Frontiers in Synaptic Neuroscience

Long-term potentiation (LTP) induces presynaptic bouton enlargement and a reduction in the number of synaptic vesicles. To understand the relationship between these events, we performed 3D analysis of serial section electron micrographs in rat hippocampal area CA1, 2 hours after LTP induction. We observed a high vesicle packing density in control boutons, contrasting with a lower density in most LTP boutons. Notably, the summed membrane area of the vesicles lost in low-density LTP boutons is comparable to the surface membrane required for the observed bouton enlargement when compared to high-density control boutons. These novel findings suggest that presynaptic vesicle density provides a new structural indicator of LTP that supports a local mechanism of bouton enlargement.

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young synGAP+/- mice. Here we show that a highly significant increase in TARP in the PSDs of young synGAP+/- rodents is present only in females and not in males. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP heterozygosity.

The development of optogenetic tools has significantly advanced our understanding of neural circuits and behavior. The medial amygdala, posterior dorsal subdivision (MeApd) is part of a distributed network controlling social behaviors such as mating and aggression. Previous work showed that activation of GABAergic neurons in mouse MeApd using channelrodopsin-2 (ChR2H134R) promoted aggression. In a recent study, Baleisyte et al. (2022) confirmed these findings using the same reagents (i.e. ChR2H134R), but also reported that a different ChR2 variant with faster kinetics--ChETA--inhibited rather than promoted aggression when high laser power, long duration photostimulation conditions were used. As ChETA is known to have a substantially lower photocurrent than ChR2 and other opsins, an improved version of ChETA (i.e. ChR2E123T/T159C; ChETATC) was subsequently developed. ChETATC has larger photocurrents than the original ChETA while maintaining fast kinetics and low plateau depolarization. Here we show that activating MeApd GABAergic neurons using the improved ChETATC promotes aggression, similar to ChR2H134R, suggesting that the results obtained using the original ChETA are not due to a difference in channel kinetics. Furthermore, we found that ChETATC is capable of driving a rapid onset of aggression within 200-300 milliseconds of stimulation, suggesting that this effect reflects direct activation of MeApd GABAergic neurons. We conclude that the different behavioral phenotypes observed using the original ChETA vs. ChETATC and ChR2 likely reflects the weaker photocurrents in ChETA vs. other opsins, and/or the long duration/high power photostimulation conditions used with ChETA. Consistent with this conclusion, the results obtained using ChR2 or ChETATC are complementary to findings from loss-of-functions experiments using optogenetic inhibition, chemogenetic inhibition, and neuronal ablation. These data support a positive-acting role of MeApd Vgat+ neurons in aggression. Our findings, in conjunction with studies of Berndt et al. (2011), suggest that the improved ChETATC should be used when faster kinetics than ChR2 offers are required.

Sleep is critically involved in strengthening memories. However, our understanding of the morphological changes underlying this process is still emerging. Recent studies suggest that specific subsets of dendritic spines are strengthened during sleep in specific neurons involved in recent learning. Contextual memories associated with traumatic experiences are involved in post-traumatic stress disorder (PTSD) and represent recent learning that may be strengthened during sleep. We tested the hypothesis that dendritic spines encoding contextual fear memories are selectively strengthened during sleep. Furthermore, we tested how sleep deprivation after initial fear learning impacts dendritic spines following re-exposure to fear conditioning. We used ArcCreERT2 mice to visualize neurons that encode contextual fear learning (Arc+ neurons), and concomitantly labeled neurons that did not encode contextual fear learning (Arc-neurons). Dendritic branches of Arc+ and Arc-neurons were sampled using confocal imaging to assess spine densities using three-dimensional image analysis from either sleep deprived (SD) or control mice allowed to sleep normally. Mushroom spines in Arc+ branches displayed decreased density in SD mice, indicating upscaling of mushroom spines during sleep following fear learning. In comparison, no changes were observed in dendritic spines from Arc-branches. When animals were re-exposed to contextual fear conditioning 4 weeks later, we observed lower density of mushroom spines in both Arc+ and Arc-branches, as well as lower density of thin spines in Arc-branches in mice that were SD following the initial fear conditioning trial. Our findings indicate that sleep strengthens dendritic spines in neurons that recently encoded fear memory, and sleep deprivation following initial fear learning impairs dendritic spine strengthening initially and following later re-exposure. SD following a traumatic experience thus may be a viable strategy in weakening the strength of contextual memories associated with trauma and PTSD.

