Biology subjects

Micheva, K. D.

Publications and source records attributed to Micheva, K. D..

7 recordsLinked to original sources

bioRxiv2024

Data-driven synapse classification reveals a logic of glutamate receptor composition

The rich diversity of synapses facilitates the capacity of neural circuits to transmit, process and store information. We used multiplex super-resolution proteometric imaging through array tomography to define features of single synapses in mouse neocortex. We find that glutamatergic synapses cluster into subclasses that parallel the distinct biochemical and functional categories of receptor subunits: GluA1/4, GluA2/3 and GluN1/GluN2B. Two of these subclasses align with physiological expectations based on synaptic plasticity: large AMPAR-rich synapses may represent potentiated synapses, whereas small NMDAR-rich synapses suggest "silent" synapses. The NMDA receptor content of large synapses correlates with spine neck diameter, and thus the potential for coupling to the parent dendrite. Overall, ultrastructural features predict receptor content of synapses better than parent neuron identity does, suggesting synapse subclasses act as fundamental elements of neuronal circuits. No barriers prevent future generalization of this approach to other species, or to study of human disorders and therapeutics.

neuroscience↗

bioRxiv2023

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. Significance StatementArray tomography (AT) is a powerful volume microscopy technique for high-dimensional analysis of complex protein populations in cells and organelles, including synapses. AT involves the use of ultrathin serial sections embedded in resin and subjected to multiple rounds of immunofluorescence antibody (Ab) labeling and imaging. AT relies on antibody-based detection of proteins but because commercial antibodies are typically validated for other applications they often fail for AT. To identify antibodies with high probability of success in AT we developed a novel screening strategy and used this to create a comprehensive database of AT-validated antibodies for neuroscience.

neuroscience↗

bioRxiv2021

Extensive structural remodeling of the axonal arbors of parvalbumin basket cells during development

Parvalbumin-containing (PV+) basket cells are specialized cortical interneurons that regulate the activity of local neuronal circuits with high temporal precision and reliability. PV+ interneuron disfunction is associated with numerous psychiatric disorders, including schizophrenia and autism spectrum disorders. To understand how the PV+ interneuron connectivity underlying their 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 postnatal week and precedes a massive synapse pruning which takes place in the 4th postnatal week leading to about three-fold reduction of synaptic contacts made by the PV+ interneuron on its postsynaptic partner. Autapses, the synapses that PV+ interneurons form on themselves, however, are not subjected to pruning. 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 PV+ basket cells. Significance statementThe 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 subsequent 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.

neuroscience↗

bioRxiv2020

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 (IPSCs). These same neurons were then imaged with immunofluorescent array tomography, the synaptic contacts 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.

neuroscience↗

bioRxiv2019

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. In layers 1 and 2/3, there is a significant decrease in the density of excitatory glutamatergic synapses, both VGluT1 and VGluT2 type, and their association with astrocytes, while the changes in inhibitory GABAergic synapses are less pronounced. Meanwhile in layer 4, there is a significant increase in the density of small VGluT1 synapses, with no changes in the astrocytic association of synapses. 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 define targets for therapeutic intervention.

neuroscience↗

bioRxiv2018

Myelinated inhibitory axons in human neocortex

Numerous myelinated axons traverse the human neocortex. In a previous paper (Micheva et al., 2016) we showed that in mouse many of these axons belong to local inhibitory neurons, the parvalbumin-positive basket cells. Here, using samples of neurosurgically-excised cortex, we confirm the presence of myelinated inhibitory axons in all layers of human neocortex. As in mouse, these axons have distinctive features, including high neurofilament content, short nodes of Ranvier, and high content of myelin basic protein in their myelin sheath. We further show that, consistent with the known high-energy demands of parvalbumin interneurons, the inhibitory myelinated axons have more mitochondria, as well more 2,3-cyclic nucleotide 3-phosphodiesterase (a protein enriched in the myelin cytoplasmic channels thought to provide access for trophic support from oligodendrocytes). The distinctive features of myelinated inhibitory axons in human cortical grey matter may have important implications for neurological disorders that involve pathologies of myelinated axons.

neuroscience↗

bioRxiv2018

Automated Antibody Characterization and Screening for Array Tomography via Probabilistic Synapse Detection

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 Screening Tool (SACT), is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. By allowing the user to define the molecular composition and size of synapses expected to contain the antigen, the method detects and characterizes puncta and synapses, and outputs automatically computed characteristics such as synapse density and target specificity ratio, which reflect the sensitivity and specificity of immunolabeling with a given 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.

neuroscience↗