Formation of Dendritic Spines with GABAergic Synapses Induced by Whisker Stimulation in Adult Mice

Neuron. Apr, 2002  |  Pubmed ID: 11970868

During development, alterations in sensory experience modify the structure of cortical neurons, particularly at the level of the dendritic spine. Are similar adaptations involved in plasticity of the adult cortex? Here we show that a 24 hr period of single whisker stimulation in freely moving adult mice increases, by 36%, the total synaptic density in the corresponding cortical barrel. This is due to an increase in both excitatory and inhibitory synapses found on spines. Four days after stimulation, the inhibitory inputs to the spines remain despite total synaptic density returning to pre-stimulation levels. Functional analysis of layer IV cells demonstrated altered response properties, immediately after stimulation, as well as four days later. These results indicate activity-dependent alterations in synaptic circuitry in adulthood, modifying the flow of sensory information into the cerebral cortex.

Long-term in Vivo Imaging of Experience-dependent Synaptic Plasticity in Adult Cortex

Nature. Dec 19-26, 2002  |  Pubmed ID: 12490942

Do new synapses form in the adult cortex to support experience-dependent plasticity? To address this question, we repeatedly imaged individual pyramidal neurons in the mouse barrel cortex over periods of weeks. We found that, although dendritic structure is stable, some spines appear and disappear. Spine lifetimes vary greatly: stable spines, about 50% of the population, persist for at least a month, whereas the remainder are present for a few days or less. Serial-section electron microscopy of imaged dendritic segments revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. Experience-dependent plasticity of cortical receptive fields was accompanied by increased synapse turnover. Our measurements suggest that sensory experience drives the formation and elimination of synapses and that these changes might underlie adaptive remodelling of neural circuits.

Morphological and Molecular Heterogeneity in Release Sites of Single Neurons

The European Journal of Neuroscience. Apr, 2003  |  Pubmed ID: 12713639

We have previously shown that labelling intensities for synaptic proteins vary strongly among synaptic boutons. Here we addressed the questions as to whether there are heterogeneous levels of integral membrane synaptic vesicle proteins at distinct active release sites of single neurons and if these sites possess the ultrastructural features of synapses. By double-immunostaining with specific antibodies against synaptophysin, synaptotagmin I, VAMP1 and VAMP2, we identified different relative levels of these integral membrane proteins of synaptic vesicles in comparison to boutons of the same rat cortical neuron. This heterogeneity could also be observed between the two isoforms VAMP1 and VAMP2. By studying pairs of these proteins implicated in neurotransmitter release, including both VAMP isoforms, we also show that the sites that contained predominantly one protein were nevertheless functional, as they internalized and released FM1-43 upon potassium stimulation. Using electron microscopy, we show that these active sites could have either synaptic specializations, or the features of vesicle-containing varicosities without a postsynaptic target. Different varicosities of the same neuron showed different intensities for synaptic vesicle proteins; some varicosities were capable of internalizing and releasing FM1-43, while others were silent. These results show that integral membrane synaptic vesicle proteins are differentially distributed among functional release sites of the same neuron.

Glial Glutamate Transporters and Maturation of the Mouse Somatosensory Cortex

Cerebral Cortex (New York, N.Y. : 1991). Oct, 2003  |  Pubmed ID: 12967927

In the adult nervous system, glutamatergic neurotransmission is tightly controlled by neuron-glia interactions through glial glutamate reuptake by the specific transporters GLT-1 and GLAST. Here, we have explored the role of these transporters in the structural and functional maturation of the somatosensory cortex of the mouse. We provide evidence that GLT-1 and GLAST are early and selectively expressed in barrels from P5 to P10. Confocal and electron microscopy confirm that the expression is restricted to the astroglial membrane. By P12, and despite an increased global expression as observed by immunoblotting, the barrel pattern of GLAST and GLT-1 staining is no longer evident. In P10 GLT-1 -/- and GLAST -/- mice, the cytoarchitectural segregation of the barrels is preserved. However, at P9-10, the functional response to whisker stimulation, measured by deoxyglucose uptake, is markedly decreased in GLT-1 -/- and GLAST -/- mice. The role of GLAST is transient since the metabolic response is already restored at P11-12 in GLAST -/- mice and remains unchanged in adulthood. However, deletion of GLT-1 seems to impair the functional metabolic response until adulthood. Our data suggest that astrocyte-neuron interactions via the glial glutamate transporters are involved in the functional maturation of the whisker representation in the somatosensory cortex.

