Education & Certifications

  • PhD, University of Montreal, Canada, Neuroscience (1996)

Service, Volunteer and Community Work

  • Neurobiology Course, Faculty, Marine Biological Laboratory (2006 - 2014)



    Woods Hole, Massachusetts


  • Kristina D. Micheva, Stephen J Smith. "United States Patent 7,767,414 Optical Imaging of molecular characteristics of biological specimen", Leland Stanford Junior University
  • Kristina D. Micheva, Stephen J Smith. "United States Patent 9,008,378 Arrangement and Imaging of Biological Samples", Leland Stanford Junior University

All Publications

  • Extensive Structural Remodeling of the Axonal Arbors of Parvalbumin Basket Cells during Development in Mouse Neocortex. The Journal of neuroscience : the official journal of the Society for Neuroscience Micheva, K. D., Kiraly, M., Perez, M. M., Madison, D. V. 2021


    Parvalbumin-containing (PV+) basket cells are specialized cortical interneurons that regulate the activity of local neuronal circuits with high temporal precision and reliability. To understand how the PV+ interneuron connectivity underlying these functional properties is established during development, we used array tomography to map pairs of synaptically connected PV+ interneurons and postsynaptic neurons from the neocortex of mice of both sexes. We focused on the axon-myelin unit of the PV+ interneuron and quantified the number of synapses onto the postsynaptic neuron, length of connecting axonal paths, and their myelination at different time points between 2weeks and 7months of age. We find that myelination of the proximal axon occurs very rapidly during the third and, to a lesser extent, fourth postnatal weeks. The number of synaptic contacts made by the PV+ interneuron on its postsynaptic partner meanwhile is significantly reduced to about one-third by the end of the first postnatal month. The number of autapses, the synapses that PV+ interneurons form on themselves, however, remains constant throughout the examined period. Axon reorganizations continue beyond postnatal month 2, with the postsynaptic targets of PV+ interneurons gradually shifting to more proximal locations, and the length of axonal paths and their myelin becoming conspicuously uniform per connection. These continued microcircuit refinements likely provide the structural substrate for the robust inhibitory effects and fine temporal precision of adult PV+ basket cells.Significance Statement:The axon of adult parvalbumin-containing (PV+) interneurons is highly specialized for fast and reliable neurotransmission. It is myelinated and forms synapses mostly onto the cell bodies and proximal dendrites of postsynaptic neurons for maximal impact. In this study, we follow the development of the PV+ interneuron axon, its myelination and synapse formation, revealing a rapid sequence of axonal reorganization, myelination of the PV+ interneuron proximal axon, and pruning of almost two-thirds of the synapses in an individual connection. This is followed by a prolonged period of axon refinement and additional myelination leading to a remarkable precision of connections in the adult mouse cortex, consistent with the temporal precision and fidelity of PV+ interneuron action.

    View details for DOI 10.1523/JNEUROSCI.0871-21.2021

    View details for PubMedID 34583957

  • Conduction Velocity Along the Local Axons of Parvalbumin Interneurons Correlates With the Degree of Axonal Myelination. Cerebral cortex (New York, N.Y. : 1991) Micheva, K. D., Kiraly, M., Perez, M. M., Madison, D. V. 2021


    Parvalbumin-containing (PV+) basket cells in mammalian neocortex are fast-spiking interneurons that regulate the activity of local neuronal circuits in multiple ways. Even though PV+ basket cells are locally projecting interneurons, their axons are myelinated. Can this myelination contribute in any significant way to the speed of action potential propagation along such short axons? We used dual whole cell recordings of synaptically connected PV+ interneurons and their postsynaptic target in acutely prepared neocortical slices from adult mice to measure the amplitude and latency of single presynaptic action potential-evoked inhibitory postsynaptic currents. These same neurons were then imaged with immunofluorescent array tomography, the synapses between them identified and a precise map of the connections was generated, with the exact axonal length and extent of myelin coverage. Our results support that myelination of PV+ basket cells significantly increases conduction velocity, and does so to a degree that can be physiologically relevant.

    View details for DOI 10.1093/cercor/bhab018

    View details for PubMedID 33704414

  • Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons. International journal of molecular sciences Kuljis, D. A., Micheva, K. D., Ray, A., Wegner, W., Bowman, R., Madison, D. V., Willig, K. I., Barth, A. L. 2021; 22 (18)


    Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABAARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.

