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The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites. [ABSTRACT FROM AUTHOR]

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin- 2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABAA receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9- dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity. [ABSTRACT FROM AUTHOR]

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that have been implicated in a plethora of neuronal processes. Nevertheless, their role in regulating brain activity in the context of sleep has so far received little attention. To test their involvement, we deleted mature miRNAs in post-mitotic neurons at two developmental ages, i.e., in early adulthood using conditional Dicer knockout (cKO) mice and in adult mice using an inducible conditional Dicer cKO (icKO) line. In both models, electroencephalographic (EEG) activity was affected and the response to sleep deprivation (SD) altered; while the rapid-eye- movement sleep (REMS) rebound was compromised in both, the increase in EEG delta (1 to 4 Hz) power during non-REMS (NREMS) was smaller in cKO mice and larger in icKO mice compared to controls. We subsequently investigated the effects of SD on the forebrain miRNA transcriptome and found that the expression of 48 miRNAs was affected, and in particular that of the activity-dependent miR-709. In vivo inhibition of miR-709 in the brain increased EEG power during NREMS in the slow-delta (0.75 to 1.75 Hz) range, particularly after periods of prolonged wakefulness. Transcriptome analysis of primary cortical neurons in vitro revealed that miR-709 regulates genes involved in glutamatergic neurotransmission. A subset of these genes was also affected in the cortices of sleep-deprived, miR-709- inhibited mice. Our data implicate miRNAs in the regulation of EEG activity and indicate that miR-709 links neuronal activity during wakefulness to brain synchrony during sleep through the regulation of glutamatergic signaling. [ABSTRACT FROM AUTHOR]

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels. [ABSTRACT FROM AUTHOR]

Resting- or baseline-state low-frequency (0.01-0.2 Hz) brain activity is observed in fMRI, EEG, and local field potential recordings. These fluctuations were found to be correlated across brain regions and are thought to reflect neuronal activity fluctuations between functionally connected areas of the brain. However, the origin of these infra-slow resting-state fluctuations remains unknown. Here, using a detailed computational model of the brain network, we show that spontaneous infra-slow (

The Ca 2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C 2 B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca 2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca 2+ -evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca 2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al. , bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using nonnegative tensor factorization and conventional state modeling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at the calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but are determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming, and fusion. It suggests a mechanism for activity-induced redistribution of synaptic efficacy. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca2+ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca2+ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca2+ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca2+ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca2+ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca2+ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission. [ABSTRACT FROM AUTHOR]

The amino-terminal domain (ATD) of AMPA receptors (AMPARs) accounts for approximately 50% of the protein, yet its functional role, if any, remains a mystery. We have discovered that the translocation of surface GluA1, but not GluA2, AMPAR subunits to the synapse requires the ATD. GluA1A2 heteromers in which the ATD of GluA1 is absent fail to translocate, establishing a critical role of the ATD of GluA1. Inserting GFP into the ATD interferes with the constitutive synaptic trafficking of GluA1, but not GluA2, mimicking the deletion of the ATD. Remarkably, long-term potentiation (LTP) can override the masking effect of the GFP tag. GluA1, but not GluA2, lacking the ATD fails to show LTP. These findings uncover a role for the ATD in subunit-specific synaptic trafficking of AMPARs, both constitutively and during plasticity. How LTP, induced postsynaptically, engages these extracellular trafficking motifs and what specific cleft proteins participate in the process remain to be elucidated.

The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.

The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation-dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations.

