribution of individual gamma-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. "Weak" inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, "strong" inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1-3 release sites, whereas stronger inhibition would require simultaneous activation of 5-70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.

  View details for Web of Science ID A1997XQ12400100

  View details for PubMedID 9238067

• Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex JOURNAL OF NEUROPHYSIOLOGY Bennett, B. D., Huguenard, J. R., Prince, D. A. 1997; 78 (1): 561-566

  Abstract

  At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.

  View details for Web of Science ID A1997XM21100054

  View details for PubMedID 9242307

• Nucleus-specific chloride homeostasis in rat thalamus JOURNAL OF NEUROSCIENCE Ulrich, D., Huguenard, J. R. 1997; 17 (7): 2348-2354

  Abstract

  Synchronous thalamic network activity occurring during slow wave sleep and paroxysmal discharges critically depends on the ability of thalamocortical relay cells and inhibitory neurons of the nucleus reticularis thalami (nRt) to fire bursts of action potentials. Inhibitory synaptic potentials (IPSPs) originating from nRt cells are crucial in deinactivating T-channels and thus promoting burst firing in relay cells, but the functional role of intra-nRt IPSPs is less well understood. A major factor that regulates the net effects of IPSP generation is the chloride equilibrium potential (ECl). Here we applied the perforated patch-clamp technique, using the cation-selective ionophore gramicidin to assess the reversal potential of chloride in nRt and relay cells in brain slices. We found that the reversal potential of GABA-induced membrane currents (EGABA) was significantly more hyperpolarized in relay (-81 +/- 2.6 mV), as compared with nRt cells (-71 +/- 2.5 mV). EGABA was not significantly different from the reversal potential of evoked IPSCs (EIPSC; -82 +/- 4.4 mV) in relay cells. In both relay and reticular neurons the chloride gradient was collapsed partially by the chloride cation cotransport blocker furosemide, suggesting an active chloride extrusion mechanism in thalamic neurons. Given the relatively hyperpolarized resting potentials (approximately -70 mV) reported for nRt and relay cells during in vitro thalamic oscillations, we conclude that under these conditions GABAA IPSPs lead to significant hyperpolarization in relay cells. By contrast, intra-nRt inhibition essentially would be shunting, i.e., would produce minimal membrane polarization but still could reduce the amplitude of excitatory events.

  View details for Web of Science ID A1997WQ58500010

  View details for PubMedID 9065495

• Peptidergic modulation of intrathalamic circuit activity in vitro: Actions of cholecystokinin JOURNAL OF NEUROSCIENCE Cox, C. L., Huguenard, J. R., Prince, D. A. 1997; 17 (1): 70-82

  Abstract

  Cholecystokinin (CCK)-mediated actions on intrathalamic rhythmic activities were examined in an in vitro rat thalamic slice preparation. Single electrical stimuli in the thalamic reticular nucleus (nRt) evoked rhythmic activity (1-15 sec duration) in nRt and the adjacent ventrobasal nucleus (VB). Low CCK concentrations (20-50 nM) suppressed rhythmic oscillations in 43% of experiments but prolonged such activities in the remaining slices. Higher CCK concentrations (100-400 nM) had a predominantly antioscillatory effect. Suppression of oscillations was associated with a relatively large membrane depolarization of nRt neurons that changed their firing mode from phasic (burst) to tonic (single-spike) output. This decreased burst discharge of nRt neurons during CCK application reduced inhibitory drive onto VB neurons from multiple peaked inhibitory postsynaptic currents (IPSCs) to single peaked inhibitory events. We hypothesize that suppression of inhibitory drive onto VB neurons decreases their probability of burst output, which, together with a reduction of nRt burst output, dampens the oscillatory activity. Low CCK concentrations, which produced little or no depolarization of nRt neurons, did not alter the firing mode of the nRt neurons. However, the probability of burst output from nRt neurons in response to subthreshold stimuli was increased in low CCK concentrations, presumably leading to an increase in the number of nRt neurons participating in the rhythmic activity. Our findings suggest that the neuropeptide CCK, by altering the firing characteristics of nRt neurons, has powerful modulatory effects on intrathalamic rhythms; the ultimate action was dependent on CCK concentration and resting state of these cells.

  View details for Web of Science ID A1997WJ67700007

  View details for PubMedID 8987737

• Two types of BK channels in immature rat neocortical pyramidal neurons JOURNAL OF NEUROPHYSIOLOGY Kang, J., Huguenard, J. R., Prince, D. A. 1996; 76 (6): 4194-4197

  Abstract

  1. The properties of large conductance Ca(2+)-activated K+ channels (BK channels) were investigaed in neocortical infragranular pyramidal neurons by the use of inside-out patch recordings. Neurons were acutely isolated from slices of newborn to 28-day-old rats (P0-P28) by using minimal protease exposure followed by trituration with a vibrating glass probe. Two types of BK channels, slow-gating and fast-gating, were observed in immature neurons (P0-P5), whereas only slow-gating BK were found in more mature neurons. Fast-gating BK channels differed in conductance, voltage dependence, and kinetics from the slow-gating ones. 2. The properties of fast-gating channels included a conductance of 145 +/- 12.9 (SE) pS; frequent openings with short mean open times that were relatively voltage-independent, mean closed times that showed a voltage-dependent increase, a voltage-dependent decrease in open probability (Po). The properties of slow-gating channels contrasted with those of the fast-gating ones, in that the former had a conductance of 181 +/- 3.9 pS, longer mean open times that showed a voltage-dependent increase, mean closed times that showed a marked voltage-dependent decrease, a voltage-dependent increase in Po, and slight inward rectification. The significance of these developmental variations in channel properties is discussed.

  View details for Web of Science ID A1996WA80300056

  View details for PubMedID 8985914

• gamma-Aminobutyric acid type B receptor-dependent burst-firing in thalamic neurons: A dynamic clamp study PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ulrich, D., Huguenard, J. R. 1996; 93 (23): 13245-13249

  Abstract

  Synchronized network responses in thalamus depend on phasic inhibition originating in the thalamic reticular nucleus (nRt) and are mediated by the neurotransmitter gamma-aminobutyric acid (GABA). A suggested role for intra-nRt connectivity in inhibitory phasing remains controversial. Recently, functional GABA type B (GABAB) receptors were demonstrated on nRt cells, and the slow time course of the GABAB synaptic response seems ideally suited to deinactivate low-threshold calcium channels. This promotes burst firing, a characteristic feature of synchronized responses. Here we investigate GABAB-mediated rebound burst firing in thalamic cells. Whole-cell current-clamp recordings were obtained from nRt cells and somatosensory thalamocortical relay cells in rat brain slices. Synthetic GABAB inhibitory postsynaptic potentials, generated by a hybrid computerneuron synapse (dynamic clamp), triggered rebound low-threshold calcium spikes in both cell types when peak inhibitory postsynaptic potential hyperpolarization was greater than -92 mV. The threshold inhibitory postsynaptic potential conductance for rebound burst generation was comparable in nRt (7 nS) and thalamocortical (5 nS) cells. However, burst onset in nRt (1 s) was considerably delayed compared with thalamocortical (0.6 s) cells. Thus, GABAB inhibitory postsynaptic potentials can elicit low-threshold calcium spikes in both relay and nRt neurons, but the resultant oscillation frequency would be faster for thalamocortical-nRt networks (3 Hz) than for nRt-nRt networks (1-2 Hz). We conclude, therefore, that fast (> 2 Hz) GABAB-dependent thalamic oscillations are maintained primarily by reciprocal connections between excitatory and inhibitory cells. These findings further indicate that when oscillatory neural networks contain both recurrent and reciprocal inhibition, then distinct population frequencies may result when one or the other type of inhibition is favored.