    View details for DOI 10.3390/ijms221810032

    View details for PubMedID 34576197

  • A synapse census for the ages. Science (New York, N.Y.) Micheva, K. D., Weinberg, R. J., Smith, S. J. 2020; 369 (6501): 253–54

    View details for DOI 10.1126/science.abc9555

    View details for PubMedID 32675362

  • Multifaceted Changes in Synaptic Composition and Astrocytic Involvement in a Mouse Model of Fragile X Syndrome. Scientific reports Simhal, A. K., Zuo, Y., Perez, M. M., Madison, D. V., Sapiro, G., Micheva, K. D. 2019; 9 (1): 13855


    Fragile X Syndrome (FXS), a common inheritable form of intellectual disability, is known to alter neocortical circuits. However, its impact on the diverse synapse types comprising these circuits, or on the involvement of astrocytes, is not well known. We used immunofluorescent array tomography to quantify different synaptic populations and their association with astrocytes in layers 1 through 4 of the adult somatosensory cortex of a FXS mouse model, the FMR1 knockout mouse. The collected multi-channel data contained approximately 1.6 million synapses which were analyzed using a probabilistic synapse detector. Our study reveals complex, synapse-type and layer specific changes in the neocortical circuitry of FMR1 knockout mice. We report an increase of small glutamatergic VGluT1 synapses in layer 4 accompanied by a decrease in large VGluT1 synapses in layers 1 and 4. VGluT2 synapses show a rather consistent decrease in density in layers 1 and 2/3. In all layers, we observe the loss of large inhibitory synapses. Lastly, astrocytic association of excitatory synapses decreases. The ability to dissect the circuit deficits by synapse type and astrocytic involvement will be crucial for understanding how these changes affect circuit function, and ultimately defining targets for therapeutic intervention.

    View details for DOI 10.1038/s41598-019-50240-x

    View details for PubMedID 31554841

  • Distinctive Structural and Molecular Features of Myelinated Inhibitory Axons in Human Neocortex. eNeuro Micheva, K. D., Chang, E. F., Nana, A. L., Seeley, W. W., Ting, J. T., Cobbs, C., Lein, E., Smith, S. J., Weinberg, R. J., Madison, D. V. 2018; 5 (5)


    Numerous types of inhibitory neurons sculpt the performance of human neocortical circuits, with each type exhibiting a constellation of subcellular phenotypic features in support of its specialized functions. Axonal myelination has been absent among the characteristics used to distinguish inhibitory neuron types; in fact, very little is known about myelinated inhibitory axons in human neocortex. Here, using array tomography to analyze samples of neurosurgically excised human neocortex, we show that inhibitory myelinated axons originate predominantly from parvalbumin-containing interneurons. Compared to myelinated excitatory axons, they have higher neurofilament and lower microtubule content, shorter nodes of Ranvier, and more myelin basic protein (MBP) in their myelin sheath. Furthermore, these inhibitory axons have more mitochondria, likely to sustain the high energy demands of parvalbumin interneurons, as well as more 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein enriched in the myelin cytoplasmic channels that are thought to facilitate the delivery of nutrients from ensheathing oligodendrocytes. Our results demonstrate that myelinated axons of parvalbumin inhibitory interneurons exhibit distinctive features that may support the specialized functions of this neuron type in human neocortical circuits.

    View details for PubMedID 30406183

  • A Computational Synaptic Antibody Characterization Tool for Array Tomography FRONTIERS IN NEUROANATOMY Simhal, A. K., Gong, B., Trimmer, J. S., Weinberg, R. J., Smith, S. J., Sapiro, G., Micheva, K. D. 2018; 12
  • A Computational Synaptic Antibody Characterization Tool for Array Tomography. Frontiers in neuroanatomy Simhal, A. K., Gong, B., Trimmer, J. S., Weinberg, R. J., Smith, S. J., Sapiro, G., Micheva, K. D. 2018; 12: 51


    Application-specific validation of antibodies is a critical prerequisite for their successful use. Here we introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). The proposed Synaptic Antibody Characterization Tool (SACT) is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. SACT automatically detects puncta of immunofluorescence labeling from candidate antibodies and determines whether a punctum belongs to a synapse. The molecular composition and size of the target synapses expected to contain the antigen is determined by the user, based on biological knowledge. Operationally, the presence of a synapse is defined by the colocalization or adjacency of the candidate antibody punctum to one or more reference antibody puncta. The outputs of SACT are automatically computed measurements such as target synapse density and target specificity ratio that reflect the sensitivity and specificity of immunolabeling with a given candidate antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications.