Dendritic spines are plastic structures exhibiting a high degree of morphological variability. Certain morphometric parameters, such as volume, positively correlate with the strength of the synapse in which they participate. Memories, too, are subject to change over time and with experiences. In particular, the presence of a reminder of a learning event can trigger the labilization of the memory trace, followed by a re-stabilization process termed reconsolidation. The underlying mechanisms behind the labilization/reconsolidation processes are of great interest, as they are thought of as possible targets for the treatment of post-traumatic stress disorders. Dendritic spines have long been considered the physical sites for memory formation and storage. Our work aimed at studying the long-term spine morphological plasticity associated with labilization/reconsolidation in the dorsal hippocampus, a brain region relevant for the formation of contextual memories. Our results suggest that labilization/reconsolidation does not affect spine density, but rather induces changes in spine morphology. Furthermore, we show that some of these changes are prevented by the inhibition of the transcription factor NF-{kappa}B inhibition. Finally, we found that NF-{kappa}B negative modulation also affects spine morphology in animals that were not exposed to recall but have undergone the training session, suggesting that there may be a late surge of NF-{kappa}B activity resulting from the consolidation itself.

Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.

The actin-binding protein synaptopodin (Synpo) regulates the cytoskeleton and intracellular Ca2+ and is important for long-term potentiation (LTP) and learning. The inconsistent onset age for LTP in mice makes their Synpo knockout (KO) a suboptimal developmental model. Hence, we generated Synpo KO rats using CRISPR-Cas9. Synpo KO rats are viable with reduced body weight and bone length after postnatal days (P)35-P45. Their basal kidney function is normal. 3D reconstruction from electron microscopy reveals the absence of the Synpo-dependent dendritic spine apparatus and cisternal organelles in the axon initial segment (AIS), which may contribute to reduced LTP in the KO rat. Inhibitory synapses in the wild-type AIS appear preferentially clustered near cisternal organelles--a pattern disrupted in the KO, where synapses appear more uniformly distributed. The consistent developmental profile of LTP in the rat makes this KO a robust model to assess Synpo function in development, synaptic plasticity, and behavior. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/690444v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@e588c0org.highwire.dtl.DTLVardef@175302eorg.highwire.dtl.DTLVardef@ae3a81org.highwire.dtl.DTLVardef@82f0a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segments (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. Given the importance of protein trafficking to the OS, less is known about the trafficking of rod synaptic proteins. Furthermore, the subcellular impact of Rho mislocalization on rod synapses (i.e., "spherules") has not been investigated. In this study we used super-resolution and electron microscopies, along with proteomics, to perform a subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP mutant mice. We discovered that mutant P23H-Rho-RFP protein mislocalized in distinct ER aggregations within the spherule cytoplasm, which we confirmed with AAV overexpression. Additionally, we found synaptic protein abundance differences in P23H-Rho-RFP mice. By comparison, Rho mislocalized along the spherule plasma membrane in WT and rd10 mutant rods, in which there was no synaptic protein disruption. Throughout the study, we also identified a network of ER membranes within WT rod presynaptic spherules. Together, our findings indicate that photoreceptor synaptic proteins are sensitive to ER dysregulation. Summary StatementThis study examines the impact of rhodopsin mislocalization on rod photoreceptor synaptic structures and synaptic protein levels using P23H rhodopsin and other retinitis pigmentosa mouse models.