  View details for Web of Science ID A1996VT05400103

  View details for PubMedID 8917576

• Development of BK channels in neocortical pyramidal neurons JOURNAL OF NEUROPHYSIOLOGY Kang, J., Huguenard, J. R., Prince, D. A. 1996; 76 (1): 188-198

  Abstract

  1. Postnatal development of a large conductance Ca(2+)-activated K+ channel (BK channel) was investigated in neocortical infragranular pyramidal neurons with inside-out and outside-out patchclamp configurations. Neurons were acutely isolated from slices of 1- to 28-day-old rats (P1-P28) by using a vibrating glass probe after preincubation with low concentrations of enzymes. Patch membrane area was estimated by measuring membrane capacitance. The density, distribution, voltage dependence, Ca2+ sensitivity, kinetics, and pharmacological properties of BK channels were examined in neurons from animals of different ages. 2. In somata, the density of BK channels was 0.056 +/- 0.011/ micron 2 in P1 neurons and 0.312 +/- 0.008/micron 2 in P28 neurons. There was an abrupt increase between P5 and P7 at a rate of approximately 0.042/ micron 2/day. Before P5 and after P7, the density of BK channels also increased but at slower rates. 3. The density of BK channels in proximal apical dendrites underwent a similar developmental sequence. There was a relatively large increase between P5 and P7 with a rate of approximately 0.021/ micron 2/day, and after P7, channel density increased more slowly (approximately 0.002/microns 2/day). In P1 neurons, channel density in apical dendrites was 0.039 +/- 0.008/micron 2, which was close to that in somata, whereas in P28 neurons, channel density (0.134 +/- 0.008/micron 2) was less than one-half of that in somata. 4. The distribution of BK channels was different in immature and mature neurons. In somata of P1 neurons, BK channels were distributed singly without evidence of clustering, whereas in P28 neurons BK channels were clustered in groups of approximately 4. 5. BK channels in both P1 and P14 neurons showed a steep increase in the probability of opening (Po) as intracellular Ca2+ concentration was raised from 50 to 100 nM, especially at positive membrane potentials. The Ca2+ dependence, as measured by the [Ca2+]i that provided half-maximal Po at a variety of membrane potentials, was not different in patches from P1 and P14 neurons. On the other hand, the voltage dependence of BK channels shifted during ontogeny such that Po was larger at negative potentials in P14 than in P1 neurons. 6. The voltage dependence of P1 BK channels was bimodally distributed with 57% of channels exhibiting an "immature" pattern consisting of a more positive V1/2 and a smaller change in voltage required to produce an e-fold increase in Po. Immature type P1 BK channels showed a longer mean closed time at negative membrane potentials than either P14 or "mature" P1 BK channels. 7. No postnatal developmental changes in pharmacological properties of BK channels were observed. In both mature and immature neurons, BK channels were partially inhibited by 30 or 100 nM charybdotoxin (ChTX) and fully blocked by 1 microM ChTX. The IC50 for ChTX was 100 nM, indicating that BK channels in neocortical pyramidal neurons are much less sensitive to ChTX than those in muscle cells and sympathetic ganglion neurons. BK channels were also inhibited by 0.5 mM tetraethylammonium chloride (TEA) and 50 microM trifluoperazine. 8. These data indicate that functional somatic and dendritic BK channels are inserted into neuronal membranes during neocortical development, with an especially rapid increment in density occurring around P5-P7. These changes, which occur at a time when other voltage-gated ion channels are known to be increasing in density, contribute to the development of neocortical excitability.

  View details for Web of Science ID A1996UY79300015

  View details for PubMedID 8836218

• GABA(B) receptor-mediated responses in GABAergic projection neurones of rat nucleus reticularis thalami in vitro JOURNAL OF PHYSIOLOGY-LONDON Ulrich, D., Huguenard, J. R. 1996; 493 (3): 845-854

  Abstract

  1. Whole-cell voltage-clamp recordings were obtained from GABAergic neurones of rat nucleus reticularis thalami (NRT) in vitro to assess pre- and postsynaptic GABAB receptor-mediated responses. Presynaptic inhibition of GABA release was studied at terminals on local axon collaterals within NRT as well as on projection fibres in the somatosensory relay nuclei. 2. The GABAB receptor agonist (R)-baclofen (10 microM) reduced monosynaptically evoked GABAA-mediated inhibitory postsynaptic currents (IPSCs) in NRT and somatosensory relay cells to 11 and 12% of control, respectively. 3. Action potential-independent miniature IPSCs (mIPSCs) were observed in both cell types. Mean mIPSC amplitude was 20 pA in both NRT and relay cells at a holding potential of 0 mV. The mean mIPSC frequencies were 0.83 and 2.2 Hz in NRT and relay cells, respectively. Baclofen decreased mIPSP frequency by about half in each cell type without affecting amplitude. 4. Paired-burst inhibition of evoked IPSCs was studied in relay and NRT cells by applying pairs of 100 Hz stimulus bursts separated by 600 ms. The mean ratio of second to first peak IPSC amplitudes was 0.77. 5. In NRT cells baclofen induced a linear postsynaptic conductance increase of 0.82 nS with an associated reversal potential of -121 mV. A small (0.14 nS) GABAB component of the evoked IPSC was detected in only a minority of NRT cells (3 of 18). 6. All pre- and postsynaptic effects of baclofen, as well as PBI, were largely reversed by the specific GABAB receptor antagonist CGP 35348 (0.5 mM). 7. We conclude that activation of GABAB receptors in NRT leads to presynaptic autoinhibition of IPSCs in both NRT and relay cells, and to direct activation of a small linear K+ conductance. In addition our experiments suggest that reciprocal connectivity within NRT can be partially mediated by a small GABAB inhibitory event.

  View details for Web of Science ID A1996UV41100020

  View details for PubMedID 8799904

• Heterogeneous axonal arborizations of rat thalamic reticular neurons in the ventrobasal nucleus JOURNAL OF COMPARATIVE NEUROLOGY Cox, C. L., Huguenard, J. R., Prince, D. A. 1996; 366 (3): 416-430

  Abstract

  The gamma-aminobutyric acid (GABA)-containing neurons of the thalamic reticular nucleus (nRt) are a major source of inhibitory innervation in dorsal thalamic nuclei. Individual nRt neurons were intracellularly recorded and labelled in an in vitro rat thalamic slice preparation to investigate their projection into ventrobasal thalamic nuclei (VB). Camera lucida reconstructions of 37 neurons indicated that nRt innervation ranges from a compact, focal projection to a widespread, diffuse projection encompassing large areas of VB. The main axons of 65% of the cells gave rise to intra-nRt collaterals prior to leaving the nucleus and, once within VB, ramified into one of three branching patterns: cluster, intermediate, and diffuse. The cluster arborization encompassed a focal region averaging approximately 25,000 mu m2 and contained a high density of axonal swellings, indicative of a topographic projection. The intermediate structure extended across an area approximately fourfold greater and also contained numerous axonal swellings. The diffuse arborization of nRt neurons covered a large region of VB and contained a relatively low density of axonal swellings. Analysis of somatic size and shape revealed that diffuse arborizations arose from significantly smaller, fusiform-shaped somata. Cytochrome oxidase reactivity or parvalbumin immunoreactivity was used to delineate a discontinuous staining pattern representing thalamic barreloids. The size of a cluster arborization closely approximated that of an individual barreloid. The heterogeneous arborizations from nRt neurons may reflect a dynamic range of inhibitory influences of nRt on dorsal thalamic activity.

  View details for Web of Science ID A1996TY11300004

  View details for PubMedID 8907356

• In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons JOURNAL OF NEUROSCIENCE Destexhe, A., CONTRERAS, D., Steriade, M., Sejnowski, T. J., Huguenard, J. R. 1996; 16 (1): 169-185

  Abstract

  Thalamic reticular (RE) neurons are involved in the genesis of synchronized thalamocortical oscillations, which depend in part on their complex bursting properties. We have investigated the intrinsic properties of RE cells using computational models based on morphological and electrophysiological data. Simulations of a reconstructed RE cells were compared directly with recordings from the same cell to obtain precise values for the passive parameters. In a first series of experiments, the low-threshold calcium current (I(Ts)) was studied via voltage clamp in acutely dissociated RE cells that lack most of their dendrites. Simulations based on a cell with truncated dendrites and Hodgkin-Huxley kinetics reproduced these recordings with a relatively low density of I(Ts). In a second series of experiments, voltage-clamp recordings obtained in intact RE cells in slices showed a higher amplitude and slower kinetics of I(Ts). These properties could be reproduced from the reconstructed cell model assuming higher densities of I(Ts) in distal dendrites. In a third series of experiments, current-clamp recordings were obtained on RE cells in vivo. The marked differences with in vitro recordings could be reconciled by simulating synaptic bombardment in the dendrites of RE cells, but only if they contained high distal densities of I(Ts). In addition, simpler models with as few as three compartments could reproduce the same behavior assuming dendritic I(Ts). These models and experiments show how intrinsic bursting properties of RE cells, as recorded in vivo and in vitro, may be explained by dendritic calcium currents.