    View details for DOI 10.3389/fnana.2018.00051

    View details for PubMedID 30065633

    View details for PubMedCentralID PMC6057115

  • Probabilistic fluorescence-based synapse detection. PLoS computational biology Simhal, A. K., Aguerrebere, C., Collman, F., Vogelstein, J. T., Micheva, K. D., Weinberg, R. J., Smith, S. J., Sapiro, G. 2017; 13 (4)


    Deeper exploration of the brain's vast synaptic networks will require new tools for high-throughput structural and molecular profiling of the diverse populations of synapses that compose those networks. Fluorescence microscopy (FM) and electron microscopy (EM) offer complementary advantages and disadvantages for single-synapse analysis. FM combines exquisite molecular discrimination capacities with high speed and low cost, but rigorous discrimination between synaptic and non-synaptic fluorescence signals is challenging. In contrast, EM remains the gold standard for reliable identification of a synapse, but offers only limited molecular discrimination and is slow and costly. To develop and test single-synapse image analysis methods, we have used datasets from conjugate array tomography (cAT), which provides voxel-conjugate FM and EM (annotated) images of the same individual synapses. We report a novel unsupervised probabilistic method for detection of synapses from multiplex FM (muxFM) image data, and evaluate this method both by comparison to EM gold standard annotated data and by examining its capacity to reproduce known important features of cortical synapse distributions. The proposed probabilistic model-based synapse detector accepts molecular-morphological synapse models as user queries, and delivers a volumetric map of the probability that each voxel represents part of a synapse. Taking human annotation of cAT EM data as ground truth, we show that our algorithm detects synapses from muxFM data alone as successfully as human annotators seeing only the muxFM data, and accurately reproduces known architectural features of cortical synapse distributions. This approach opens the door to data-driven discovery of new synapse types and their density. We suggest that our probabilistic synapse detector will also be useful for analysis of standard confocal and super-resolution FM images, where EM cross-validation is not practical.

    View details for DOI 10.1371/journal.pcbi.1005493

    View details for PubMedID 28414801

  • Array tomography of physiologically-characterized CNS synapses JOURNAL OF NEUROSCIENCE METHODS Valenzuela, R. A., Micheva, K. D., Kiraly, M., Li, D., Madison, D. V. 2016; 268: 43-52


    The ability to correlate plastic changes in synaptic physiology with changes in synaptic anatomy has been very limited in the central nervous system because of shortcomings in existing methods for recording the activity of specific CNS synapses and then identifying and studying the same individual synapses on an anatomical level.We introduce here a novel approach that combines two existing methods: paired neuron electrophysiological recording and array tomography, allowing for the detailed molecular and anatomical study of synapses with known physiological properties.The complete mapping of a neuronal pair allows determining the exact number of synapses in the pair and their location. We have found that the majority of close appositions between the presynaptic axon and the postsynaptic dendrite in the pair contain synaptic specializations. The average release probability of the synapses between the two neurons in the pair is low, below 0.2, consistent with previous studies of these connections. Other questions, such as receptor distribution within synapses, can be addressed more efficiently by identifying only a subset of synapses using targeted partial reconstructions. In addition, time sensitive events can be captured with fast chemical fixation.Compared to existing methods, the present approach is the only one that can provide detailed molecular and anatomical information of electrophysiologically-characterized individual synapses.This method will allow for addressing specific questions about the properties of identified CNS synapses, even when they are buried within a cloud of millions of other brain circuit elements.

    View details for DOI 10.1016/j.jneumeth.2016.04.017

    View details for Web of Science ID 000379104400006

    View details for PubMedID 27141856

  • 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


    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

  • A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons. eLife Micheva, K. D., Wolman, D., Mensh, B. D., Pax, E., Buchanan, J., Smith, S. J., Bock, D. D. 2016; 5


    Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits.

    View details for DOI 10.7554/eLife.15784

    View details for PubMedID 27383052

    View details for PubMedCentralID PMC4972537

  • A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons. eLife Micheva, K. D., Wolman, D., Mensh, B. D., Pax, E., Buchanan, J., Smith, S. J., Bock, D. D. 2016; 5


    Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits.

    View details for DOI 10.7554/eLife.15784

    View details for PubMedID 27383052

    View details for PubMedCentralID PMC4972537

  • Knowing a synapse when you see one FRONTIERS IN NEUROANATOMY Burette, A., Collman, F., Micheva, K. D., Smith, S. J., Weinberg, R. J. 2015; 9


    Recent years have seen a rapidly growing recognition of the complexity and diversity of the myriad individual synaptic connections that define brain synaptic networks. It has also become increasingly apparent that the synapses themselves are a major key to understanding the development, function and adaptability of those synaptic networks. In spite of this growing appreciation, the molecular, structural and functional characteristics of individual synapses and the patterning of their diverse characteristics across functional networks have largely eluded quantitative study with available imaging technologies. Here we offer an overview of new computational imaging methods that promise to bring single-synapse analysis of synaptic networks to the fore. We focus especially on the challenges and opportunities associated with quantitative detection of individual synapses and with measuring individual synapses across network scale populations in mammalian brain.

    View details for DOI 10.3389/fnana.2015.00100

    View details for Web of Science ID 000360431900001

    View details for PubMedCentralID PMC4517447

  • Mapping synapses by conjugate light-electron array tomography. journal of neuroscience Collman, F., Buchanan, J., Phend, K. D., Micheva, K. D., Weinberg, R. J., Smith, S. J. 2015; 35 (14): 5792-5807


    Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex.

