Gordon Wang

All Publications

• 3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension. Circulation research Ichimura, K., Boehm, M., Andruska, A. M., Zhang, F., Schimmel, K., Bonham, S., Kabiri, A., Kheyfets, V. O., Ichimura, S., Reddy, S., Mao, Y., Zhang, T., Wang, G., Santana, E. J., Tian, X., Essafri, I., Vinh, R., Tian, W., Nicolls, M. R., Yajima, S., Shudo, Y., MacArthur, J. W., Woo, Y. J., Metzger, R. J., Spiekerkoetter, E. 2024

  Abstract

  Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure.Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function.Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments.3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

  View details for DOI 10.1161/CIRCRESAHA.123.323546

  View details for PubMedID 38770652

• Translational modulator ISRIB alleviates synaptic and behavioral phenotypes in Fragile X syndrome. iScience Coulson, R. L., Frattini, V., Moyer, C. E., Hodges, J., Walter, P., Mourrain, P., Zuo, Y., Wang, G. X. 2024; 27 (4): 109259

  Abstract

  Fragile X syndrome (FXS) is caused by the loss of fragile X messenger ribonucleoprotein (FMRP), a translational regulator that binds the transcripts of proteins involved in synaptic function and plasticity. Dysregulated protein synthesis is a central effect of FMRP loss, however, direct translational modulation has not been leveraged in the treatment of FXS. Thus, we examined the effect of the translational modulator integrated stress response inhibitor (ISRIB) in treating synaptic and behavioral symptoms of FXS. We show that FMRP loss dysregulates synaptic protein abundance, stabilizing dendritic spines through increased PSD-95 levels while preventing spine maturation through reduced glutamate receptor accumulation, thus leading to the formation of dense, immature dendritic spines, characteristic of FXS patients and Fmr1 knockout (KO) mice. ISRIB rescues these deficits and improves social recognition in Fmr1 KO mice. These findings highlight the therapeutic potential of targeting core translational mechanisms in FXS and neurodevelopmental disorders more broadly.

  View details for DOI 10.1016/j.isci.2024.109259

  View details for PubMedID 38510125

  View details for PubMedCentralID PMC10951902

• Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model. Journal of visualized experiments : JoVE Nernekli, K., Mangarova, D. B., Shi, Y., Varniab, Z. S., Chang, E., Tikenogullari, O. Z., Pisani, L., Tikhomirov, G., Wang, G., Daldrup-Link, H. E. 2024

  Abstract

  The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

  View details for DOI 10.3791/66304

  View details for PubMedID 38497657

• The intersection of sleep and synaptic translation in synaptic plasticity deficits in neurodevelopmental disorders. Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology Coulson, R. L., Mourrain, P., Wang, G. X. 2024

  Abstract

  Individuals with neurodevelopmental disorders experience persistent sleep deficits, and there is increasing evidence that sleep dysregulation is an underlying cause, rather than merely an effect, of the synaptic and behavioral defects observed in these disorders. At the molecular level, dysregulation of the synaptic proteome is a common feature of neurodevelopmental disorders, though the mechanism connecting these molecular and behavioral phenotypes is an ongoing area of investigation. A role for eIF2α in shifting the local proteome in response to changes in the conditions at the synapse has emerged. Here, we discuss recent progress in characterizing the intersection of local synaptic translation and sleep and propose a reciprocal mechanism of dysregulation in the development of synaptic plasticity defects in neurodevelopmental disorders.

  View details for DOI 10.1007/s00360-023-01531-3

  View details for PubMedID 38396062

  View details for PubMedCentralID 2969179

• Sleep deficiency as a driver of cellular stress and damage in neurological disorders. Sleep medicine reviews Coulson, R. L., Mourrain, P., Wang, G. X. 2022; 63: 101616

  Abstract

  Neurological disorders encompass an extremely broad range of conditions, including those that present early in development and those that progress slowly or manifest with advanced age. Although these disorders have distinct underlying etiologies, the activation of shared pathways, e.g., integrated stress response (ISR) and the development of shared phenotypes (sleep deficits) may offer clues toward understanding some of the mechanistic underpinnings of neurologic dysfunction. While it is incontrovertibly complex, the relationship between sleep and persistent stress in the brain has broad implications in understanding neurological disorders from development to degeneration. The convergent nature of the ISR could be a common thread linking genetically distinct neurological disorders through the dysregulation of a core cellular homeostasis pathway.