  View details for Web of Science ID A1996TL99000019

  View details for PubMedID 8613783

• Low-threshold calcium currents in central nervous system neurons ANNUAL REVIEW OF PHYSIOLOGY Huguenard, J. R. 1996; 58: 329-348

  Abstract

  The low-threshold calcium current, or T current, has recently been demonstrated with voltage-clamp recordings in a variety of central nervous system (CNS) neurons. It is especially prominent in the soma and dendrites of neurons with robust calcium-dependent burst firing behaviors such as thalamic relay neurons and cerebellar Purkinje cells. Single-channel and macroscopic current behavior have been carefully investigated and kinetic schemes devised to completely describe the activation and inactivation processes. The kinetic properties of T current lead to activation of low-threshold spikes subsequent to transient membrane hyperpolarizations. Putative functional roles for T current include generation of low-threshold spikes that lead to burst firing, promotion of intrinsic oscillatory behavior, boosting of calcium entry, and synaptic potentiation.

  View details for Web of Science ID A1996UB98400016

  View details for PubMedID 8815798

• Excitability changes in thalamic and neocortical neurons after injury. Epilepsy research. Supplement Huguenard, J. R., Chung, J. M., Prince, D. A. 1996; 12: 129-135

  View details for PubMedID 9302511

• A model of spike initiation in neocortical pyramidal neurons NEURON Mainen, Z. F., Joerges, J., Huguenard, J. R., Sejnowski, T. J. 1995; 15 (6): 1427-1439

  Abstract

  Neocortical pyramidal cells possess voltage-dependent dendritic sodium channels that promote propagation of action potentials into the dendritic tree but paradoxically may fail to originate dendritic spikes. A biophysical model was constructed to reconcile these observations with known anatomical and physiological properties. When dendritic and somatic sodium channel densities compatible with electrophysiological measurements were combined with much higher densities in the axon initial segment then, regardless of the site of stimulation, spikes initiated at the initial segment and subsequently invaded the dendrites. The lower initial segment threshold arose from high current density and electrical isolation from the soma. Failure of dendritic channels to initiate spikes was due to inactivation and source-load considerations, which were more favorable for conduction of back-propagated spikes.

  View details for Web of Science ID A1995TL67800020

  View details for PubMedID 8845165

• PURINERGIC INHIBITION OF GABA AND GLUTAMATE RELEASE IN THE THALAMUS - IMPLICATIONS FOR THALAMIC NETWORK ACTIVITY NEURON Ulrich, D., Huguenard, J. R. 1995; 15 (4): 909-918

  Abstract

  Adenosine is a CNS depressant with both pre- and postsynaptic actions. Presynaptically, adenosine decreases neurotransmitter release in the hippocampus but only at excitatory terminals. In the thalamus, however, we show that, in addition to its actions at excitatory synapses, adenosine strongly suppresses monosynaptic inhibitory currents both in relay cells of the thalamic ventrobasal complex (VB) and in inhibitory neurons of the nucleus reticularis thalami (nRt). A concomitant increase in transmission failures and results coefficient of variation analysis are both consistent with a presynaptic mechanism. Pharmacological manipulations support an A1 receptor-mediated process. Slow thalamic oscillations induced in vitro by extracellular stimulation and recorded with extracellular multiunit electrodes in VB and nRt are dampened by adenosine without affecting their periodicity. We conclude that adenosine can presynaptically down-regulate inhibitory postsynaptic responses in thalamus and exert robust antioscillatory effects, likely by synergistic depression of both excitatory and inhibitory neurotransmitter release.

  View details for Web of Science ID A1995TC64000019

  View details for PubMedID 7576639

• CHOLECYSTOKININ DEPOLARIZES RAT THALAMIC RETICULAR NEURONS BY SUPPRESSING A K+ CONDUCTANCE JOURNAL OF NEUROPHYSIOLOGY Cox, C. L., Huguenard, J. R., Prince, D. A. 1995; 74 (3): 990-1000

  Abstract

  1. The thalamic reticular nucleus (nRt) is innervated by cholecystokinin (CCK)-containing neurons and contains CCK binding sites. We used tight-seal, whole cell recording techniques with in vitro rat thalamic slices to investigate the action of CCK on neurons in nRt and ventrobasal thalamus (VB). 2. Brief applications of the CCK agonist cholecystokinin octapeptide (26-33) sulfated (CCK8S) evoked prolonged spike discharges in nRt neurons but had no direct effects on VB neuron activity. This selective excitatory action of CCK8S in nRt resulted from a long-lasting membrane depolarization (2-10 min) associated with an increased input resistance. Voltage-clamp recordings revealed that CCK8S reduced membrane conductance by 0.6-3.8 nS, which amounted to 5-54% of the resting conductance of these neurons. 3. The conductance blocked by CCK8S was linear over the range of -50 to -100 mV and reversed near the potassium equilibrium potential. Modifications of extracellular K+ concentration altered the reversal potential of the conductance as predicted by the Nernst equation. The K+ channel blocker Cs+, applied either intracellularly or combined intra- and extracellularly, blocked the response to CCK8S. 4. The CCK8S-induced depolarization persisted after suppression of synaptic transmission by either tetrodotoxin or a low-Ca2+, high-Mg2+ extracellular solution, indicating that the depolarization was primarily due to activation of postsynaptic CCK receptors and not mediated through the release of other neurotransmitters. 5. The selective CCKA antagonists L364,718 and Cam-1481 attenuated the CCK8S-induced depolarization, whereas the CCKB antagonist L365,260 had little or no effect on the depolarization. 6. Our findings indicate that CCK8S, acting via CCKA-type receptors, reduces a K+ leak current, resulting in a long-lasting membrane depolarization that can presumably modify the firing mode of nRt neurons. Through this effect, CCK actions in nRt may strongly influence thalamocortical function.

  View details for Web of Science ID A1995RU95200007

  View details for PubMedID 7500167

• INTRATHALAMIC RHYTHMICITY STUDIED IN-VITRO - NOMINAL T-CURRENT MODULATION CAUSES ROBUST ANTIOSCILLATORY EFFECTS JOURNAL OF NEUROSCIENCE Huguenard, J. R., Prince, D. A. 1994; 14 (9): 5485-5502

  Abstract

  Thalamocortical oscillations mediate both physiological and pathophysiological behaviors including sleep and generalized absence epilepsy (GA). Reciprocal intrathalamic circuitry and robust burst firing, dependent on underlying transient Ca current (IT) in thalamic neurons, support generation of such rhythms. In order to study the regulation of intrathalamic rhythm generation and the effects of GA anticonvulsants previously shown to reduce IT in acutely isolated thalamic neurons, we developed an in vitro rat thalamic slice preparation that retains sufficient intrathalamic circuitry to support evoked oscillations (range = 2.0-4.6 Hz, average = 2.7, n = 38), associated with burst firing in the thalamic reticular nucleus (nRt) and thalamic relay neurons. Extracellular stimulation of nRt evoked in relay neurons a biphasic inhibitory response with prominent GABAA and GABAB receptor-mediated components. The GABAA component was picrotoxin sensitive, outwardly rectifying and Cl- dependent, with a very negative reversal potential (-94 mV), indicating that an active extrusion mechanism exists in these cells to keep [Cl-]i < 5 mM. The GABAB component had a linear conductance, a reversal potential of -103 mV, and was quite long lasting (about 300 msec) so that rebound bursts often were generated on its decay phase, presumably leading to reexcitation of nRt through known excitatory connections. GABAB-mediated responses thus provide a timing mechanism for promoting slow intrathalamic oscillations. Reduction of IT (30-40%) by succinimides slightly increased the threshold for burst generation in relay and nRt cells, but there was little effect on either number of spikes/burst or intraburst frequency, and there were no other direct effects on other measures of cellular excitability. Intrathalamic oscillations were significantly reduced by these agents through a slight decrease in burst probability of thalamic neurons. We conclude that interactions between the intrinsic properties of thalamic neurons and intrathalamic circuitry lead to generation of slow oscillations. A similar mechanism may underlie the pathophysiological 3 Hz spike and wave EEG activity that characterizes GA. Furthermore, anti-GA drugs such as ethosuximide probably exert their action by reducing the burst-firing probability of neurons within populations of reciprocally interconnected relay and nRt neurons, thus producing a desynchronization of the thalamic circuit that prevents spike/wave generation.

