Extensive Structural Remodeling of the Axonal Arbors of Parvalbumin Basket Cells during Development in Mouse Neocortex.

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 2 weeks and 7 months 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.

Conduction Velocity Along the Local Axons of Parvalbumin Interneurons Correlates With the Degree of Axonal Myelination.

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.

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons.

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.

Probabilistic fluorescence-based synapse detection.

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.

Deep molecular diversity of mammalian synapses: why it matters and how to measure it.

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.

Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target.

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.

Mapping synapses by conjugate light-electron array tomography.

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.

Array tomography: trails to discovery.

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody-based imaging techniques rely on reagents whose performance may be application-specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody (Ab)-based imaging techniques rely on reagents whose performance may be application specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here, we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.

Large-scale automated histology in the pursuit of connectomes.

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.

Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.

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.

Volume electron microscopy.

Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.

Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.

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.

Multifaceted Changes in Synaptic Composition and Astrocytic Involvement in a Mouse Model of Fragile X Syndrome.

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.

Retrograde regulation of synaptic vesicle endocytosis and recycling.

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.

A Computational Synaptic Antibody Characterization Tool for Array Tomography.

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.

Distinctive Structural and Molecular Features of Myelinated Inhibitory Axons in Human Neocortex.

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.

Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals.

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.

Array tomography of physiologically-characterized CNS synapses.

BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. COMPARISON WITH EXISTING METHODS: Compared to existing methods, the present approach is the only one that can provide detailed molecular and anatomical information of electrophysiologically-characterized individual synapses. CONCLUSIONS: 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.