  View details for DOI 10.1016/j.smrv.2022.101616

  View details for PubMedID 35381445

• Hyperexcitable arousal circuits drive sleep instability during aging. Science (New York, N.Y.) Li, S. B., Damonte, V. M., Chen, C., Wang, G. X., Kebschull, J. M., Yamaguchi, H., Bian, W. J., Purmann, C., Pattni, R., Urban, A. E., Mourrain, P., Kauer, J. A., Scherrer, G., de Lecea, L. 2022; 375 (6583): eabh3021

  Abstract

  Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

  View details for DOI 10.1126/science.abh3021

  View details for PubMedID 35201886

• Non-REM and REM/paradoxical sleep dynamics across phylogeny. Current opinion in neurobiology Jaggard, J. B., Wang, G. X., Mourrain, P. 2021; 71: 44-51

  Abstract

  All animals carefully studied sleep, suggesting that sleep as a behavioral state exists in all animal life. Such evolutionary maintenance of an otherwise vulnerable period of environmental detachment suggests that sleep must be integral in fundamental biological needs. Despite over a century of research, the knowledge of what sleep does at the tissue, cellular or molecular levels remain cursory. Currently, sleep is defined based on behavioral criteria and physiological measures rather than at the cellular or molecular level. Physiologically, sleep has been described as two main states, non-rapid eye moment (NREM) and REM/paradoxical sleep (PS), which are defined in the neocortex by synchronous oscillations and paradoxical wake-like activity, respectively. For decades, these two sleep states were believed to be defining characteristics of only mammalian and avian sleep. Recent work has revealed slow oscillation, silencing, and paradoxical/REM-like activities in reptiles, fish, flies, worms, and cephalopods suggesting that these sleep dynamics and associated physiological states may have emerged early in animal evolution. Here, we discuss these recent developments supporting the conservation of neural dynamics (silencing, oscillation, paradoxical activity) of sleep states across phylogeny.

  View details for DOI 10.1016/j.conb.2021.08.004

  View details for PubMedID 34583217

• Sleep: DNA Repair Function for Better Neuronal Aging? Current biology : CB Mourrain, P., Wang, G. X. 2019; 29 (12): R585–R588

  Abstract

  A novel potential role of sleep is neuronal DNA repair. Live imaging of chromosome dynamics in zebrafish neurons has uncovered how sleep can repair DNA breaks accumulated during wake to maintain genome integrity and likely slow down neuronal aging.

  View details for DOI 10.1016/j.cub.2019.05.018

  View details for PubMedID 31211981

• Neural signatures of sleep in zebrafish. Nature Leung, L. C., Wang, G. X., Madelaine, R. n., Skariah, G. n., Kawakami, K. n., Deisseroth, K. n., Urban, A. E., Mourrain, P. n. 2019; 571 (7764): 198–204

  Abstract

  Slow-wave sleep and rapid eye movement (or paradoxical) sleep have been found in mammals, birds and lizards, but it is unclear whether these neuronal signatures are found in non-amniotic vertebrates. Here we develop non-invasive fluorescence-based polysomnography for zebrafish, and show-using unbiased, brain-wide activity recording coupled with assessment of eye movement, muscle dynamics and heart rate-that there are at least two major sleep signatures in zebrafish. These signatures, which we term slow bursting sleep and propagating wave sleep, share commonalities with those of slow-wave sleep and paradoxical or rapid eye movement sleep, respectively. Further, we find that melanin-concentrating hormone signalling (which is involved in mammalian sleep) also regulates propagating wave sleep signatures and the overall amount of sleep in zebrafish, probably via activation of ependymal cells. These observations suggest that common neural signatures of sleep may have emerged in the vertebrate brain over 450 million years ago.