  View details for Web of Science ID A1994PJ12600028

  View details for PubMedID 8083749

• CLONAZEPAM SUPPRESSES GABA(B)-MEDIATED INHIBITION IN THALAMIC RELAY NEURONS THROUGH EFFECTS IN NUCLEUS-RETICULARIS JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Prince, D. A. 1994; 71 (6): 2576-2581

  Abstract

  1. Experiments were carried out using patch-clamp techniques in rat thalamic slices, maintained in vitro, to examine the effects of the benzodiazepine compound, clonazepam (CZP), on intrathalamic inhibition. Bath-applied CZP reduced the gamma-aminobutyric acid-B (GABAB) component of inhibitory postsynaptic potentials and currents (IPSPs and IPSCs, respectively) evoked in rat thalamic somatosensory relay neurons by stimulation of nucleus reticularis thalami (nRt), without consistently affecting the GABAA IPSP. Secondary IPSPs, which occur as a result of intrathalamic oscillations, were dramatically reduced. 2. Voltage-clamp experiments combined with local or bath perfusion of the GABAA antagonist bicuculline methiodide (BMI), demonstrated that nRt is a site of GABAA-mediated postsynaptic inhibition that affects inhibitory output onto relay neurons. BMI enhanced both GABAA and GABAB postsynaptic inhibition in relay neurons when applied to nRt. Focal applications in the ventrobasal relay nucleus near the recording electrode blocked the GABAA-mediated IPSP but had no effects on GABAB inhibitory potentials. 3. Results suggest that CZP acts to facilitate recurrent inhibition in nRt and decrease its inhibitory output onto relay neurons. Intra-nRt GABAA-mediated inhibition thus has an important role in controlling thalamic excitability and in the anti-absence actions of CZP.

  View details for Web of Science ID A1994NQ73400048

  View details for PubMedID 7931539

• NEUROTRANSMITTER CONTROL OF NEOCORTICAL NEURONAL-ACTIVITY AND EXCITABILITY CEREBRAL CORTEX McCormick, D. A., Wang, Z., Huguenard, J. 1993; 3 (5): 387-398

  Abstract

  The pattern of activity and excitability of cortical neurons and neuronal circuits is dependent upon the interaction between glutamatergic and GABAergic fast-activating transmitter systems as well as the state of the more slowly acting transmitters such as ACh, norepinephrine, 5-HT, and histamine. Through the activation of GABAA receptors, GABAergic neurons regulate the amplitude and duration of EPSPs and, in so doing, control the level of functional activation of NMDA receptors. In contrast, activation of muscarinic, adrenergic, serotoninergic, histaminergic, and glutamate metabotropic receptors controls the excitability and pattern of action potential generation in identified pyramidal cells through increases or decreases in various K+ conductances. Activation of muscarinic, alpha 1-adrenergic, or glutamate metabotropic receptors on layer V burst-generating corticotectal or corticopontine neurons results in depolarization through a reduction in a K+ conductance and a switch in the firing mode from repetitive burst firing to single-spike activity. In contrast, activation of muscarinic, beta-adrenergic, H2-histaminergic, and serotoninergic receptors on regular-spiking layer II/III, V, and/or VI corticogeniculate pyramidal cells results in a decrease in spike frequency adaptation and increased responsiveness to depolarizing inputs through a reduction in a slow Ca(2+)-activated K+ current IAHP, and/or a voltage-dependent K+ current, IM. Through these, and other, mechanisms the spatial and temporal pattern of activity generated in cortical circuits is regulated by both intracortical and extracortical neurotransmitter systems.

  View details for Web of Science ID A1993MA56700004

  View details for PubMedID 7903176

• TRANSIENT CA2+ CURRENTS IN NEURONS ISOLATED FROM RAT LATERAL HABENULA JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Gutnick, M. J., Prince, D. A. 1993; 70 (1): 158-166

  Abstract

  1. The properties of the low-voltage-activated transient Ca2+ current (LVA, IT) that underlies rhythmic burst firing in neurons of the lateral habenula (LHb) were examined to further our understanding of mechanisms that promote rhythmogenesis in the CNS. We compared these properties with those of IT in thalamic ventrobasal relay neurons (IVB) and of the more slowly inactivating ITs of thalamic reticular neurons (InRt). 2. Patch-clamp techniques were used to record whole cell Ca2+ currents in LHb cells acutely isolated from rats ranging in age from postnatal days 6 to 34 (P6-P34). The LVA current in LHb (ILHb) had a number of properties similar to those of IVB, including activation threshold (near -65 mV) and voltage-dependent steady-state activation [half-activation voltage (V1/2) = -58.5 mV, slope = 3.4 mV-1] and inactivation (V1/2 = -83.5 mV, slope = 5.0 mV-1) functions. 3. ILHb was characterized by biphasic inactivation, with a fast, voltage-dependent time constant (20-50 ms) similar to that of IVB and a slower, voltage-independent decay phase (time constant approximately 120 ms) that was much more prominent than in IVB. Recovery of ILHb from inactivation was monophasic (time constant, 507 ms at -90 mV), and was slower than for IVB and about the same as for InRt. 4. ILHb was relatively insensitive to equimolar substitution of Ba2+ for Ca2+, in contrast to IVB, which was decreased, and InRt, which was enhanced. 5. In computer simulations, these results could not be accounted for by a mixture of the two previously described IT types (IVB and InRt) in individual LHb cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1993LM76000013

  View details for PubMedID 8395572

• TRANSIENT ENHANCEMENT OF LOW-THRESHOLD CALCIUM CURRENT IN THALAMIC RELAY NEURONS AFTER CORTICECTOMY JOURNAL OF NEUROPHYSIOLOGY Chung, J. M., Huguenard, J. R., Prince, D. A. 1993; 70 (1): 20-27

  Abstract

  1. The alterations of voltage-sensitive calcium currents produced in thalamic cells by injury were investigated under voltage clamp using patch-clamp recordings in the whole-cell configuration. 2. One day after unilateral cortical ablation in immature rats (postnatal day 7), low-threshold transient calcium (T) currents in acutely isolated thalamic relay neurons (RNs) were increased by 68% compared with contralateral controls (P < 0.001). Three days after the operation, T currents in injured neurons were at 44% of control levels (P < 0.001). On the other hand, high-threshold (L) calcium currents in RNs did not change over the same interval. 3. To investigate the mechanism for the increase of T current, both kinetics and voltage dependency of activation and inactivation were examined. At a test voltage of -40 mV, the activation time constant decreased from 4.1 to 3.2 ms (P < 0.05); however, this small change was insufficient to explain the large increase in T current. Time constants for both fast and slow inactivation did not change significantly, nor did voltage dependence of activation or inactivation of thalamic T currents. 4. Methyl-phenyl-succinimide (MPS, 1 mM), a compound known to block T currents, was used to examine possible alterations in the pharmacological properties of T channels after injury. MPS was more effective in reducing T currents in normal versus injured RNs (24 and 20% reductions, respectively; P < 0.05), suggesting that pharmacological properties of T channels in the injured RNs may be different from those of the normal RNs.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1993LM76000003

  View details for PubMedID 8395574

• SIMULATION OF THE CURRENTS INVOLVED IN RHYTHMIC OSCILLATIONS IN THALAMIC RELAY NEURONS JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., McCormick, D. A. 1992; 68 (4): 1373-1383

  Abstract

  1. To perform simulations of the various modes of action potential generation in thalamic relay neurons, we developed Hodgkin-and-Huxley style mathematical equations that describe the voltage dependence and kinetics of activation and inactivation of four different currents, including the transient, low-voltage-activated Ca2+ current (IT), the rapidly inactivating transient K+ current (IA), the slowly inactivating K+ current (IK2), and the hyperpolarization-activated, mixed cationic current (Ih). The modeled currents were derived either from acutely dissociated rat thalamic relay neurons (IT, IA, IK2), or from guinea pig thalamic relay cells maintained in slices in vitro (Ih). 2. The voltage dependence of steady-state activation and inactivation of IT, IA, and IK2 and the activation of Ih could be modeled with Boltzmann-style equations. Modeling of the behavior of IT to depolarizing steps in voltage clamp required the use of the constant field equation to relate permeability to T-current amplitude. The time constant of activation of IT was described by a continuous bell-shaped function with a maximum near 15 ms at threshold for activation (-75 mV) and 23 degrees C. Mathematical description of the kinetics of inactivation and removal of inactivation of this current required two separate functions. 3. The rapidly activating and inactivating K+ current IA was modeled by assuming two components with different time constants of inactivation. The kinetics of activation was described as a continuous function of voltage with the slowest time constant, near 2.5 ms, at threshold for activation (-60 mV) and 23 degrees C. In contrast, the kinetics of inactivation of both components were described as voltage independent, consistent with experimental data. The rate or removal of inactivation of both components of IA was described as continuously increasing with the degree of hyperpolarization. 4. The slowly inactivating K+ current IK2 was also modeled by assuming two components with different rates of inactivation. The kinetics of activation were described by a bell-shaped function with a maximum time constant near 80 ms at -40 mV and 23 degrees C, whereas threshold for activation was approximately -60 mV. Inactivation of both components was modeled as relatively independent of voltage, whereas removal of inactivation was described as a continuous function of membrane potential. 5. The hyperpolarization-activation cationic current, Ih, was modeled by assuming that the current activates with a single exponential relation and does not inactivate.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1992JV73300033