    View details for DOI 10.1523/JNEUROSCI.4274-14.2015

    View details for PubMedID 25855189

    View details for PubMedCentralID PMC4388933

  • Knowing a synapse when you see one. Frontiers in neuroanatomy Burette, A., Collman, F., Micheva, K. D., Smith, S. J., Weinberg, R. J. 2015; 9: 100-?


    Recent years have seen a rapidly growing recognition of the complexity and diversity of the myriad individual synaptic connections that define brain synaptic networks. It has also become increasingly apparent that the synapses themselves are a major key to understanding the development, function and adaptability of those synaptic networks. In spite of this growing appreciation, the molecular, structural and functional characteristics of individual synapses and the patterning of their diverse characteristics across functional networks have largely eluded quantitative study with available imaging technologies. Here we offer an overview of new computational imaging methods that promise to bring single-synapse analysis of synaptic networks to the fore. We focus especially on the challenges and opportunities associated with quantitative detection of individual synapses and with measuring individual synapses across network scale populations in mammalian brain.

    View details for DOI 10.3389/fnana.2015.00100

    View details for PubMedID 26283929

  • Deep molecular diversity of mammalian synapses: why it matters and how to measure it NATURE REVIEWS NEUROSCIENCE O'Rourke, N. A., Weiler, N. C., Micheva, K. D., Smith, S. J. 2012; 13 (6): 365-379


    Pioneering studies in the middle of the twentieth century revealed substantial diversity among mammalian chemical synapses and led to a widely accepted classification of synapse type on the basis of neurotransmitter molecule identity. Subsequently, powerful new physiological, genetic and structural methods have enabled the discovery of much deeper functional and molecular diversity within each traditional neurotransmitter type. Today, this deep diversity continues to pose both daunting challenges and exciting new opportunities for neuroscience. Our growing understanding of deep synapse diversity may transform how we think about and study neural circuit development, structure and function.

    View details for DOI 10.1038/nrn3170

    View details for Web of Science ID 000304197000007

    View details for PubMedID 22573027

    View details for PubMedCentralID PMC3670986

  • The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure CURRENT OPINION IN NEUROBIOLOGY Micheva, K. D., Bruchez, M. P. 2012; 22 (1): 94-100


    The rapid accumulation of neuroproteomics data in recent years has prompted the emergence of novel antibody-based imaging methods that aim to understand the anatomical and functional context of the multitude of identified proteins. The pioneering field of ultrastructural multiplexed proteomic imaging now includes a number of high resolution methods, such as array tomography, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy and automated transmission electron microscopy, which allow a detailed molecular characterization of individual synapses and subsynaptic structures within brain tissues for the first time. While all of these methods still face considerable limitations, a combined complementary approach building on the respective strengths of each method is possible and will enable fascinating research into the proteomic diversity of the nervous system.

    View details for DOI 10.1016/j.conb.2011.08.004

    View details for Web of Science ID 000301874400013

    View details for PubMedID 21944260

    View details for PubMedCentralID PMC3265692

  • Large-Scale Automated Histology in the Pursuit of Connectomes JOURNAL OF NEUROSCIENCE Kleinfeld, D., Bharioke, A., Blinder, P., Bock, D. D., Briggman, K. L., Chklovskii, D. B., Denk, W., Helmstaedter, M., Kaufhold, J. P., Lee, W. A., Meyer, H. S., Micheva, K. D., Oberlaender, M., Prohaska, S., Reid, R. C., Smith, S. J., Takemura, S., Tsai, P. S., Sakmann, B. 2011; 31 (45): 16125-16138


    How does the brain compute? Answering this question necessitates neuronal connectomes, annotated graphs of all synaptic connections within defined brain areas. Further, understanding the energetics of the brain's computations requires vascular graphs. The assembly of a connectome requires sensitive hardware tools to measure neuronal and neurovascular features in all three dimensions, as well as software and machine learning for data analysis and visualization. We present the state of the art on the reconstruction of circuits and vasculature that link brain anatomy and function. Analysis at the scale of tens of nanometers yields connections between identified neurons, while analysis at the micrometer scale yields probabilistic rules of connection between neurons and exact vascular connectivity.

    View details for DOI 10.1523/JNEUROSCI.4077-11.2011

    View details for Web of Science ID 000296799700012

    View details for PubMedID 22072665

  • Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and Markers NEURON Micheva, K. D., Busse, B., Weiler, N. C., O'Rourke, N., Smith, S. J. 2010; 68 (4): 639-653


    A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

    View details for DOI 10.1016/j.neuron.2010.09.024

    View details for Web of Science ID 000285079500005

    View details for PubMedID 21092855

    View details for PubMedCentralID PMC2995697

  • Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain PLOS ONE Li, L., Tasic, B., Micheva, K. D., Ivanov, V. M., Spletter, M. L., Smith, S. J., Luo, L. 2010; 5 (7)


    Proper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.In this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.