  View details for DOI 10.1038/s41586-019-1336-7

  View details for PubMedID 31292557

• Blimp1 induces transient metastatic heterogeneity in pancreatic cancer. Cancer discovery Chiou, S. H., Risca, V. I., Wang, G. X., Yang, D. n., Grüner, B. M., Kathiria, A. S., Ma, R. K., Vaka, D. n., Chu, P. n., Kozak, M. n., Castellini, L. n., Graves, E. E., Kim, G. E., Mourrain, P. n., Koong, A. C., Giaccia, A. J., Winslow, M. M. 2017

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. Using a novel genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor Blimp1 as a key driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes and hypoxia-mediated induction of Blimp1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which up-regulation of Blimp1 links microenvironmental cues to a metastatic stem cell character.

  View details for PubMedID 28790031

• Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses. eLife Wang, G. X., Smith, S. J., Mourrain, P. 2016; 5

  Abstract

  The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and super-resolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed sub-synaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers.

  View details for DOI 10.7554/eLife.20560

  View details for PubMedID 27770568

  View details for PubMedCentralID PMC5098911

• Sleep-Dependent Structural Synaptic Plasticity of Inhibitory Synapses in the Dendrites of Hypocretin/Orexin Neurons. Molecular neurobiology Elbaz, I., Zada, D., Tovin, A., Braun, T., Lerer-Goldshtein, T., Wang, G., Mourrain, P., Appelbaum, L. 2016: -?

  Abstract

  Sleep is tightly regulated by the circadian clock and homeostatic mechanisms. Although the sleep/wake cycle is known to be associated with structural and physiological synaptic changes that benefit the brain, the function of sleep is still debated. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate various functions including feeding, reward, sleep, and wake. Continuous imaging of single neuronal circuits in live animals is vital to understanding the role of sleep in regulating synaptic dynamics, and the transparency of the zebrafish model enables time-lapse imaging of single synapses during both day and night. Here, we use the gephyrin (Gphnb) protein, a central inhibitory synapse organizer, as a fluorescent post-synaptic marker of inhibitory synapses. Double labeling showed that Gphnb-tagRFP and collybistin-EGFP clusters co-localized in dendritic inhibitory synapses. Using a transgenic hcrt:Gphnb-EGFP zebrafish, we showed that the number of inhibitory synapses in the dendrites of Hcrt neurons was increased during development. To determine the effect of sleep on the inhibitory synapses, we performed two-photon live imaging of Gphnb-EGFP in Hcrt neurons during day and night, under light/dark and constant light and dark conditions, and following sleep deprivation (SD). We found that synapse number increased during the night under light/dark conditions but that these changes were eliminated under constant light or dark conditions. SD reduced synapse number during the night, and the number increased during post-deprivation daytime sleep rebound. These results suggest that rhythmic structural plasticity of inhibitory synapses in Hcrt dendrites is independent of the circadian clock and is modulated by consolidated wake and sleep.

  View details for PubMedID 27734337

• 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

• Fmr1 KO and Fenobam Treatment Differentially Impact Distinct Synapse Populations of Mouse Neocortex NEURON Wang, G. X., Smith, S. J., Mourrain, P. 2014; 84 (6): 1273-1286

  Abstract

  Cognitive deficits in fragile X syndrome (FXS) are attributed to molecular abnormalities of the brain's vast and heterogeneous synapse populations. Unfortunately, the density of synapses coupled with their molecular heterogeneity presents formidable challenges in understanding the specific contribution of synapse changes in FXS. We demonstrate powerful new methods for the large-scale molecular analysis of individual synapses that allow quantification of numerous specific changes in synapse populations present in the cortex of a mouse model of FXS. Analysis of nearly a million individual synapses reveals distinct, quantitative changes in synaptic proteins distributed across over 6,000 pairwise metrics. Some, but not all, of these synaptic alterations are reversed by treatment with the candidate therapeutic fenobam, an mGluR5 antagonist. These patterns of widespread, but diverse synaptic protein changes in response to global perturbation suggest that FXS and its treatment must be understood as a networked system at the synapse level.