  View details for PubMedID 1279135

• A MODEL OF THE ELECTROPHYSIOLOGICAL PROPERTIES OF THALAMOCORTICAL RELAY NEURONS JOURNAL OF NEUROPHYSIOLOGY McCormick, D. A., Huguenard, J. R. 1992; 68 (4): 1384-1400

  Abstract

  1. A model of the electrophysiological properties of single thalamocortical relay neurons in the rodent and cat dorsal lateral geniculate nucleus was constructed, based in part on the voltage dependence and kinetics of ionic currents detailed with voltage-clamp techniques. The model made the simplifying assumption of a single uniform compartment and incorporated a fast and transient Na+ current, INa; a persistent, depolarization-activated Na+ current, INap; a low-threshold Ca2+ current, I(T); a high-threshold Ca2+ current, IL; a Ca(2+)-activated K+ current, IC; a transient and depolarization-activated K+ current, IA; a slowly inactivating and depolarization-activated K+ current, IK2; a hyperpolarization-activated cation current, Ih; and K+ and Na+ leak currents IKleak and INaleak. 2. The effects of the various ionic currents on the electrophysiological properties of thalamocortical relay neurons were initially investigated through examining the effect of each current individually on passive membrane responses. The two leak currents, IKleak and INaleak, determined in large part the resting membrane potential and the apparent input resistance of the model neuron. Addition of IA resulted in a delay in the response of the model cell to a depolarizing current pulse, whereas addition of IK2, or IL combined with IC, resulted in a marked and prolonged decrease in the response to depolarization. Addition of Ih resulted in a depolarizing "sag" in response to hyperpolarization, whereas addition of IT resulted in a large rebound Ca2+ spike after hyperpolarization. Finally, addition of INap resulted in enhancement of depolarization. 3. The low-threshold Ca2+ spike of rodent neurons was successfully modeled with the active currents I(T), IL, IA, IC, and IK2. The low-threshold Ca2+ current I(T) generated the low-threshold Ca2+ spike. The transient K+ current IA slowed the rate of rise and reduced the peak amplitude of the low-threshold Ca2+ spike, whereas the slowly inactivating K+ current IK2 contributed greatly to the repolarization of the Ca2+ spike. Activation of IL during the peak of the Ca2+ spike led to activation of IC, which also contributed to the repolarization of the Ca2+ spike. Reduction of any one of the K+ currents resulted in an increase in the other two, thereby resulting in substantially smaller changes in the Ca2+ spike than would be expected on the basis of the amplitude of each ionic current alone.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1992JV73300034

  View details for PubMedID 1331356

• A NOVEL T-TYPE CURRENT UNDERLIES PROLONGED CA2+-DEPENDENT BURST FIRING IN GABAERGIC NEURONS OF RAT THALAMIC RETICULAR NUCLEUS JOURNAL OF NEUROSCIENCE Huguenard, J. R., Prince, D. A. 1992; 12 (10): 3804-3817

  Abstract

  The inhibitory GABAergic projection of thalamic nucleus reticularis (nRt) neurons onto thalamocortical relay cells (TCs) is important in generating the normal thalamocortical rhythmicity of slow wave sleep, and may be a key element in the production of abnormal rhythms associated with absence epilepsy. Both TCs and nRt cells can generate prominent Ca(2+)-dependent low-threshold spikes, which evoke bursts of Na(+)-dependent fast spikes, and are influential in rhythm generation. Substantial differences in the pattern of burst firing in TCs versus nRt neurons led us to hypothesize that there are distinct forms of transient Ca2+ current (I(T)) underlying burst discharges in these two cell types. Using whole-cell voltage-clamp recordings, we analyzed I(T) in acutely isolated TCs and nRt neurons and found three key differences in biophysical properties. (1) The transient Ca2+ current in nRt neurons inactivated much more slowly than I(T) in TCs. This slow current is thus termed I(Ts). (2) The rate of inactivation for I(Ts) was nearly voltage independent. (3) Whole-cell I(Ts) amplitude was increased when Ba2+ was substituted for Ca2+ as the charge carrier. In addition, activation kinetics were slower for I(Ts) and the activation range was depolarized compared to that for I(T). Other properties of I(Ts) and I(T) were similar, including steady-state inactivation and sensitivities to blockade by divalent cations, amiloride, and antiepileptic drugs. Our findings demonstrate that subtypes of transient Ca2+ current are present in two different classes of thalamic neurons. The properties of I(Ts) lead to generation of long-duration calcium-dependent spike bursts in nRt cells. The resultant prolonged periods of GABA release onto TCs would play a critical role in maintaining rhythmicity by inducing TC hyperpolarization and promoting generation of low-threshold calcium spikes within relay nuclei.

  View details for Web of Science ID A1992JT19700010

  View details for PubMedID 1403085

• A FAST TRANSIENT POTASSIUM CURRENT IN THALAMIC RELAY NEURONS - KINETICS OF ACTIVATION AND INACTIVATION JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Coulter, D. A., Prince, D. A. 1991; 66 (4): 1304-1315

  Abstract

  1. Whole-cell voltage-clamp techniques were used to record K+ currents in relay neurons (RNs) that had been acutely isolated from rat thalamic ventrobasal complex and maintained at 23 degrees C in vitro. Tetrodoxin (TTX; 0.5 microM) was used to block Na+ currents, and reduced extracellular levels of Ca2+ (1 mM) were used to minimize contributions from Ca2+ current (ICa). 2. In RNs, depolarizing commands activate K+ currents characterized by a substantial rapidly inactivating (time constant approximately 20 ms) component, the features of which correspond to those of the transient K+ current (IA) in other preparations, and by a smaller, more slowly activating K+ current, "IK". IA was reversibly blocked by 4-aminopyridine (4-AP, 5 mM), and the reversal potential varied with [K+]o as predicted by the Nernst equation. 3. IA was relatively insensitive to blockade by tetraethylammonium [TEA; 50%-inhibitory concentration (IC50) much much greater than 20 mM]; however, two components of IK were blocked with IC50S of 30 microM and 3 mM. Because 20 mM TEA blocked 90% of the sustained current while reducing IA by less than 10%, this concentration was routinely used in experiments in which IA was isolated and characterized. To further minimize contamination by other conductances, 4-AP was added to TEA-containing solutions and the 4-AP-sensitive current was obtained by subtraction. 4. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzman function with a slope factor (k) of -6.5 and half-inactivation (V1/2) occurring at -75 mV. Activation of IA was characterized by a Boltzman curve with V1/2 = -35 mV and k = 10.8. 5. IA activation and inactivation kinetics were best fitted by the Hodgkin-Huxley m4h formalism. The rate of activation was voltage dependent, with tau m decreasing from 2.3 ms at -40 mV to 0.5 ms at +50 mV. Inactivation was relatively voltage independent and nonexponential. The rate of inactivation was described by two exponential decay processes with time constants (tau h1 and tau h2) of 20 and 60 ms. Both components were steady-state inactivated with similar voltage dependence. 6. Temperature increases within the range of 23-35 degrees C caused IA activation and inactivation rates to become faster, with temperature coefficient (Q10) values averaging 2.8. IA amplitude also increased as a function of temperature, albeit with a somewhat lower Q10 of 1.6. 7. Several voltage-dependent properties of IA closely resemble those of the transient inward Ca2+ current, IT. (ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1991GK79300015

  View details for PubMedID 1662262

• SLOW INACTIVATION OF A TEA-SENSITIVE K-CURRENT IN ACUTELY ISOLATED RAT THALAMIC RELAY NEURONS JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Prince, D. A. 1991; 66 (4): 1316-1328