    View details for DOI 10.1371/journal.pone.0011503

    View details for Web of Science ID 000279715300014

    View details for PubMedID 20634890

    View details for PubMedCentralID PMC2901335

  • Classical MHCI Molecules Regulate Retinogeniculate Refinement and Limit Ocular Dominance Plasticity NEURON Datwani, A., McConnell, M. J., Kanold, P. O., Micheva, K. D., Busse, B., Shamloo, M., Smith, S. J., Shatz, C. J. 2009; 64 (4): 463-470


    Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.

    View details for DOI 10.1016/j.neuron.2009.10.015

    View details for PubMedID 19945389

  • Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Koffie, R. M., Meyer-Luehmann, M., Hashimoto, T., Adams, K. W., Mielke, M. L., Garcia-Alloza, M., Micheva, K. D., Smith, S. J., Kim, M. L., Lee, V. M., Hyman, B. T., Spires-Jones, T. L. 2009; 106 (10): 4012-4017


    Synapse loss correlates with a cognitive decline in Alzheimer's disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid beta (Abeta) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric Abeta surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric Abeta. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric Abeta surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 microm from a plaque in an approximately linear fashion (linear regression, r(2) = 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric Abeta associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric Abeta. The proportion of excitatory synapses associated with Abeta correlates with decreasing density (correlation, -0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric Abeta, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of "plaque" vs. "oligomeric Abeta" as the synaptotoxic species in the brain of AD patients.

    View details for DOI 10.1073/pnas.0811698106

    View details for Web of Science ID 000264036900066

    View details for PubMedID 19228947

    View details for PubMedCentralID PMC2656196

  • The classical complement cascade mediates CNS synapse elimination CELL Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., Micheva, K. D., Mehalow, A. K., Huberman, A. D., Stafford, B., Sher, A., Litke, A. M., Lambris, J. D., Smith, S. J., John, S. W., Barres, B. A. 2007; 131 (6): 1164-1178


    During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

    View details for DOI 10.1016/j.cell.2007.10.036

    View details for PubMedID 18083105

  • Array tomography: A new tool for Imaging the molecular architecture and ultrastructure of neural circuits NEURON Micheva, K. D., Smith, S. J. 2007; 55 (1): 25-36


    Many biological functions depend critically upon fine details of tissue molecular architecture that have resisted exploration by existing imaging techniques. This is particularly true for nervous system tissues, where information processing function depends on intricate circuit and synaptic architectures. Here, we describe a new imaging method, called array tomography, which combines and extends superlative features of modern optical fluorescence and electron microscopy methods. Based on methods for constructing and repeatedly staining and imaging ordered arrays of ultrathin (50-200 nm), resin-embedded serial sections on glass microscope slides, array tomography allows for quantitative, high-resolution, large-field volumetric imaging of large numbers of antigens, fluorescent proteins, and ultrastructure in individual tissue specimens. Compared to confocal microscopy, array tomography offers the advantage of better spatial resolution, in particular along the z axis, as well as depth-independent immunofluorescent staining. The application of array tomography can reveal important but previously unseen features of brain molecular architecture.

    View details for DOI 10.1016/j.neuron.2007.06.014

    View details for Web of Science ID 000248010700006

    View details for PubMedID 17610815

    View details for PubMedCentralID PMC2080672

  • Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons MOLECULAR PHARMACOLOGY Micheva, K. D., Taylor, C. P., Smith, S. J. 2006; 70 (2): 467-476


    Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

    View details for DOI 10.1124/mol.106.023309

    View details for Web of Science ID 000239117900007

    View details for PubMedID 16641316

  • Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals JOURNAL OF NEUROSCIENCE Micheva, K. D., Smith, S. J. 2005; 25 (33): 7481-7488


    Most cellular processes are known to be strongly temperature dependent. Nevertheless, a large fraction of studies of mammalian synaptic function have been and are performed near room temperature (i.e., at least 10 degrees C below physiological temperature). Here, we examined the effects of temperature on presynaptic function in primary cultures of rat hippocampal neurons. FM dyes, VAMP (vesicle-associated membrane protein)-GFP (green fluorescent protein) transfection, and HRP uptake were used to quantify various aspects of synaptic vesicle recycling. Our results show that there are very substantial differences in synaptic vesicle recycling at physiological temperature as opposed to the common, lower experimental temperatures. At 37 degrees C, compared with 23 degrees C, the speed of both exocytosis and endocytosis was higher. The size of the recycling vesicle pool (in both number of vesicles and spatial extent) was twofold larger at 37 degrees C. In addition, although repeated 10 Hz electrical stimulation caused an NMDA receptor-dependent enlargement (averaging 170%) of the measurable recycling vesicle pool at 23 degrees C, the same stimulus repetition had no effect at 37 degrees C. These results show that it is potentially misleading to extend conclusions drawn about vesicle function or presynaptic plasticity at lowered experimental temperature to physiological conditions and that much new experimental work at the higher physiological temperature range will be needed to understand the true parameters of presynaptic functions.

