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Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an \"average\" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large ‘rosehip’-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1–SST–CALB2–PVALB–) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

Emerging evidence indicates that liquid–liquid phase separation, the formation of a condensed molecular assembly within another diluted aqueous solution, is a means for cells to organize highly condensed biological assemblies (also known as biological condensates or membraneless compartments) with very broad functions and regulatory properties in different subcellular regions. Molecular machineries dictating synaptic transmissions in both presynaptic boutons and postsynaptic densities of neuronal synapses may be such biological condensates. Here we review recent developments showing how phase separation can build dense synaptic molecular clusters, highlight unique features of such condensed clusters in the context of synaptic development and signaling, discuss how aberrant phase-separation-mediated synaptic assembly formation may contribute to dysfunctional signaling in psychiatric disorders, and present some challenges and opportunities of phase separation in synaptic biology.Phase separation is emerging as a versatile means for cellular sub-compartment formation. Chen et al. review recent advances of dense synaptic assembly formation via phase separation and discuss implications of phase separation in synaptic physiology.

Combining patch-clamp recordings and subsequent STORM imaging of individual cells, the authors show that the axon terminals of perisomatically- and dendritically-projecting GABAergic interneurons show differences in CB 1 receptor number, active zone complexity, and receptor:effector ratio. Chronic exposure to THC evoked a dose-dependent and long-lasting downregulation of CB 1 at these synapses. A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type– and subcellular compartment–specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB 1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ 9 -tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB 1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ 9 -tetrahydrocannabinol treatment. These findings indicate that cell type–specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

The authors uncovered a pathway from the lateral amygdala to the auditory cortex (ACx) of mice that is essential for auditory fear memory retrieval. Simultaneous imaging of pre- and postsynaptic structures in ACx in vivo revealed an increased rate of synapse formation in this pathway after auditory fear conditioning. Neural circuits underlying auditory fear conditioning have been extensively studied. Here we identified a previously unexplored pathway from the lateral amygdala (LA) to the auditory cortex (ACx) and found that selective silencing of this pathway using chemo- and optogenetic approaches impaired fear memory retrieval. Dual-color in vivo two-photon imaging of mouse ACx showed pathway-specific increases in the formation of LA axon boutons, dendritic spines of ACx layer 5 pyramidal cells, and putative LA–ACx synaptic pairs after auditory fear conditioning. Furthermore, joint imaging of pre- and postsynaptic structures showed that essentially all new synaptic contacts were made by adding new partners to existing synaptic elements. Together, these findings identify an amygdalocortical projection that is important to fear memory expression and is selectively modified by associative fear learning, and unravel a distinct architectural rule for synapse formation in the adult brain.

Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially “tethered” to the PM by a variable number of protein densities (∼10 to 20 nm long)with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the “primed” vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.

Neurons in the thalamic reticular nucleus (TRN) are a primary source of inhibition to the dorsal thalamus and, as they are innervated in part by the cortex, are a means of corticothalamic regulation. Previously, cortical inputs to the TRN were thought to originate solely from layer 6 (L6), but we recently reported the presence of putative synaptic terminals from layer 5 (L5) neurons in multiple cortical areas in the TRN [J. A. Prasad, B. J. Carroll, S. M. Sherman, J. Neurosci. 40, 5785−5796 (2020)]. Here, we demonstrate with electron microscopy that L5 terminals from multiple cortical regions make bona fide synapses in the TRN. We further use light microscopy to localize these synapses relative to recently described TRN subdivisions and show that L5 terminals target the edges of the somatosensory TRN, where neurons reciprocally connect to higher-order thalamus, and that L5 terminals are scarce in the core of the TRN, where neurons reciprocally connect to first-order thalamus. In contrast, L6 terminals densely innervate both edge and core subregions and are smaller than those from L5. These data suggest that a sparse but potent input from L5 neurons of multiple cortical regions to the TRN may yield transreticular inhibition targeted to higherorder thalamus.

Using functional and morphological analysis, this study demonstrates that in CA3 pyramidal cell recurrent axon terminals, the release probability and the number of voltage-gated calcium channels are linearly correlated with the size of the active zone. Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca 2+ ] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca 2+ imaging–derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca 2+ ] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.

Dopamine is an essential neuromodulator in the brain, involved in reward and motor control. Dopaminergic (DA) neurons project to most brain areas, with particularly dense innervation in the striatum. DA varicosities bind to target striatal synapses, forming dopamine hub synapses (DHS). However, the basic features of dopamine release sites are still largely unknown. Here we studied the ultrastructure of fluorescent DA and glutamatergic (GLU) synaptosomes isolated from the striatum of adult mice with cryo-correlative light and electron microscopy and cryo-electron tomography. We observed that DA synaptosomes display ~10 times fewer vesicles than GLU ones. DA vesicles are bigger and less round. The nanoscale organization of vesicles indicates that most GLU synaptosomes have tethered and primed vesicles, indicative of a readily releasable pool, while only 37% of DA synaptosomes have tethered vesicles, which appear not to be primed. In addition, GLU terminals contacted by DA terminals in DHS have more primed vesicles than others. While DA varicosities do not form genuine synapses, their adhesion to cortico-striatal synapses may convey a local regulation of synaptic release properties. The basic features of dopamine release sites are still largely unknown. Here, the authors determine the ultrastructure of fluorescent dopaminergic and glutamatergic synaptosomes in mouse striatum using cryo-correlative light and electron microscopy.

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1 M146L /APP 751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.