  Abstract

  1. Voltage-gated K currents were studied in relay neurons (RNs) acutely isolated from somatosensory (VB) thalamus of 7- to 14-day-old rats. In addition to a rapidly activated, transient outward current, IA, depolarizations activated slower K+ currents, which were isolated through the use of appropriate ionic and pharmacological conditions and measured via whole-cell voltage-clamp. 2. At least two slow components of outward current were observed, both of which were sensitive to changes in [K+]o, as expected for K conductances. The first, IK1, had an amplitude that was insensitive to holding potential and a relatively small conductance of 150 pS/pF. It was blocked by submillimolar levels of tetraethylammonium [TEA, 50%-inhibitory concentration (IC50 = 30 microM)] and 4-aminopyridine (4-AP, 40 microM). In the absence of intracellular Ca2+ buffering, the amplitude of IK1 was both larger and dependent on holding potential, as expected for a Ca(2+)-dependent current. Replacement of [Ca2+]o by Co2+ reduced IK1, although the addition of Cd2+ to Ca(2+)-containing solutions had no effect. 3. The second component, IK2, had a normalized conductance of 2.0 nS/pF and was blocked by millimolar concentrations of TEA (IC50 = 4 mM) but not by 4AP. The kinetics of IK2 were analogous to (but much slower than) those of IA in that both currents displayed voltage-dependent activation and voltage-independent inactivation. IK2 was not reduced by the addition of Cd2+ to Ca(2+)-containing solutions or by replacement of Ca2+ by Co2+. 4. IK2 had a more depolarized activation threshold than IA and attained peak amplitude with a latency of approximately 100 ms at room temperature. IK2 decay was nonexponential and could be described as the sum of two components with time constants (tau) near 1 and 10 s. 5. IK2 was one-half steady-state inactivated at a membrane potential of -63 mV, near the normal resting potential for these cells. The slope factor of the Boltzman function describing steady-state inactivation was 13 mV-1, which indicates that IK2 varies in availability across a broad voltage range between -100 and -20 mV. 6. Activation kinetics of IK2 were voltage dependent, with peak latency shifting from 300 to 50 ms in the voltage range -50 to +30 mV. Deinactivation and deactivation were also voltage dependent, in contrast to inactivation, which showed little dependence on membrane potential. Increase in temperature sped the kinetics of IK2, with temperature coefficient (Q10) values near 3 for activation and inactivation. Heating increased the amplitude of IK2 with a Q10 value near 2.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1991GK79300016

  View details for PubMedID 1761985

• Patch-Clamp Studies of Voltage-Gated Currents in Identified Neurons of the Rat Cerebral Cortex CEREBRAL CORTEX Hamill, O. P., Huguenard, J. R., Prince, D. A. 1991; 1 (1): 48-61

  Abstract

  In the cerebral cortex, neurons can be classified into 2 broad morphological classes, referred to as pyramidal and nonpyramidal (stellate) cells, which correspond to functional classes of projection neurons and local circuit interneurons, respectively. In this study, we demonstrate that specific morphological, immunohistochemical, and physiological features, that allow class distinction of neurons in situ, are retained in acutely isolated neocortical neurons. Furthermore, voltage-clamp analysis with patch-clamp techniques indicate the differences in functional properties in adult neurons, reflect cell-specific, developmental changes in the density and type of specific classes of Na+, K+ and Ca2+ channels expressed. The differences in channel properties contribute to the different input-output relations of neocortical neurons, which enable inhibitory neurons to follow excitatory inputs faithfully and projection neurons to have more integrative roles.

  View details for DOI 10.1093/cercor/1.1.48

  View details for Web of Science ID 000208047200003

  View details for PubMedID 1668364

• DIFFERENTIAL-EFFECTS OF PETIT-MAL ANTICONVULSANTS AND CONVULSANTS ON THALAMIC NEURONS - GABA CURRENT BLOCKADE BRITISH JOURNAL OF PHARMACOLOGY Coulter, D. A., Huguenard, J. R., Prince, D. A. 1990; 100 (4): 807-813

  Abstract

  1. Currents evoked by applications of gamma-aminobutyric acid (GABA) to acutely dissociated thalamic neurones were analysed by voltage-clamp techniques, and the effects of the anticonvulsant succinimides ethosuximide (ES) and alpha-methyl-alpha-phenylsuccinimide (MPS) and the convulsants tetramethylsuccinimide (TMS), picrotoxin, pentylenetetrazol (PTZ), and bicuculline methiodide were assessed. 2. TMS (1 microM-10 microM) reduced responses to iontophoretically applied GABA, as did picrotoxin (0.1-100 microM), PTZ (1-100 mM) and bicuculline (1-100 microM). 3. ES, in high concentrations (1-10 mM), reduced GABA responses to a lesser extent, and also occluded the reductions in GABA-evoked currents produced by TMS, picrotoxin, and PTZ. ES did not occlude the effects of bicuculline on GABA responses. Therefore, we propose that ES acts as a partial agonist at the picrotoxin GABA-blocking receptor. 4. MPS had no effect on GABA responses (at a concentration of 1 mM), and, like ES, occluded the GABA-blocking actions of TMS, apparently acting as a full antagonist. 5. The anticonvulsant actions of ES and MPS against TMS and PTZ-induced seizures may thus involve two independent mechanisms: (1) the occlusion of TMS and PTZ GABA-blocking effects; and (2) the previously described specific effect of ES and MPS on low-threshold calcium current of thalamic neurones. The latter cellular mechanism may be more closely related to petit mal anticonvulsant activity.

  View details for Web of Science ID A1990DQ01800025

  View details for PubMedID 2119843

• DIFFERENTIAL-EFFECTS OF PETIT-MAL ANTICONVULSANTS AND CONVULSANTS ON THALAMIC NEURONS - CALCIUM CURRENT REDUCTION BRITISH JOURNAL OF PHARMACOLOGY Coulter, D. A., Huguenard, J. R., Prince, D. A. 1990; 100 (4): 800-806

  Abstract

  1. Succinimide derivatives can be either convulsant (tetramethylsuccinimide (TMS)), or anticonvulsant (ethosuximide (ES); alpha-methyl-alpha-phenylsuccinimide (MPS)). ES, an anticonvulsant succinimide, has previously been shown to block calcium currents of thalamic neurones, while the convulsant succinimide TMS blocks gamma-aminobutyric acid (GABA) responses in a similar fashion to the convulsant pentylenetetrazol (PTZ). 2. Using voltage-clamp techniques, we analysed the effects of the anticonvulsant succinimides ES and MPS and the convulsants TMS and PTZ on calcium currents of acutely isolated thalamic relay neurones of the rat. 3. MPS and ES reduced low-threshold calcium current (LTCC) in a voltage-dependent manner, without affecting steady-state inactivation. MPS was less potent than ES (IC50 of 1100 vs 200 microM) but greater in efficacy (100% maximal reduction vs 40% for ES). 4. PTZ had no effect on calcium currents, and TMS only reduced LTCC at very high concentrations, and did not occlude MPS effects when applied concurrently. 5. These results, which demonstrate that anticonvulsant, but not convulsant, succinimides block LTCC, provide additional support for the hypothesis that LTCC reduction is a mechanism of action of the anticonvulsant succinimides related to their effects in petit mal epilepsy.

  View details for Web of Science ID A1990DQ01800024

  View details for PubMedID 2169941

• PHYSIOLOGY OF THALAMIC RELAY NEURONS - PROPERTIES OF CALCIUM CURRENTS INVOLVED IN BURST-FIRING SYMP ON GENERALIZED EPILEPSY : CELLULAR, MOLECULAR AND PHARMACOLOGICAL APPROACHES Huguenard, J. R., Coulter, D. A., Prince, D. A. BIRKHAUSER BOSTON. 1990: 181–189

  View details for Web of Science ID A1990BS16U00013

• CELLULAR ACTIONS OF PETIT-MAL ANTICONVULSANTS - IMPLICATION OF THALAMIC LOW-THRESHOLD CALCIUM CURRENT IN GENERATION OF SPIKE-WAVE DISCHARGE SYMP ON GENERALIZED EPILEPSY : CELLULAR, MOLECULAR AND PHARMACOLOGICAL APPROACHES Coulter, D. A., Huguenard, J. R., Prince, D. A. BIRKHAUSER BOSTON. 1990: 425–435

  View details for Web of Science ID A1990BS16U00030

• CALCIUM CURRENTS IN RAT THALAMOCORTICAL RELAY NEURONS - KINETIC-PROPERTIES OF THE TRANSIENT, LOW-THRESHOLD CURRENT JOURNAL OF PHYSIOLOGY-LONDON Coulter, D. A., Huguenard, J. R., Prince, D. A. 1989; 414: 587-604