    View details for DOI 10.1523/JNEUROSCI.1801-05.2005

    View details for PubMedID 16107635

  • Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. 2005; 102 (24): 8740-8745


    Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.

    View details for Web of Science ID 000229807200061

    View details for PubMedID 15939876

    View details for PubMedCentralID PMC1143584

  • Fragmentation of the Golgi apparatus induced by the overexpression of wild-type and mutant human tau forms in neurons AMERICAN JOURNAL OF PATHOLOGY Liazoghli, D., Perreault, S., Micheva, K. D., Desjardins, M., Leclerc, N. 2005; 166 (5): 1499-1514


    Tau is a microtubule-associated protein enriched in the axonal compartment. In several neurodegenerative diseases including Alzheimer's disease, hyperphosphorylated tau accumulates in the somatodendritic compartment, self-aggregates, and forms neurofibrillary tangles. A fragmentation of the neuronal Golgi apparatus (GA) was also observed in Alzheimer's disease. In the present study, we examined the effect of overexpressing human tau on the organization of the neuronal GA in rat hippocampal cultures and in JNPL3 mice expressing tau mutant P301L. GA fragmentation was noted in a significantly higher percentage of hippocampal neurons overexpressing wild-type human tau than in control neurons over-expressing green fluorescent protein (GFP) alone. Most importantly, in neurons overexpressing mutant forms of human tau (P301L, V337M, or R406W), the percentage of neurons with a fragmented GA was 10% higher than that of neurons overexpressing wild-type human tau. In JNPL3 mice, a significantly higher percentage of motor neurons presented a fragmented GA compared to control mice. Interestingly, fragmentation of the GA was more frequent in neurons containing an accumulation and aggregation of hyperphosphorylated tau in the cell body than in neurons without these features. In both primary hippocampal neurons and JNPL3 mice, the tau-induced GA fragmentation was not caused by apoptosis. The pre-sent results implicate tau in GA fragmentation and show that this event occurs before the formation of neurofibrillary tangles.

    View details for Web of Science ID 000228658500020

    View details for PubMedID 15855649

  • Retrograde regulation of synaptic vesicle endocytosis and recycling NATURE NEUROSCIENCE Micheva, K. D., Buchanan, J., Holz, R. W., Smith, S. J. 2003; 6 (9): 925-932


    Sustained release of neurotransmitter depends upon the recycling of synaptic vesicles. Until now, it has been assumed that vesicle recycling is regulated by signals from the presynaptic bouton alone, but results from rat hippocampal neurons reported here indicate that this need not be the case. Fluorescence imaging and pharmacological analysis show that a nitric oxide (NO) signal generated postsynaptically can regulate endocytosis and at least one later step in synaptic vesicle recycling. The proposed retrograde pathway involves an NMDA receptor (NMDAR)-dependent postsynaptic production of NO, diffusion of NO to a presynaptic site, and a cGMP-dependent increase in presynaptic phosphatidylinositol 4,5-biphosphate (PIP2). These results indicate that the regulation of synaptic vesicle recycling may integrate a much broader range of neural activity signals than previously recognized, including postsynaptic depolarization and the activation of NMDARs at both immediate and nearby postsynaptic active zones.

    View details for DOI 10.1038/nn1114

    View details for PubMedID 12910242

  • Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity JOURNAL OF CELL BIOLOGY Micheva, K. D., Holz, R. W., Smith, S. J. 2001; 154 (2): 355-368


    Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

    View details for Web of Science ID 000170136100015

    View details for PubMedID 11470824

    View details for PubMedCentralID PMC2150764

  • beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum EUROPEAN JOURNAL OF NEUROSCIENCE Micheva, K. D., Vallee, A., Beaulieu, C., Herman, I. M., Leclerc, N. 1998; 10 (12): 3785-3798


    Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle gamma- and beta-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that gamma-actin is expressed at a constant level throughout development, while the level of beta-actin expression rapidly decreases with age. At the light microscopic level, gamma-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, beta-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, gamma-actin is found in the cell body, neurites and growth cones, while beta-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565-9572; Bassell et al., (1998), J. Neuroscience, 18, 251-265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of gamma-actin, and also reveals an increased concentration of gamma-actin in dendritic spines in the adult. During development, beta-actin accumulation is observed in actively growing structures, e.g., growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, beta-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.