Altered Synapse Formation in the Adult Somatosensory Cortex of Brain-derived Neurotrophic Factor Heterozygote Mice

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2004  |  Pubmed ID: 15014114

Increased sensory stimulation in the adult whisker-to-barrel pathway induces the expression of BDNF as well as synapse formation in cortical layer IV. Here, we investigated whether BDNF plays a role in the alterations of connectivity between neurons by analyzing the ultrastructure of the BDNF heterozygote mouse, characterized by a reduced level of BDNF expression. Using serial section electron microscopy, we measured synapse density, spine morphology, and synaptic vesicle distribution to show that mice with a reduced level of BDNF have a barrel neuropil that is indistinguishable from wild-type controls. After 24 hr of whisker stimulation, however, there is no indication of synapse formation in the heterozygous mouse. Whereas the balance between excitatory and inhibitory synapses is modified in the controls, it remains constant in the heterozygotes. The distribution of synaptic vesicles in excitatory synapses is the same in heterozygous and wild-type mice and is not influenced by the stimulation paradigm. Spine volume, however, is unchanged by stimulation in the wild-type animals, but does increase significantly in the heterozygous animal. These results provide evidence that, in vivo, BDNF plays an important role in the structural rearrangement of adult cortical circuitry as a consequence of an increased sensory input.

Induction of Spine Growth and Synapse Formation by Regulation of the Spine Actin Cytoskeleton

Neuron. Oct, 2004  |  Pubmed ID: 15473970

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.

Subcellular Domain-restricted GABAergic Innervation in Primary Visual Cortex in the Absence of Sensory and Thalamic Inputs

Nature Neuroscience. Nov, 2004  |  Pubmed ID: 15475951

Distinct classes of GABAergic synapses target restricted subcellular domains, thereby differentially regulating the input, integration and output of principal neurons, but the underlying mechanism for such synapse segregation is unclear. Here we show that the distributions of two major classes of GABAergic synapses along the perisomatic and dendritic domains of pyramidal neurons were indistinguishable between primary visual cortex in vivo and cortical organotypic cultures. Therefore, subcellular synapse targeting is independent of thalamic input and probably involves molecular labels and experience-independent forms of activity.

Experience and Activity-dependent Maturation of Perisomatic GABAergic Innervation in Primary Visual Cortex During a Postnatal Critical Period

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2004  |  Pubmed ID: 15509747

The neocortical GABAergic network consists of diverse interneuron cell types that display distinct physiological properties and target their innervations to subcellular compartments of principal neurons. Inhibition directed toward the soma and proximal dendrites is crucial in regulating the output of pyramidal neurons, but the development of perisomatic innervation is poorly understood because of the lack of specific synaptic markers. In the primary visual cortex, for example, it is unknown whether, and to what extent, the formation and maturation of perisomatic synapses are intrinsic to cortical circuits or are regulated by sensory experience. Using bacterial artificial chromosome transgenic mice that label a defined class of perisomatic synapses with green fluorescent protein, here we show that perisomatic innervation developed during a protracted postnatal period after eye opening. Maturation of perisomatic innervation was significantly retarded by visual deprivation during the third, but not the fifth, postnatal week, implicating an important role for sensory input. To examine the role of cortical intrinsic mechanisms, we developed a method to visualize perisomatic synapses from single basket interneurons in cortical organotypic cultures. Characteristic perisomatic synapses formed through a stereotyped process, involving the extension of distinct terminal branches and proliferation of perisomatic boutons. Neuronal spiking in organotypic cultures was necessary for the proliferation of boutons and the extension, but not the maintenance, of terminal branches. Together, our results suggest that although the formation of perisomatic synapses is intrinsic to the cortex, visual experience can influence the maturation and pattern of perisomatic innervation during a postnatal critical period by modulating the level of neural activity within cortical circuits.