  Abstract

  1. Calcium currents were recorded with whole-cell voltage-clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low-threshold and high-threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than -60 mV. A transient current, analogous to the T-current in sensory neurones, was activated at low threshold near -65 mV and was completely inactivating at command steps up to -35 mV. Voltage steps to more depolarized levels activated a high-threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high-threshold current contained both non-inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A 'T-type' current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high-threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T-current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T-current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of -60 to -10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than -35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady-state inactivation of T-current could be well fitted by a Boltzman equation with slope factor of 6.3 and half-inactivated voltage of -83.5 mV. 7. Recovery from inactivation of T-current was not exponential. The major component of recovery (70-80% of total) was not very voltage sensitive at potentials more negative than -90 mV, with tau r of 251 ms at -92 mV and 23 degrees C, compared to 225 ms at -112 mV. A smaller, voltage-sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T-current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1989AH41500033

  View details for PubMedID 2607443

• CHARACTERIZATION OF ETHOSUXIMIDE REDUCTION OF LOW-THRESHOLD CALCIUM CURRENT IN THALAMIC NEURONS ANNALS OF NEUROLOGY Coulter, D. A., Huguenard, J. R., Prince, D. A. 1989; 25 (6): 582-593

  Abstract

  The mechanism by which ethosuximide reduces thalamic low-threshold calcium current (LTCC) was analyzed using voltage-clamp techniques in acutely isolated ventrobasal complex neurons from rats and guinea pigs. The ethosuximide-induced reduction of LTCC was voltage dependent: it was most pronounced at more-hyperpolarized potentials and did not affect the time course of activation or inactivation of the current. Ethosuximide reduced LTCC without altering the voltage dependence of steady-state inactivation or the time course of recovery from inactivation. Dimethadione reduced LTCC by a similar mechanism, while valproic acid had no effect on LTCC. We conclude that ethosuximide reduction of LTCC in thalamic neurons is consistent with a reduction in the number of available LTCC channels or in the single LTCC channel conductance, perhaps indicating a direct channel-blocking action of this drug. Given the importance of LTCC in thalamic oscillatory behavior, a reduction in this current by ethosuximide would be a mechanism of action compatible with the known anticonvulsant effects of this drug in typical absence seizures.

  View details for Web of Science ID A1989AA44400009

  View details for PubMedID 2545161

• SODIUM-CHANNELS IN DENDRITES OF RAT CORTICAL PYRAMIDAL NEURONS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huguenard, J. R., Hamill, O. P., Prince, D. A. 1989; 86 (7): 2473-2477

  Abstract

  The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.

  View details for Web of Science ID A1989U042300073

  View details for PubMedID 2538843

• SPECIFIC PETIT MAL ANTICONVULSANTS REDUCE CALCIUM CURRENTS IN THALAMIC NEURONS NEUROSCIENCE LETTERS Coulter, D. A., Huguenard, J. R., Prince, D. A. 1989; 98 (1): 74-78

  Abstract

  Low-threshold calcium current (LTCC) in thalamic neurons is important in generation of normal thalamocortical rhythms, and may be involved in the genesis of abnormal activities such as spike-wave discharges that characterize petit mal epilepsy. Ethosuximide and dimethadione, anticonvulsants effective in petit mal, reduced the LTCC when applied to thalamic neurons at clinically relevant concentrations. Therapeutic concentrations of phenytoin and carbamazepine, drugs ineffective in the control of petit mal, had minimal effects on calcium conductances. Reduction in LTCC may be an important mechanism of action by which specific petit mal anticonvulsants depress spike-wave activity.

  View details for Web of Science ID A1989T655000014

  View details for PubMedID 2710401

• DEVELOPMENT OF GABA RESPONSIVENESS IN EMBRYONIC TURTLE CORTICAL-NEURONS NEUROSCIENCE LETTERS Shen, J. M., Huguenard, J. R., Kriegstein, A. R. 1988; 89 (3): 335-341

  Abstract

  The whole-cell patch-clamp method was used to study the development of functional GABA receptors in cortical neurons dissociated from embryonic turtles. GABA elicited an increase in membrane conductance, even from cells obtained from the earliest stages of corticogenesis. The GABA-mediated conductance had a mean value 7.4 times greater than membrane 'leak' conductance and increased with developmental age. In all stages studied, the response inverted polarity at a value approximating ECl- and was blocked by applications of bicuculline, suggesting that it was mediated by GABAA receptors. GABA receptors are thus present and functional very early in corticogenesis, preceding electrogenesis, synaptogenesis, and full neuronal differentiation.

  View details for Web of Science ID A1988P213200014

  View details for PubMedID 3419632

• DEVELOPMENTAL-CHANGES IN NA+ CONDUCTANCES IN RAT NEOCORTICAL NEURONS - APPEARANCE OF A SLOWLY INACTIVATING COMPONENT JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Hamill, O. P., Prince, D. A. 1988; 59 (3): 778-795

  Abstract

  1. Na+ conductances have been characterized in rat neocortical neurons from the sensorimotor area. Neurons were obtained by acute dissociation from animals at developmental stages from embryonic day 16 (E16) to postnatal day 50 (P50) to quantify any developmental changes in the kinetic properties of the Na+ conductance. 2. Neurons were divided into two classes, based on morphology, to determine whether there are any cell-type specific differences in Na+ conductances that contribute to the different action potential morphologies seen in current-clamp recordings in vitro. 3. Upon isolation, neurons were voltage clamped using the whole-cell variation of the patch-clamp technology. Both cell types, pyramidal and nonpyramidal, demonstrate large increases in Na+ current density during this developmental period (E16-P50). Normalized conductances were near 10 pS/micron2 in neurons from embryonic animals, and increased 6- to 10-fold during the first 2 wk postnatal. The final conductance reached in pyramidal neurons was higher than in non-pyramidal neurons. 4. We found no differences between the two cell types, pyramidal and nonpyramidal, in the voltage dependence of activation, inactivation kinetics, voltage dependence of steady-state inactivation, and recovery from inactivation. 5. The time course of Na+ current in immature neurons were fit with classical Hodgkin-Huxley kinetics. However, in more mature neurons the kinetics of inactivation became more complicated such that two decay components were required to obtain good fit. The slowly decaying component had a time course 5 to 10 times slower than the fast component. 6. Several procedures were used to reduce the magnitude of Na+ conductance in mature neurons to ensure graded, voltage-dependent inward currents. These included reduced extracellular [Na+], submaximal tetrodotoxin concentrations, and reduced holding potential. Under each of these conditions we were able to verify the observation that Na+ current inactivation occurs with two exponentials. 7. Single-channel Na+ currents were obtained from cell-attached patches. The membrane density of active Na+ channels increases with development, and ensemble averages from mature neurons demonstrated two inactivation processes. The slow inactivation process was accounted for by long-latency single-channel openings of the same amplitude as the short-latency openings. 8. We conclude that there are no kinetic differences in the Na+ channels between cell types. Differences in action potentials are then not explained by differences in Na+ current kinetics, but might be partially explained by the different densities.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1988M531600006

  View details for PubMedID 2452862

• WHOLE-CELL VOLTAGE-CLAMP STUDY OF THE FADING OF GABA-ACTIVATED CURRENTS IN ACUTELY DISSOCIATED HIPPOCAMPAL-NEURONS JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., Alger, B. E. 1986; 56 (1): 1-18

  Abstract

  The lability of the responses of mammalian central neurons to gamma-aminobutyric acid (GABA) was studied using neurons acutely dissociated from the CA1 region of the adult guinea pig hippocampus as a model system. GABA was applied to the neuronal somata by pressure ejection and the resulting current (IGABA) recorded under whole-cell voltage clamp. In initial experiments we examined several basic properties of cells in this preparation. Our data confirm that passive and active membrane properties are similar to those which characterize cells in other preparations. In addition, GABA-dependent conductance (gGABA), reversal potential (EGABA), and the interaction of GABA with pentobarbital and bicuculline all appeared to be normal. Dendritic GABA application could cause depolarizing GABA responses, and somatic GABA application caused hyperpolarizations due to chloride (Cl-) movements. Repetitive brief applications (5-15 ms) of GABA (10(-5) to 10(-3) M) at a frequency of 0.5 Hz led to fading of successive peaks of IGABA until, at a given holding potential, a steady state was reached in which IGABA no longer changed. Imposing voltage steps lasting seconds during a train of steady-state GABA responses led initially to increased IGABA that then diminished with maintenance of the step voltage. The rate of decrease of IGABA at each new holding potential was independent of the polarity of the step in holding potential but was highly dependent on the rate of GABA application. Application rates as low as 0.05 Hz led to fading of IGABA, even with activation of relatively small conductances (5-15 nS). Since IGABA evoked by somatic GABA application in these cells is carried by Cl-, the Cl- equilibrium potential (ECl) is equal to the reversal potential for IGABA, i.e., to EGABA. The fading of IGABA with changes in holding potential can be almost entirely accounted for by a shift in ECl resulting from transmembrane flux of Cl- through the GABA-activated conductance. Maneuvers that prevent changes in the intracellular concentration of Cl-ions, [Cl-]i, including holding the membrane potential at EGABA during repetitive GABA application or buffering [Cl-]i with high pipette [Cl-], prevent changes in EGABA. Desensitization of the GABA response (an actual decrease in gGABA) occurs in these neurons during prolonged application of GABA (greater than 1 s) but with a slower time course than changes in EGABA. Whole-cell voltage-clamp techniques applied to tissue-cultured spinal cord neurons indicated that rapid shifts in EGABA result from repetitive GABA application in these cells as well.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1986D341600001