    View details for Web of Science ID 000077815400021

    View details for PubMedID 9875357

  • Increased number and size of dendritic spines in ipsilateral barrel field cortex following unilateral whisker trimming in postnatal rat JOURNAL OF COMPARATIVE NEUROLOGY Vees, A. M., Micheva, K. D., Beaulieu, C., Descarries, L. 1998; 400 (1): 110-124


    The barrel field area of the primary somatosensory cortex of rodents is a fertile ground for investigating experience-dependent plasticity and its mechanisms, because the neurons in its layer IV are distributed in groups (barrels) which correspond somatotopically to the vibrissae of the contralateral facial pad. After removal of three rows of whiskers from the right facial pad of young rats during the first two postnatal months, we looked for eventual changes in dendritic spine number and morphology in the corresponding barrels ipsi- and contralateral to the deprivation. Intact littermate controls were also examined. Spine number was determined by means of the unbiased disector method in electron micrographs from serial thin sections processed for post-embedding gamma-aminobutyric acid (GABA) immunocytochemistry. The volume and surface area of spine head, surface area of postsynaptic density and length of spine neck were measured from computerized three-dimensional reconstructions. Even though there was no significant side-to-side difference in the numerical density of dendritic spines in the experimental animals, the total number of spines in the ipsilateral barrels had increased by 67%, in view of the greater thickness of layer IV on this side. Moreover, spine head volume and surface area of postsynaptic densities were increased, and the length of spine neck was reduced in the ipsilateral compared to the contralateral cortex, and similar differences were noticeable between ipsilateral and control cortex. These changes apparently involved not only the predominant population of relatively small, dendritic spines innervated by asymmetrical synaptic terminals, but also the relatively small contingent of larger spines receiving symmetrical synapses formed by GABA terminals. The most likely explanation for such ipsilateral changes was an increased use of the intact (contralateral) facial pad during postnatal life, in keeping with the notion that activation of a peripheral sensory apparatus during the early postnatal period may have profound effects on the neuronal morphology and structural design of the primary somatosensory cortex. A possible mechanism in this case might be the excessive early activation of thalamic afferents, resulting in increased production of trophic factors, such as brain-derived nerve growth factor.

    View details for Web of Science ID 000076059800008

    View details for PubMedID 9762870

  • Development and plasticity of the inhibitory neocortical circuitry with an emphasis on the rodent barrel field cortex: A review Symposium of the Centre-de-Recherche-en-Sciences-Neurologiques-of-the-Universite-de-Montreal on GABA Mechanisms in the Cerebral Cortex Micheva, K. D., Beaulieu, C. NATL RESEARCH COUNCIL CANADA-N R C RESEARCH PRESS. 1997: 470–78


    The present paper reviews current knowledge of the development and plasticity of the inhibitory gamma-aminobutyric acid (GABA) containing circuitry of the cerebral neocortex, in particular, the rat somatosensory barrel field cortex. Recent studies reveal a delayed and protracted maturation of the inhibitory compared with the excitatory cortical system, both at the neuronal and synaptic levels. This characteristic developmental pattern leaves a longer time window during which behaviourally relevant activity coming from the periphery can influence the organization of the GABA system. Indeed, sensory deprivation experiments confirm the involvement of the GABA system in phenomena of experience-dependent cortical plasticity. Changing the pattern and level of afferent activity of in the rat somatosensory system during development by removing vibrissae results in a significant decrease in the number of GABA neurons and synapses in the thalamocortical recipient layer IV. Particularly affected are GABA synapses contacting dendritic spines, the number of which decreases by almost two-thirds. The involvement of the GABA system in events of experience-dependent plasticity contributes to the adequate functioning of the cerebral cortex in the conditions of constantly changing environment and varying individual experience.

    View details for Web of Science ID A1997XM24600014

    View details for PubMedID 9250380

  • Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry JOURNAL OF COMPARATIVE NEUROLOGY Micheva, K. D., Beaulieu, C. 1996; 373 (3): 340-354


    The postnatal establishment of cortical connectivity was studied by estimating the number (numerical density, synapse-to-neuron ratio, and total number) of the overall synaptic population and its distribution into gamma-aminobutyric acid (GABA)-immunopositive and GABA-immunonegative synaptic contacts in the developing rat somatosensory cortex. These numerical data were obtained using the unbiased disector method in combination with GABA postembedding immunocytochemistry. The numerical density of both synaptic populations was low in the early postnatal period (postnatal days 5 and 10, P5, P10) after which it abruptly increased between P10 and P15 to approach adult values. However, since cortical volume continues to increase after this age, the number of synapses per neuron and the total number of synapses reached adult values only by P30. There was no evidence of overproduction of either GABA or non-GABA synapses. Direct comparison between the two synaptic populations revealed a similar developmental pattern with the exception of the period around P20 when the production of GABA synapses slowed down. Thus, while the formation of non-GABA synapses proceeded in a continuous manner throughout the first month of life, GABA synapse production was accomplished in two consecutive waves. We suggest that the second delayed wave of GABA synapse formation is related to the great developmental plasticity of the cortical inhibitory system.