Kalirin-7 (Kal7) is a Rac1/RhoG GEF and multidomain scaffold localized to the postsynaptic density which plays an important role in synaptic plasticity. Behavioral and physiological phenotypes observed in the Kal7 knockout mouse are quite specific: genetics of breeding, growth, strength and coordination are normal; Kal7 knockout animals self-administer cocaine far more than normal mice, show exaggerated locomotor responses to cocaine, but lack changes in dendritic spine morphology seen in wildtype mice; Kal7 knockout mice have depressed surface expression of GluN2B receptor subunits and exhibit marked suppression of long-term potentiation and depression in hippocampus, cerebral cortex, and spinal cord; and Kal7 knockout mice have dramatically blunted perception of pain. To address the underlying cellular and molecular mechanisms which are deranged by loss of Kal7, we administered intracellular blocking peptides to acutely change Kal7 function at the synapse, to determine if plasticity deficits in Kal7-/-mice are the product of developmental processes since conception, or could be detected on a much shorter time scale. We found that specific disruption of the interactions of Kal7 with PSD-95 or GluN2B resulted in significant suppression of long-term potentiation and long-term depression. Biochemical approaches indicated that Kal7 interacted with PSD-95 at multiple sites within Kal7.\n\nGraphical Table of ContentsThe postsynaptic density is an integral player in receiving, interpreting and storing signals transmitted by presynaptic terminals. The correct molecular composition is crucial for successful expression of synaptic plasticity. Key components of the postsynaptic density include ligand-gated ion channels, structural and binding proteins, and multidomain scaffolding plus enzymatic proteins. These studies address whether the multiple components of the synaptic density bind together in a static or slowly adapting molecular complex, or whether critical interactions are fluid on a minute-to-minute basis.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC=\"FIGDIR/small/744508v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (55K):\norg.highwire.dtl.DTLVardef@a65aeborg.highwire.dtl.DTLVardef@19b175borg.highwire.dtl.DTLVardef@11a70ceorg.highwire.dtl.DTLVardef@e403b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Presynaptic modulation is a fundamental process regulating synaptic transmission. Striatal indirect pathway projections originate from A2A- expressing spiny projection neurons (iSPNs), targeting the globus pallidus external segment (GPe) and control the firing of the tonically active GPe neurons via GABA release. It is unclear if and how the presynaptic GPCRs, GABAB and CB1 receptors, modulate iSPN-GPe projections. Here we used an optogenetic platform to study presynaptic Ca2+ and GABAergic transmission at iSPN projections, using a genetic strategy to express the calcium sensor GCaMP6f or the excitatory channelrhodopsin (hChR2) on iSPNs. We found that P/Q-type calcium channels are the primary VGCC-subtype controlling presynaptic calcium and GABA release at iSPN-GPe projections. N-type and L-type VGCCs contribute to GABA release at iSPN-GPe synapses. GABAB receptor activation resulted in a reversible inhibition of presynaptic Ca2+ transients (PreCaTs) and an inhibition of GABAergic transmission at iSPN-GPe synapses. CB1 receptor activation did not inhibit PreCaTs while inhibiting GABAergic transmission at iSPN-GPe projections. CB1 effects on GABAergic transmission persisted in experiments where NaV and KV1 were blocked, indicating a VGCC- and KV1 independent presynaptic mechanism of action of CB1 receptors. Taken together, presynaptic modulation of iSPN-GPe projections by CB1 and GABAB receptors is mediated by distinct mechanisms. Key PointsP/Q-type are the predominant VGCC controlling presynaptic Ca2+ and GABA release on the striatal indirect pathway projections GABAB receptor modulate of iSPN-GPe projections via a VGCC- dependent mechanism CB1 receptors modulate iSPN-GPe projections via a VGCC- independent mechanism

BackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems. New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets. ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level. Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems. ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.

Although many of the proteins of secretory granules have been identified, little is known about their molecular organization and diffusion characteristics. Granule-plasma membrane fusion can only occur when proteins that enable fusion are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may be an important determinant of fusion pore formation and expansion. To address this issue, we measured the mobility of (fluorophore-tagged) vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field ([~]80 nm exponential decay constant) produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, thereby enabling the measurement of diffusion coefficients. The studies revealed that VAMP2, Syt1, and Syt7 are relatively immobile in chromaffin granules membranes with diffusion constants of [≤] 3 x 10-10 cm2/s. Utilizing these diffusion parameters and the known density of VAMP2 and Syt 1 on synaptic vesicles, we estimated the time required for these proteins to arrive at a nascent fusion site to be tens of milliseconds. We propose that the mobilities of secretory granule SNARE and Syt proteins, heretofore unappreciated factors, influence the kinetics of exocytosis and protein discharge. Significance StatementIn eukaryotic cells, secretory vesicles fuse with the plasma membrane to secrete chemical transmitters, hormones and proteins that enable diverse physiological functions including neurotransmission. Fusion proteins need to be assembled at the fusion site in sufficient number in order to enable membrane fusion. However, the diffusion characteristics of fusogenic proteins on secretory vesicles remained unknown. Here we used a novel method not limited by standard optical resolution to measure the diffusion of VAMP2 and synaptotagmins on chromaffin granule membranes. We found they have limited mobility. The time required for these proteins to reach the granule-plasma membrane contact site suggests that their limited mobility likely influences the kinetics of membrane fusion and subsequent fusion pore expansion.

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from P21 to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared to neighbouring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favour synchronous activity in CA1 networks.

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed "homeostatic synaptic plasticity." We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo, where an increase in postsynaptic receptors does not contribute (Wang et al., 2005; Wang et al., 2011). It is unknown whether this form of Rab3A-dependent homeostatic plasticity at the neuromuscular junction shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that the increase in postsynaptic AMPAR levels in wild type cultures was more variable than that of mEPSC amplitudes, which might be explained by a presynaptic contribution, but we cannot rule out variability in the measurement. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that a protein thought to function presynaptically is required for homeostatic synaptic plasticity of quantal size in central neurons.