  View details for DOI 10.1016/j.neuron.2014.11.016

  View details for Web of Science ID 000346574500018

  View details for PubMedID 25521380

• Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature Chung, W., Clarke, L. E., Wang, G. X., Stafford, B. K., Sher, A., Chakraborty, C., Joung, J., Foo, L. C., Thompson, A., Chen, C., Smith, S. J., Barres, B. A. 2013; 504 (7480): 394-400

  Abstract

  To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

  View details for DOI 10.1038/nature12776

  View details for PubMedID 24270812

• Accelerated Experience-Dependent Pruning of Cortical Synapses in Ephrin-A2 Knockout Mice NEURON Yu, X., Wang, G., Gilmore, A., Yee, A. X., Li, X., Xu, T., Smith, S. J., Chen, L., Zuo, Y. 2013; 80 (1): 64-71

  Abstract

  Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using in vivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

  View details for DOI 10.1016/j.neuron.2013.07.014

  View details for Web of Science ID 000326305300008

  View details for PubMedID 24094103

  View details for PubMedCentralID PMC3792401

• Human Neural Stem Cells Enhance Synaptic Structural Remodeling in the Ischemic Brain. Hiu, T., Bliss, T. M., Manley, N., Wang, E. H., Wang, G., Micheva, K. D., Olson, A., Smith, S. J., Steinberg, G. K. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000330540201322

• Increaed GABA(A) Mediated Synaptic Activity and Structural Remodeling in Peri-infarct Cortex Layer 5 in the Post-stroke Rodent Brain. Hiu, T., Bliss, T. M., Farzampour, Z., Paz, J. T., Olson, A., Micheva, K. D., Wang, E. H., Wang, G., Manley, N., Nishiyama, Y., Arac, A., O'Rourke, N., Huguenard, J. R., Smith, S. J., Steinberg, G. K., Tran, K. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000330540200438

• Imaging zebrafish neural circuitry from whole brain to synapse. Frontiers in neural circuits Leung, L. C., Wang, G. X., Mourrain, P. 2013; 7: 76-?

  Abstract

  Recent advances in imaging tools are inspiring zebrafish researchers to tackle ever more ambitious questions in the neurosciences. Behaviorally fundamental conserved neural networks can now be potentially studied using zebrafish from a brain-wide scale to molecular resolution. In this perspective, we offer a roadmap by which a zebrafish researcher can navigate the course from collecting neural activities across the brain associated with a behavior, to unraveling molecular identities and testing the functional relevance of active neurons. In doing so, important insights will be gained as to how neural networks generate behaviors and assimilate changes in synaptic connectivity.

  View details for DOI 10.3389/fncir.2013.00076

  View details for PubMedID 23630470

  View details for PubMedCentralID PMC3634052

• Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens PLOS COMPUTATIONAL BIOLOGY Wang, G., Smith, S. J. 2012; 8 (8)

  Abstract

  Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA(2) (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is -220 nm and -600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50-100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.

  View details for DOI 10.1371/journal.pcbi.1002671

  View details for Web of Science ID 000308553500046

  View details for PubMedID 22956902

  View details for PubMedCentralID PMC3431294

• Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors NATURE Allen, N. J., Bennett, M. L., Foo, L. C., Wang, G. X., Chakraborty, C., Smith, S. J., Barres, B. A. 2012; 486 (7403): 410-?

  Abstract

  In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

  View details for DOI 10.1038/nature11059

  View details for Web of Science ID 000305466800045

  View details for PubMedID 22722203

  View details for PubMedCentralID PMC3383085

• Bidirectional Regulation of Dendritic Voltage-Gated Potassium Channels by the Fragile X Mental Retardation Protein NEURON Lee, H. Y., Ge, W., Huang, W., He, Y., Wang, G. X., Rowson-Baldwin, A., Smith, S. J., Jan, Y. N., Jan, L. Y. 2011; 72 (4): 630-642

  Abstract

  How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.

  View details for DOI 10.1016/j.neuron.2011.09.033

  View details for Web of Science ID 000297180100013

  View details for PubMedID 22099464

  View details for PubMedCentralID PMC3433402

• Synaptic plasticity in sleep: learning, homeostasis and disease TRENDS IN NEUROSCIENCES Wang, G., Grone, B., Colas, D., Appelbaum, L., Mourrain, P. 2011; 34 (9): 452-463

  Abstract

  Sleep is a fundamental and evolutionarily conserved aspect of animal life. Recent studies have shed light on the role of sleep in synaptic plasticity. Demonstrations of memory replay and synapse homeostasis suggest that one essential role of sleep is in the consolidation and optimization of synaptic circuits to retain salient memory traces despite the noise of daily experience. Here, we review this recent evidence and suggest that sleep creates a heightened state of plasticity, which may be essential for this optimization. Furthermore, we discuss how sleep deficits seen in diseases such as Alzheimer's disease and autism spectrum disorders might not just reflect underlying circuit malfunction, but could also play a direct role in the progression of those disorders.