Transient and Persistent Dendritic Spines in the Neocortex in Vivo

Neuron. Jan, 2005  |  Pubmed ID: 15664179

Dendritic spines were imaged over days to months in the apical tufts of neocortical pyramidal neurons (layers 5 and 2/3) in vivo. A fraction of thin spines appeared and disappeared over a few days, while most thick spines persisted for months. In the somatosensory cortex, from postnatal day (PND) 16 to PND 25 spine retractions exceeded additions, resulting in a net loss of spines. The fraction of persistent spines (lifetime > or = 8 days) grew gradually during development and into adulthood (PND 16-25, 35%; PND 35-80, 54%; PND 80-120, 66%; PND 175-225, 73%), providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods. In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.

Primary Sensory Afferent Innervation of the Developing Superficial Dorsal Horn in the South American Opossum Monodelphis Domestica

The Journal of Comparative Neurology. Mar, 2006  |  Pubmed ID: 16432898

The development of the primary sensory innervation of the superficial dorsal horn (SDH) was studied in postnatal opossums Monodelphis domestica by using DiI labelling of primary afferents and with GSA-IB(4) lectin binding and calcitonin gene-related peptide (CGRP) immunoreactivity to label primary afferent subpopulations. We also compared the timing of SDH innervation in the cervical and lumbar regions of the spinal cord. The first primary afferent projections to SDH emerge from the most lateral part of the dorsal root entry zone at postnatal day 5 and project around the lateral edge of the SDH toward lamina V. Innervation of the SDH occurs slowly over the second and third postnatal weeks, with the most dorsal aspect becoming populated by mediolaterally oriented varicose fibers before the rest of the dorsoventral thickness of the SDH becomes innervated by fine branching varicose fibers. Labelling with GSA-IB(4) lectin also labelled fibers at the lateral edge of the dorsal horn and SDH at P5, indicating that the GSA-IB(4) is expressed on SDH/lamina V primary afferents at the time when they are making their projections into the spinal cord. In contrast, CGRP-immunoreactive afferents were not evident until postnatal day 7, when a few short projections into the lateral dorsal horn were observed. These afferents then followed a pattern similar to the development of GSA-IB(4) projects but with a latency of several days. The adult pattern of labelling by GSA-IB(4) is achieved by about postnatal day 20, whereas the adult pattern of CGRP labelling was not seen until postnatal day 30. Electron microscopy revealed a few immature synapses in the region of the developing SDH at postnatal day 10, and processes considered to be precursors of glomerular synapses (and thus of primary afferent origin) were first seen at postnatal day 16 and adopted their definitive appearance between postnatal days 28 and 55. Although structural and functional development of forelimbs of neonatal Monodelphis is more advanced than the hindlimbs, we found little evidence of a significant delay in the invasion of the spinal cord by primary afferents in cervical and lumbar regions. These observations, together with the broadly similar maturational appearance of histological sections of rostral and caudal spinal cord, suggest that, unlike the limbs they innervate, the spinal regions do not exhibit a large rostrocaudal gradient in their maturation.

Cell Type-specific Structural Plasticity of Axonal Branches and Boutons in the Adult Neocortex

Neuron. Mar, 2006  |  Pubmed ID: 16543134

We imaged axons in layer (L) 1 of the mouse barrel cortex in vivo. Axons from thalamus and L2/3/5, or L6 pyramidal cells were identified based on their distinct morphologies. Their branching patterns and sizes were stable over times of months. However, axonal branches and boutons displayed cell type-specific rearrangements. Structural plasticity in thalamocortical afferents was mostly due to elongation and retraction of branches (range, 1-150 microm over 4 days; approximately 5% of total axonal length), while the majority of boutons persisted for up to 9 months (persistence over 1 month approximately 85%). In contrast, L6 axon terminaux boutons were highly plastic (persistence over 1 month approximately 40 %), and other intracortical axon boutons showed intermediate levels of plasticity. Retrospective electron microscopy revealed that new boutons make synapses. Our data suggest that structural plasticity of axonal branches and boutons contributes to the remodeling of specific functional circuits.