Lysergic acid diethylamide (LSD) is the prototypical psychedelic drug, but its effects on the human brain have never been studied before with modern neuroimaging. Here, three complementary neuroimaging techniques: arterial spin labeling (ASL), blood oxygen level-dependent (BOLD) measures, and magnetoencephalography (MEG), implemented during resting state conditions, revealed marked changes in brain activity after LSD that correlated strongly with its characteristic psychological effects. Increased visual cortex cerebral blood flow (CBF), decreased visual cortex alpha power, and a greatly expanded primary visual cortex (V1) functional connectivity profile correlated strongly with ratings of visual hallucinations, implying that intrinsic brain activity exerts greater influence on visual processing in the psychedelic state, thereby defining its hallucinatory quality. LSD's marked effects on the visual cortex did not significantly correlate with the drug's other characteristic effects on consciousness, however. Rather, decreased connectivity between the parahippocampus and retrosplenial cortex (RSC) correlated strongly with ratings of "ego-dissolution" and "altered meaning," implying the importance of this particular circuit for the maintenance of "self" or "ego" and its processing of "meaning." Strong relationships were also found between the different imaging metrics, enabling firmer inferences to be made about their functional significance. This uniquely comprehensive examination of the LSD state represents an important advance in scientific research with psychedelic drugs at a time of growing interest in their scientific and therapeutic value. The present results contribute important new insights into the characteristic hallucinatory and consciousness-altering properties of psychedelics that inform on how they can model certain pathological states and potentially treat others.

How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1-C2B-MUN-C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to "primed" ready-release vesicles in which SNAREpins are clamped and ready to release (state 3). [ABSTRACT FROM AUTHOR]

Homeostatic plasticity of intrinsic excitability goes hand in hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging, and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2 to 3 d induces an up-regulation of both synaptic transmission at CA3-CA3 connections and intrinsic excitability of CA3 pyramidal neurons. Intrinsic plasticity was found to be mediated by a reduction of Kv1.1 channel density at the axon initial segment. In activity-deprived circuits, CA3-CA3 synapses were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a reduction in presynaptic Kv1.1 function. Further support for the down-regulation of axonal Kv1.1 channels in activity-deprived neurons was the broadening of action potentials measured in the axon. We conclude that regulation of the axonal Kv1.1 channel constitutes a major mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission. [ABSTRACT FROM AUTHOR]

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

The functional interaction between the brain's two hemispheres includes a unique set of connections between corresponding regions in opposite hemispheres (i.e., homotopic regions) that are consistently reported to be exceptionally strong compared with other interhemispheric (i.e., heterotopic) connections. The strength of homotopic functional connectivity (FC) is thought to be mediated by the regions' shared functional roles and their structural connectivity. Recently, homotopic FC was reported to be stable over time despite the presence of dynamic FC across both intrahemispheric and heterotopic connections. Here we build on this work by considering whether homotopic FC is also stable across conditions. We additionally test the hypothesis that strong and stable homotopic FC is supported by the underlying structural connectivity. Consistent with previous findings, interhemispheric FC between homotopic regions were significantly stronger in both humans and macaques. Across conditions, homotopic FC was most resistant to change and therefore was more stable than heterotopic or intrahemispheric connections. Across time, homotopic FC had significantly greater temporal stability than other types of connections. Temporal stability of homotopic FC was facilitated by direct anatomical projections. Importantly, temporal stability varied with the change in conductive properties of callosal axons along the anterior-posterior axis. Taken together, these findings suggest a notable role for the corpus callosum in maintaining stable functional communication between hemispheres.

Transmembrane AMPA receptor regulatory proteins (TARPs) play an essential role in excitatory synaptic transmission throughout the central nervous system (CNS) and exhibit subtype-specific effects on AMPA receptor (AMPAR) trafficking, gating, and pharmacology. The function of TARPs has largely been determined through work on canonical type I TARPs such as stargazin (TARP γ-2), absent in the ataxic stargazer mouse. Little is known about the function of atypical type II TARPs, such as TARP γ-7, which exhibits variable effects on AMPAR function. Because γ-2 and γ-7 are both strongly expressed in multiple cell types in the cerebellum, we examined the relative contribution of γ-2 and γ-7 to both synaptic transmission in the cerebellum and motor behavior by using both the stargazer mouse and a γ-7 knockout (KO) mouse. We found that the loss of γ-7 alone had little effect on climbing fiber (cf) responses in Purkinje neurons (PCs), yet the additional loss of γ-2 all but abolished cf responses. In contrast, γ-7 failed to make a significant contribution to excitatory transmission in stellate cells and granule cells. In addition, we generated a PC-specific deletion of γ-2, with and without γ-7 KO background, to examine the relative contribution of γ-2 and γ-7 to PC-dependent motor behavior. Selective deletion of γ-2 in PCs had little effect on motor behavior, yet the additional loss of γ-7 resulted in a severe disruption in motor behavior. Thus, γ-7 is capable of supporting a component of excitatory transmission in PCs, sufficient to maintain essentially normal motor behavior, in the absence of γ-2.