The ventral hippocampus plays a crucial role in regulating anxiety- and fear-related behaviors. Previously, we demonstrated that diazepam reduces anxiety-like behavior by inhibiting the dentate gyrus and CA3 principal neurons via 2-GABAARs, while inhibition of CA1 pyramidal neurons is necessary to suppress fear-related responses. This study investigated the role of inputs from ventral CA3 (vCA3) and entorhinal cortex to ventral CA1 (vCA1) in anxiety- and fear-like behavior using bidirectional optogenetic modulations. Adult C57BL/6J male and female mice were subjected to bilateral stereotaxic injection of a viral vector expressing channelrhodopsin or halorhodopsin into vCA3 or into layers II-III of lateral entorhinal cortex, followed by bilateral implantation of fiberoptic ferrules into vCA1. After four weeks of recovery, mice were assessed for anxiety-like behavior in the novel open field, elevated plus maze, and Vogel conflict tests, and by contextual and trace fear conditioning for fear. The behavior of the mice was recorded under laser ON and OFF conditions in all experiments. The activation of vCA3 to vCA1 projections (i.e., Schaffer collateral pathway) increased anxiety- and fear-related behaviors, whereas inhibition reduced such behaviors. In contrast, optogenetic activation or inhibition of EC to vCA1 projections (i.e., temporoammonic pathway) had no effect on anxiety-related behavior but positively or negatively modulated fear-related behavior, respectively. These results suggest that while fear-related behavior is modulated by both inputs to vCA1, modulation of anxiety-related behavior is input-specific for the vCA3 to vCA1 projection. In summary, this study offers mechanistic insights into the complex organization of hippocampal circuitry underlying fear and anxiety. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/690696v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a3a0d7org.highwire.dtl.DTLVardef@194b96forg.highwire.dtl.DTLVardef@11a62b7org.highwire.dtl.DTLVardef@1471977_HPS_FORMAT_FIGEXP M_FIG C_FIG

Structural plasticity of dendritic spines is considered to be the basis of synaptic plasticity, learning and memory. Here, we performed ultrastructural analysis of spines undergoing LTP using a novel high throughput correlative light-electron microscopy approach. We found that the PSD displays rapid (< 3 min) reorganization of its nanostructure, including perforation and segmentation. This increased structural complexity is maintained over intermediate and late phases of LTP (20 and 120 min). In a few spines, segmented PSDs are connected to different presynaptic terminals, producing a multi-innervated spine in the intermediate and late phases. In addition, the area of extrasynaptic axon-spine interface (eASI) displayed a pronounced, rapid and sustained increase. Finally, presynaptic vesicle number increased slowly and became significantly higher at late phases of LTP. These rapid ultrastructural changes in PSD and surrounding membrane, together with the slow increase in presynaptic vesicle number, likely support the rapid and sustained increase in synaptic transmission during LTP.

The medial habenula (MHb)-interpeduncular nucleus (IPN) pathway plays an important role in information transferring between the forebrain and the midbrain. The MHb-IPN pathway has been implicated in the regulation of fear behavior and nicotine addiction. The synapses between the ventral MHb and the IPN show a unique property, i.e., an enhancement of synaptic transmission upon activation of GABAB receptors. This GABAB receptor-mediated potentiation of ventral MHb-IPN synaptic transmission has been implicated in regulating fear memory. Although IPN is known to contain parvalbumin (PV) and somatostatin (SST) GABAergic neurons and vesicular glutamate transporter 3 (VGLUT3)-expressing neurons, it is unknown how GABAB receptor activation affects ventral MHb-mediated glutamatergic transmission onto these three subtypes of IPN neurons. Our studies show robust glutamatergic connectivity from ventral MHb to PV and SST neurons in the IPN, while the ventral MHb-mediated glutamatergic transmission in IPN VGLUT3 neurons is weak. Although activation of GABAB receptors produces a robust potentiation of ventral MHb-mediated glutamatergic transmission in PV neurons, we observed a modest effect in IPN SST neurons. Despite the diminished basal synaptic transmission between ventral MHb and IPN VGLUT3 neurons, activation of GABAB receptors causes transient conversion of non-responding ventral MHb synapses into active synapses in some IPN VGLUT3 neurons. Thus, our results show strong ventral MHb connectivity to GABAergic IPN neurons compared to VGLUT3-expressing IPN neurons. Furthermore, GABAB receptor activation produces a differential effect on ventral MHb-mediated glutamatergic transmission onto PV, SST, and VGLUT3 neurons in the IPN.