  View details for DOI 10.1016/j.tins.2011.07.005

  View details for Web of Science ID 000294941300002

  View details for PubMedID 21840068

  View details for PubMedCentralID PMC3385863

• Asymmetric PI(3,4,5)P₃ and Akt Signaling Mediates Chemotaxis of Axonal Growth Cones JOURNAL OF NEUROSCIENCE Henle, S. J., Wang, G., Liang, E., Wu, M., Poo, M., Henley, J. R. 2011; 31 (19): 7016-7027

  Abstract

  The action of many extracellular guidance cues on axon pathfinding requires Ca2+ influx at the growth cone (Hong et al., 2000; Nishiyama et al., 2003; Henley and Poo, 2004), but how activation of guidance cue receptors leads to opening of plasmalemmal ion channels remains largely unknown. Analogous to the chemotaxis of amoeboid cells (Parent et al., 1998; Servant et al., 2000), we found that a gradient of chemoattractant triggered rapid asymmetric PI(3,4,5)P3 accumulation at the growth cone's leading edge, as detected by the translocation of a GFP-tagged binding domain of Akt in Xenopus laevis spinal neurons. Growth cone chemoattraction required PI(3,4,5)P3 production and Akt activation, and genetic perturbation of polarized Akt activity disrupted axon pathfinding in vitro and in vivo. Furthermore, patch-clamp recording from growth cones revealed that exogenous PI(3,4,5)P3 rapidly activated TRP (transient receptor potential) channels, and asymmetrically applied PI(3,4,5)P3 was sufficient to induce chemoattractive growth cone turning in a manner that required downstream Ca2+ signaling. Thus, asymmetric PI(3,4,5)P3 elevation and Akt activation are early events in growth cone chemotaxis that link receptor activation to TRP channel opening and Ca2+ signaling. Altogether, our findings reveal that PI(3,4,5)P3 elevation polarizes to the growth cone's leading edge and can serve as an early regulator during chemotactic guidance.

  View details for DOI 10.1523/JNEUROSCI.0216-11.2011

  View details for Web of Science ID 000290514400010

  View details for PubMedID 21562263

  View details for PubMedCentralID PMC3133771

• Circadian and Homeostatic Regulation of Structural Synaptic Plasticity in Hypocretin Neurons NEURON Appelbaum, L., Wang, G., Yokogawa, T., Skariah, G. M., Smith, S. J., Mourrain, P., Mignot, E. 2010; 68 (1): 87-98

  Abstract

  Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.

  View details for DOI 10.1016/j.neuron.2010.09.006

  View details for Web of Science ID 000283704200010

  View details for PubMedID 20920793

  View details for PubMedCentralID PMC2969179

• Sleep-wake regulation and hypocretin-melatonin interaction in zebrafish PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Appelbaum, L., Wang, G. X., Maro, G. S., Mori, R., Tovin, A., Marin, W., Yokogawa, T., Kawakami, K., Smith, S. J., Gothilf, Y., Mignot, E., Mourrain, P. 2009; 106 (51): 21942-21947

  Abstract

  In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr-/-). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr-/- pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr-/- fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr-/- fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

  View details for DOI 10.1073/pnas.906637106

  View details for Web of Science ID 000272994200086

  View details for PubMedID 19966231

  View details for PubMedCentralID PMC2799794

• Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones NATURE Wang, G. X., Poo, M. M. 2005; 434 (7035): 898-904

  Abstract

  Ion channels formed by the TRP (transient receptor potential) superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. Here, we report that TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induced a membrane depolarization, part of which remained after all major voltage-dependent channels were blocked. Furthermore, the membrane depolarization was sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolished the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones.

  View details for DOI 10.1038/nature03478

  View details for Web of Science ID 000228327600041

  View details for PubMedID 15758951