A scalable and high-throughput method to identify precise subcellular localization of endogenous proteins is essential for integrative understanding of a cell at the molecular level. Here, we ...developed a simple and generalizable technique to image endogenous proteins with high specificity, resolution, and contrast in single cells in mammalian brain tissue. The technique, single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (SLENDR), uses in vivo genome editing to insert a sequence encoding an epitope tag or a fluorescent protein to a gene of interest by CRISPR-Cas9-mediated homology-directed repair (HDR). Single-cell, HDR-mediated genome editing was achieved by delivering the editing machinery to dividing neuronal progenitors through in utero electroporation. We demonstrate that SLENDR allows rapid determination of the localization and dynamics of many endogenous proteins in various cell types, regions, and ages in the brain. Thus, SLENDR provides a high-throughput platform to map the subcellular localization of endogenous proteins with the resolution of micro- to nanometers in the brain.
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•SLENDR enables in vivo protein labeling via CRISPR-Cas9-mediated HDR in the brain•Rapid and high-resolution mapping of subcellular localization of endogenous proteins•SLENDR is scalable to many endogenous proteins and cell types widely in the brain•SLENDR enables GFP knockin and monitoring of endogenous proteins in live tissue
A simple, rapid, and generalizable technique, single-cell labeling of endogenous proteins by CRISPR-Cas9-mediated homology-directed repair (SLENDR), enables high-resolution mapping of the subcellular localization of a broad spectrum of endogenous proteins in the mammalian brain.
The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single ...dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.
To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.
Postsynaptic mitochondria are critical for the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We ...examined a correlative dataset from ferret visual cortex with in vivo two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally and structurally characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with a mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons.
Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for ...immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.
Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a ...quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local Ca2+ imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.
In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance ...between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses.
The points of contact between nerve cells are called synapses, and nerve cells communicate across synapses via chemicals known as neurotransmitters. These chemical messengers are initially stored within bubble-like packages called synaptic vesicles that are released after they fuse with the membrane of the nerve cell at a specialized site referred to as the “active zone”.
Calcium ions are one of the major factors that lead to the release of synaptic vesicles. Ion channel proteins in the membrane of the nerve cell control the flow of calcium ions into the cell. There are often many different ion channels at a synapse, but one type called CaV2.1 most effectively triggers the release of synaptic vesicles when a nerve impulse reaches the synapse. Various proteins at the active zone can bind directly to parts of the CaV2.1 channel that are identified by a short sequence of amino acids – the building blocks of all proteins. Several researchers have proposed that the interactions with some of these short sequences, which are also known as motifs, control how much of this ion channel is in the synapse and how it interacts with synaptic vesicles to regulate the release of neurotransmitters. However, other researchers do not agree with this proposed explanation.
Lübbert, Goral et al. set out to determine which parts in a specific part of the CaV2.1 channel (called the “α1 subunit C-terminus”) are critical for its interaction with synaptic vesicles. The experiments revealed a new motif that regulates how many synaptic vesicles could be released in response to electrical impulses travelling along nerve cells from mice. The same motif also regulates the total number of synaptic vesicles at the active zone.
Lübbert, Goral et al. went on to show that binding to known active proteins at most played a minor role in controlling the abundance of the CaV2.1 channels and how close they were to the synaptic vesicles. As such, these findings counter prevailing views of the roles of certain motifs in the α1 subunit of the CaV2.1 channel. Thus, it may be necessary to re-think how the CaV2.1 channel regulates the release of synaptic vesicles.
Ion channels are vital to the activity of all nerve cells, and working out how the numbers and organization of CaV2.1 and related ion channels are regulated will be fundamental to understanding how information is encoded in brain. In addition, problems with these kinds of ion channel may result in disorders such as migraines and epilepsy. Therefore, the new findings may help to guide further studies investigating possible ways to treat these disorders.
The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory ...information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.
Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.
In axons, an action potential (AP) is thought to be broadcast as an unwavering binary pulse over its arbor, driving neurotransmission uniformly at release sites. Yet by recording from axons of ...cerebellar stellate cell (SC) interneurons, we show that AP width varies between presynaptic bouton sites, even within the same axon branch. The varicose geometry of SC boutons alone does not impose differences in spike duration. Rather, axonal patching revealed heterogeneous peak conductance densities of currents mediated mainly by fast-activating Kv3-type potassium channels, with clustered hotspots at boutons and restricted expression at adjoining shafts. Blockade of Kv channels at individual boutons indicates that currents immediately local to a release site direct spike repolarization at that location. Thus, the clustered arrangement and variable expression density of Kv3 channels at boutons are key determinants underlying compartmentalized control of AP width in a near synapse-by-synapse manner, multiplying the signaling capacity of these structures.
•Spike duration varies between presynaptic boutons within an individual SC axon•The varicose geometry of presynaptic boutons does not alter spike duration•Fast-activating K+ channels are clustered at boutons and restricted from shafts•AP duration is determined by Kv3 channels immediately local to an individual bouton
Rowan et al. find that APs are not uniformly represented across the axon arbor of SCs. Rather, spike duration is subject to local variation, determined at individual release sites, by clustering of fast-activating K+ channels, thus contributing to release heterogeneity.
Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections ...with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit.
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