Visual deprivation by dark rearing in kittens and monkeys delays visual pathway development and prolongs the critical period. In contrast, receptive fields (RFs) in superior colliculus (SC) of Syrian hamsters (Mesocricetus auratus) refine normally with spontaneous activity alone, requiring only brief juvenile visual experience to maintain refined RFs in adulthood (Carrasco et al., 2005). Extending dark rearing past puberty leads to lower GAD and GABA levels due to reduced BDNF-TrkB signaling, resulting in RF re-enlargement (Carrasco et al., 2011; Mudd et al., 2019). Previous studies in kittens and monkeys have reported that dark rearing is associated with changes in both GABA ligand and GABAA receptor levels. Given the reduced GABA levels in SC of dark reared adult hamsters, we asked if dark rearing also causes changes in GABAA receptor levels. We examined expression of GABAA receptor subunits, their anchoring protein gephyrin, and the cation-chloride co-transporters KCC2 and NKCC1 in dark reared hamsters. Surprisingly, we found that dark rearing from birth until puberty had no effect on the levels of any of these postsynaptic elements, revealing a new form of maladaptive, presynaptic only inhibitory plasticity in which, rather than extending the critical period as seen in kittens and monkeys, hamster receptive fields refine normally and then lose refinement in adulthood. These results suggest that attempts to increase plasticity in adulthood for rehabilitation or recovery from injury should consider the possibility of unintended negative consequences. In addition, our results demonstrate the interesting finding that changes in neurotransmitter levels are not necessarily coordinated with changes in postsynaptic components.

Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system, most presynaptic terminals are CaV2.1 dominant with a developmental reduction in CaV2.2 and CaV2.3 levels, and CaV2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV2 subtype levels are largely unsolved. Since the CaV2 1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV2.1 1 subunits containing swapped motifs with the CaV2.2 and CaV2.3 1 subunit on a CaV2.1/CaV2.2 null background at the calyx of Held presynaptic terminal. We found that expression of CaV2.1 1 subunit chimeras containing the CaV2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV2.1 1 subunit. However, the Ca2+ current sensitivity to the CaV2.1 blocker Agatoxin-IVA, was the same between the chimeras and the CaV2.1 1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV2.1 loop II-III and CT do not individually regulate CaV2.1 preference, but these motifs control CaV2.1 levels and the CaV2.3 CT contains motifs that negatively regulate presynaptic CaV2.3 levels. We propose that the motifs controlling presynaptic CaV2.1 preference are distinct from those regulating CaV2.1 levels and may act synergistically to impact pathways regulating CaV2.1 preference and abundance. Key points summaryO_LIPresynaptic CaV2 subtype abundance regulates neuronal circuit properties, however the mechanisms regulating presynaptic CaV2 subtype abundance and preference remains enigmatic. C_LIO_LIThe CaV 1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. C_LIO_LIThe CaV2.1 1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV2.1 abundance but do not regulate preference. C_LIO_LIThe CaV2.3 1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV2 subtype abundance but not preference while the CaV2.2 1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV2 subtype abundance or preference. C_LIO_LIThe CaV2 1 subunit motifs determining the presynaptic CaV2 preference are distinct from abundance. C_LI

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system, with inhibitory synaptic transmission playing an important role. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT) to selectively label GABAergic (i.e., inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single inhibitory vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that inhibitory synaptic vesicles traveled a short distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed a close correlation between the release probability of inhibitory vesicles and both the proximity to their fusion site and the total travel length. Finally, we found that inhibitory vesicles have a higher prevalence of kiss-and-run fusion compared than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and fusion properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated balance between excitation and inhibition in the central nervous system. SignificanceDespite playing an important role in maintaining brain function, the dynamics of inhibitory synaptic vesicles are poorly understood. Here, we tracked the three-dimensional position of single inhibitory vesicles up to the moment of exocytosis in real time by loading single inhibitory vesicle with QDs-conjugated to antibodies against the luminal domain of the vesicular GABA transporter (VGAT). We found that inhibitory synaptic vesicles have a smaller total travel length before fusion, a shorter fusion time, and a higher prevalence of kiss-and-run than synaptotagmin-1-lableled vesicles. Our findings provide the first evidence that inhibitory vesicles have a unique set of dynamics and exocytosis properties to support rapid inhibitory synaptic transmission.