    View details for Web of Science ID A1996VK43300003

    View details for PubMedID 8889932



    The objective of this study was to examine the influence of sensory experience on the synaptic circuitry of the cortex. For this purpose, the quantitative distribution of the overall and of the gamma-aminobutyric acid (GABA) population of synaptic contacts was investigated in each layer of the somatosensory barrel field cortex of rats which were sensory deprived from birth by continuously removing rows of whiskers. Whereas there were no statistically significant changes in the quantitative distribution of the overall synaptic population, the number and proportion of GABA-immunopositive synaptic contacts were profoundly altered in layer IV of the somatosensory cortex of sensory-deprived animals. These changes were attributable to a specific loss of as many as two-thirds of the GABA contacts targeting dendritic spines. Thus, synaptic contacts made by GABA terminals in cortical layer IV and, in particular, those targeting dendritic spines represent a structural substrate of experience-dependent plasticity. Furthermore, since in this model of cortical plasticity the neuronal receptive-field properties are known to be affected, we propose that the inhibitory control of dendritic spines is essential for the elaboration of these functional properties.

    View details for Web of Science ID A1995TJ22200106

    View details for PubMedID 8524859

    View details for PubMedCentralID PMC40497



    Modified activity of the rat vibrissae from birth to adulthood induces profound alterations of the responsiveness and selectivity of neurons in the contralateral somatosensory barrel field cortex of adult rats. Because these functional properties are under the control of the intracortical inhibitory mechanisms, we investigated the effects of unilateral removal of face pad vibrissae on the quantitative distribution of intracortical gamma-aminobutyric acid (GABA)-immunoreactive neurons in the rat contralateral and ipsilateral barrel field cortices. This distribution was then compared to a population of control animals. For the entire cortical depth, no significant changes in the density (7,700/mm3 vs. 7,400/mm3), proportion (13.6% vs. 14.4%), or size (11.7 microns vs. 12.5 microns) of GABA-immunoreactive neurons were found in the left contralateral vs. the right ipsilateral barrel field cortex. However, in cortical layer IV, contralateral to the deprivation, the density and proportion of GABA-immunoreactive neurons were lower (6,300/mm3 vs. 13,900/mm3, 6.0% vs. 13.6%; P < 0.01), and these neurons were larger (mean projected height of 15.1 microns vs. 10.8 microns; P < 0.01) than in the ipsilateral barrel field cortex, suggesting a specific loss of GABA expression in a subpopulation of small intracortical neurons. In addition to changes in the contralateral layer IV, GABA-immunoreactive neurons located in the ipsilateral granular layer were also affected. Indeed, their numerical density (13,900/mm3) and proportion (13.6%) were higher (P < 0.01) than in both hemispheres of control animals (average of 10,050/mm3 and 9.4%). On the other hand, GABA-immunoreactive neurons in the ipsilateral layer V were less numerous (5,600/mm3, 15.0%) than in both sides of the controls (average of 10,300/mm3, 22.0%; P < 0.01). Thus, our results show that a unilateral sensory deprivation induces highly selective changes in the intracortical GABA inhibitory circuitry of both hemispheres. These changes are located directly at the input of thalamic afferents and at an output layer of corticofugal and commissural axons and could result in a profound reorganization of the excitatory and inhibitory drives of both sides of the sensory-deprived barrel field cortex.

    View details for Web of Science ID A1995TC35700002

    View details for PubMedID 8576415



    As an estimate of the numerical importance of GABA-containing neurons during development, their quantitative distribution was analysed in the primary somatosensory cortex of rats between postnatal days (P) 5 and 60, using the dissector method and GABA postembedding immunocytochemistry. In relation to the overall number of neurons in the barrel field cortex, the proportion of GABA neurons showed an early significant decrease between P5 and P10 from 14 to 11%, most likely due to termination of transient expression of GABA by some cells. It then remained stable until P20, after which it started slowly but steadily to increase, reaching 14% of the total at P60. The absolute number of GABA neurons also increased by nearly 50% during that period, whereas the number of all neurons remained constant. These changes are seemingly due to a subpopulation of neurons, shown to be of small size, which express GABA late in development. Thus, anatomical adjustments of the cortical GABA system may be observed at least until the end of the second postnatal month, reflecting both delayed maturation and adaptation of this inhibitory circuitry. We suggest the existence of three subpopulations of cortical GABA neurons depending on the time of onset and the regulation of their GABA expression: (i) neurons which express GABA before completion of migration and thus provide for its neurotrophic influence, (ii) neurons which express GABA immediately after completion of migration and build up the cortical inhibitory circuitry, and (iii) neurons which express GABA later in development and represent a substrate of experience-dependent plasticity.

    View details for Web of Science ID A1995QR63400009

    View details for PubMedID 7773439