Ciliary Neurotrophic Factor Activates Astrocytes, Redistributes Their Glutamate Transporters GLAST and GLT-1 to Raft Microdomains, and Improves Glutamate Handling in Vivo

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2006  |  Pubmed ID: 16738240

To study the functional role of activated astrocytes in glutamate homeostasis in vivo, we used a model of sustained astrocytic activation in the rat striatum through lentiviral-mediated gene delivery of ciliary neurotrophic factor (CNTF). CNTF-activated astrocytes were hypertrophic, expressed immature intermediate filament proteins and highly glycosylated forms of their glutamate transporters GLAST and GLT-1. CNTF overexpression produced a redistribution of GLAST and GLT-1 into raft functional membrane microdomains, which are important for glutamate uptake. In contrast, CNTF had no detectable effect on the expression of a number of neuronal proteins and on the spontaneous glutamatergic transmission recorded from striatal medium spiny neurons. These results were replicated in vitro by application of recombinant CNTF on a mixed neuron/astrocyte striatal culture. Using microdialysis in the rat striatum, we found that the accumulation of extracellular glutamate induced by quinolinate (QA) was reduced threefold with CNTF. In line with this result, CNTF significantly increased QA-induced [(18)F]-fluoro-2-deoxyglucose uptake, an indirect index of glutamate uptake by astrocytes. Together, these data demonstrate that CNTF activation of astrocytes in vivo is associated with marked phenotypic and molecular changes leading to a better handling of increased levels of extracellular glutamate. Activated astrocytes may therefore be important prosurvival agents in pathological conditions involving defects in glutamate homeostasis.

Experience-dependent and Cell-type-specific Spine Growth in the Neocortex

Nature. Jun, 2006  |  Pubmed ID: 16791195

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size and the number of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.

Spine Growth Precedes Synapse Formation in the Adult Neocortex in Vivo

Nature Neuroscience. Sep, 2006  |  Pubmed ID: 16892056

Dendritic spines appear and disappear in an experience-dependent manner. Although some new spines have been shown to contain synapses, little is known about the relationship between spine addition and synapse formation, the relative time course of these events, or whether they are coupled to de novo growth of axonal boutons. We imaged dendrites in barrel cortex of adult mice over 1 month, tracking gains and losses of spines. Using serial section electron microscopy, we analyzed the ultrastructure of spines and associated boutons. Spines reconstructed shortly after they appeared often lacked synapses, whereas spines that persisted for 4 d or more always had synapses. New spines had a large surface-to-volume ratio and preferentially contacted boutons with other synapses. In some instances, two new spines contacted the same axon. Our data show that spine growth precedes synapse formation and that new synapses form preferentially onto existing boutons.

Plasticity of Astrocytic Coverage and Glutamate Transporter Expression in Adult Mouse Cortex

PLoS Biology. Oct, 2006  |  Pubmed ID: 17048987

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton-spine interface and an increase in glutamate transporter expression in astrocytic processes.

GAD67-mediated GABA Synthesis and Signaling Regulate Inhibitory Synaptic Innervation in the Visual Cortex

Neuron. Jun, 2007  |  Pubmed ID: 17582330

The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.

Dendritic Spine Plasticity--current Understanding from in Vivo Studies

Brain Research Reviews. Aug, 2008  |  Pubmed ID: 18353441

Changes in sensory experience modify the function of the adult brain's neuronal circuits. This flexibility is reliant on the neurons' ability to change the strength of their connections. Most excitatory connections in the adult cerebral cortex are found on dendritic spines, protrusions from the dendrite that in vitro, and histological analyses, have been shown to be implicated in neuronal plasticity. The recent development of in vivo imaging techniques now provides the ability to explore experience-dependent structural plasticity in the adult brain; the changes in neuronal connectivity that accompany functional reorganizations of the sensory maps. Multi-photon laser scanning microscopy in transgenic mice expressing fluorescent proteins in cortical neurons shows that although the majority of spines is present throughout the imaging period of weeks a proportion of spines is transient, appearing and disappearing on a daily basis. A small fraction appears and stabilizes forming synapses, and this proportion can be affected by changes in sensory experience. This synapse formation occurs preferentially through the initial generation of a filopodial-like spine contacting an axonal bouton already present in the neuropil. The results implicate these specialized compartments of synaptic transmission as playing a central role in the functional reorganization of neuronal circuits.

Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam Milling

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2008  |  Pubmed ID: 18353998

PSD-95 Promotes Synaptogenesis and Multiinnervated Spine Formation Through Nitric Oxide Signaling

The Journal of Cell Biology. Dec, 2008  |  Pubmed ID: 19075115

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

A Protocol for Preparing GFP-labeled Neurons Previously Imaged in Vivo and in Slice Preparations for Light and Electron Microscopic Analysis

Nature Protocols. 2009  |  Pubmed ID: 19617886

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

Rapid Functional Maturation of Nascent Dendritic Spines

Neuron. Jan, 2009  |  Pubmed ID: 19186167

Spine growth and retraction with synapse formation and elimination plays an important role in shaping brain circuits during development and in the adult brain, yet the temporal relationship between spine morphogenesis and the formation of functional synapses remains poorly defined. We imaged hippocampal pyramidal neurons to identify spines of different ages. We then used two-photon glutamate uncaging, whole-cell recording, and Ca(2+) imaging to analyze the properties of nascent spines and their older neighbors. New spines expressed glutamate-sensitive currents that were indistinguishable from mature spines of comparable volumes. Some spines exhibited negligible AMPA receptor-mediated responses, but the occurrence of these "silent" spines was uncorrelated with spine age. In contrast, NMDA receptor-mediated Ca(2+) accumulations were significantly lower in new spines. New spines reconstructed using electron microscopy made synapses. Our data support a model in which outgrowth and enlargement of nascent spines is tightly coupled to formation and maturation of glutamatergic synapses.

Long-term, High-resolution Imaging in the Mouse Neocortex Through a Chronic Cranial Window

Nature Protocols. 2009  |  Pubmed ID: 19617885

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (approximately 0.8-12 mm(2)). The surgical procedure can be completed within approximately 1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed.

Automated Detection and Segmentation of Synaptic Contacts in Nearly Isotropic Serial Electron Microscopy Images

PloS One. 2011  |  Pubmed ID: 22031814

We describe a protocol for fully automated detection and segmentation of asymmetric, presumed excitatory, synapses in serial electron microscopy images of the adult mammalian cerebral cortex, taken with the focused ion beam, scanning electron microscope (FIB/SEM). The procedure is based on interactive machine learning and only requires a few labeled synapses for training. The statistical learning is performed on geometrical features of 3D neighborhoods of each voxel and can fully exploit the high z-resolution of the data. On a quantitative validation dataset of 111 synapses in 409 images of 1948×1342 pixels with manual annotations by three independent experts the error rate of the algorithm was found to be comparable to that of the experts (0.92 recall at 0.89 precision). Our software offers a convenient interface for labeling the training data and the possibility to visualize and proofread the results in 3D. The source code, the test dataset and the ground truth annotation are freely available on the website http://www.ilastik.org/synapse-detection.

GABA Signaling Promotes Synapse Elimination and Axon Pruning in Developing Cortical Inhibitory Interneurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2012  |  Pubmed ID: 22219294

Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses.

Supervoxel-based Segmentation of Mitochondria in Em Image Stacks with Learned Shape Features

IEEE Transactions on Medical Imaging. Feb, 2012  |  Pubmed ID: 21997252

It is becoming increasingly clear that mitochondria play an important role in neural function. Recent studies show mitochondrial morphology to be crucial to cellular physiology and synaptic function and a link between mitochondrial defects and neuro-degenerative diseases is strongly suspected. Electron microscopy (EM), with its very high resolution in all three directions, is one of the key tools to look more closely into these issues but the huge amounts of data it produces make automated analysis necessary. State-of-the-art computer vision algorithms designed to operate on natural 2-D images tend to perform poorly when applied to EM data for a number of reasons. First, the sheer size of a typical EM volume renders most modern segmentation schemes intractable. Furthermore, most approaches ignore important shape cues, relying only on local statistics that easily become confused when confronted with noise and textures inherent in the data. Finally, the conventional assumption that strong image gradients always correspond to object boundaries is violated by the clutter of distracting membranes. In this work, we propose an automated graph partitioning scheme that addresses these issues. It reduces the computational complexity by operating on supervoxels instead of voxels, incorporates shape features capable of describing the 3-D shape of the target objects, and learns to recognize the distinctive appearance of true boundaries. Our experiments demonstrate that our approach is able to segment mitochondria at a performance level close to that of a human annotator, and outperforms a state-of-the-art 3-D segmentation technique.