  View details for PubMedID 3746390

• Membrane currents underlying bursting pacemaker activity and spike frequency adaptation in invertebrates. Advances in neurology Lewis, D. V., Huguenard, J. R., Anderson, W. W., Wilson, W. A. 1986; 44: 235-261

  Abstract

  Invertebrate systems have proved to be quite useful for the development of an understanding of some processes in the central nervous system (CNS). An understanding of the basic mechanisms of epilepsy will result from understanding not only how populations of neurons interact but also how the physiological processes in individual neurons are altered in epileptogenesis. Because invertebrate neurons have been so accessible to experimentation, it has been possible to explore in detail the basic mechanisms controlling neuronal excitability using these cells and to make some useful predictions about electrophysiological mechanisms that may be present in central neurons. This chapter deals with two electrophysiological processes that have been observed in invertebrate neurons and that may have some relevance to understanding the basic mechanisms of epilepsy. We review first the past and current studies of invertebrate burst firing neurons. It appears that the electrophysiological mechanisms producing burst firing may be present in CNS neurons participating in epileptogenesis. With caution, the information gleaned from invertebrate studies may be applicable to higher systems. The second process we consider is the phenomenon of spike frequency adaptation seen in invertebrates. Spike frequency adaptation is the process by which the firing rate of the neuron declines despite the maintenance of a constant stimulus. This process is not so thoroughly studied as burst firing, but it appears to represent a cellular mechanism designed to suppress prolonged periods of repetitive firing. Clearly, the suppression of such a process would produce excessive neuronal excitability, while its enhancement might have some anticonvulsant effects. The extreme sensitivity of spike frequency adaptation to barbiturates suggests such a possibility. These two electrophysiological processes are interesting in themselves and also because they may underlie the genesis or control of seizures. However, the greater significance is that, to understand the basic mechanisms of epilepsy, we may be well advised to examine neuronal processes in systems not considered to have seizure susceptibility.

  View details for PubMedID 2422894

• SUPPRESSION OF REPETITIVE FIRING OF NEURONS BY DIPHENYLBARBITURIC ACID JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Huguenard, J. R., Wilson, W. A. 1985; 232 (1): 228-231

  Abstract

  A slow outward current has been demonstrated in Aplysia giant neurons which serves to suppress repetitive firing. The barbiturates phenobarbital and pentobarbital enhance the slow outward current and the suppression of repetitive firing. In this study, the effects of diphenylbarbituric acid, which shows anticonvulsant activity in mice and rats but possesses minimal sedative properties, were tested on slow outward current and firing behavior. Diphenylbarbituric acid enhances slow outward current and suppresses repetitive firing at lower concentrations than phenobarbital and pentobarbital. Because diphenylbarbituric acid is effective at enhancing slow outward current but does not produce sedation, this property of barbiturates is apparently not associated with the sedative properties of these drugs, but rather is important for the anticonvulsant effects.

  View details for Web of Science ID A1985AAG5200036

  View details for PubMedID 3965694

• BARBITURATE-INDUCED ALTERATIONS IN THE KINETIC-PARAMETERS OF SLOW OUTWARD CURRENT IN APLYSIA GIANT-NEURONS JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Huguenard, J. R., Wilson, W. A. 1985; 234 (3): 821-829

  Abstract

  Regulation of spike frequency adaptation has been postulated to be a neuronal mechanism of action of the barbiturates (Zbicz and Wilson, 1981). A barbiturate sensitive slow outward current is present in Aplysia giant neurons, and this current is very effective at regulating adaptation. In the current study a quantitative method of determining the effect of barbiturates was developed using a single-electrode voltage clamp. This method, tail current analysis, involved measuring the exponential decay of the slow outward current at the end of a depolarizing voltage-clamp command. The tail currents were made up of two decay phases with average half-times of 7 and 70 sec, and the tail current amplitude accurately reflected the magnitude of slow outward current. Barbiturates were found to cause a concentration-related, saturable and stereospecific increase in one component of the tail currents, the amplitude of the slow phase. With moderate concentrations of barbiturates there were no changes in the other parameters describing the exponential decay current. The rank order of potency of barbiturates for the enhancement of slow outward current was not well correlated with the lipid solubility of these drugs, indicating that slow outward current enhancement is not due to a nonspecific mechanism, but rather may be receptor mediated.

  View details for Web of Science ID A1985AQP5100041

  View details for PubMedID 4032291

• THE IONIC MECHANISM OF THE SLOW OUTWARD CURRENT IN APLYSIA NEURONS JOURNAL OF NEUROPHYSIOLOGY Huguenard, J. R., ZBICZ, K. L., Lewis, D. V., Evans, G. J., Wilson, W. A. 1985; 54 (2): 449-461

  Abstract

  A slow outward current associated with spike frequency adaptation has been studied in the giant Aplysia neurons R2 and LP1. The current was observed during 60-s voltage clamp commands to potentials just below spike threshold. The slow outward current shows a marked voltage dependence at membrane potential less negative than -40 mV. The slow outward current is associated with increased membrane conductance. The K+ sensitivity of the slow outward current was studied by varying the extracellular K+ concentration and also by measuring potassium efflux with a K+-sensitive electrode. Both procedures indicated that the slow outward current was K+ dependent. Tail currents following the activation of the slow outward current were examined. They were shown to have a similar potassium sensitivity as the slow outward current and had a reversal potential near the potassium equilibrium potential for these cells. The sensitivity of the slow outward current to known blockers of K+ currents, tetraethylammonium and 4-aminopyridine, was tested. The sensitivity was much less than that reported for other K+ currents. The sensitivity of the slow outward current to changes of the extracellular concentrations of Na+ and Cl- ions, as well as electrogenic pump inhibitors, was tested. The results indicate that the slow outward current is much less sensitive to these changes than to the manipulations of the extracellular K+ ion concentration. We tested the sensitivity of this current to manipulations of intracellular and extracellular Ca2+ ion concentrations. We found that the current persisted at a slightly reduced level in the absence of extracellular calcium or in the presence of calcium blocking agents, cobalt and lanthanum. Intracellular injection of the calcium chelator EGTA at a concentration sufficient to block the Ca2+-dependent K+ current, seen after a brief (1.4-s) burst of action potentials, had minimal effects on the slow outward current. Procedures thought to increase intracellular Ca2+ were tested. We found that exposure of the cell to solutions containing elevated Ca2+ concentrations for prolonged periods increased the slow outward current. Also, treatment with drugs thought to elevate intracellular Ca2+ increased the slow outward current. In conclusion, the slow outward current results from an increased K+ conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1985ANY3000018

  View details for PubMedID 2411885

• ROLE OF ORNITHINE DECARBOXYLASE IN CARDIAC GROWTH AND HYPERTROPHY SCIENCE Bartolome, J., Huguenard, J., Slotkin, T. A. 1980; 210 (4471): 793-794

  Abstract

  Inhibition of cardiac ornithine decarboxylase (ODC) by alpha-difluoromethylornithine (DFMO) did not prevent normal cardiac growth in mature rats but attenuated isoproterenol-induced hypertrophy. Hypertrophy caused by triiodothyronine was not prevented by DFMO. There appear to be both ODC-dependent and ODC-independent processes contributing to the subcellular mechanisms associated with growth, which must be considered in the potential laboratory and clinical use of DFMO.

  View details for Web of Science ID A1980KP31500026

  View details for PubMedID 6449079