The dementia of Alzheimer's disease (AD) results primarily from degeneration of neurons that furnish glutamatergic corticocortical connections that subserve cognition. Although neuron death is minimal in the absence of AD, age-related cognitive decline does occur in animals as well as humans, and it decreases quality of life for elderly people. Age-related cognitive decline has been linked to synapse loss and/or alterations of synaptic proteins that impair function in regions such as the hippocampus and prefrontal cortex. These synaptic alterations are likely reversible, such that maintenance of synaptic health in the face of aging is a critically important therapeutic goal. Here, we show that riluzole can protect against some of the synaptic alterations in hippocampus that are linked to age-related memory loss in rats. Riluzole increases glutamate uptake through glial transporters and is thought to decrease glutamate spillover to extrasynaptic NMDA receptors while increasing synaptic glutamatergic activity. Treated aged rats were protected against age-related cognitive decline displayed in nontreated aged animals. Memory performance correlated with density of thin spines on apical dendrites in CA1, although not with mushroom spines. Furthermore, riluzole-treated rats had an increase in clustering of thin spines that correlated with memory performance and was specific to the apical, but not the basilar, dendrites of CA1. Clustering of synaptic inputs is thought to allow nonlinear summation of synaptic strength. These findings further elucidate neuroplastic changes in glutamatergic circuits with aging and advance therapeutic development to prevent and treat age-related cognitive decline.

Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that "long thin" but not "mushroom" spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

Precise spike times carry information and are important for synaptic plasticity. Synchronizing oscillations such as gamma bursts could coordinate spike times, thus regulating information transmission in the cortex. Oscillations are driven by inhibitory neurons and are modulated by sensory stimuli and behavioral states. How their power and frequency are regulated is an open question. Using a model cortical circuit, we propose a regulatory mechanism that depends on the activity balance of monosynaptic and disynaptic pathways to inhibitory neurons: Monosynaptic input causes more powerful oscillations whereas disynaptic input increases the frequency of oscillations. The balance of stimulation to the two pathways modulates the overall distribution of spikes, with stronger disynaptic stimulation (e.g., preferred stimuli inside visual receptive fields) producing high firing rates and weak oscillations; in contrast, stronger monosynaptic stimulation (e.g., suppressive contextual stimulation from outside visual receptive fields) generates low firing rates and strong oscillatory regulation of spike timing, as observed in alert cortex processing complex natural stimuli. By accounting for otherwise paradoxical experimental findings, our results demonstrate how the frequency and power of oscillations, and hence spike times, can be modulated by both sensory input and behavioral context, with powerful oscillations signifying a cortical state under inhibitory control in which spikes are sparse and spike timing is precise.

Humans and nonhuman primates are vulnerable to age- and menopause-related decline in working memory, a cognitive function reliant on the energy-demanding recurrent excitation of neurons within Brodmann's Area 46 of the dorsolateral prefrontal cortex (dlPFC). Here, we tested the hypothesis that the number and morphology (straight, curved, or donut-shaped) of mitochondria in dlPFC presynaptic boutons are altered with aging and menopause in rhesus monkeys (Macaca mulatta) and that these metrics correlate with delayed response (DR) accuracy, a well-characterized measure of dlPFC-dependent working memory. Although presynaptic bouton density or size was not significantly different across groups distinguished by age or menses status, DR accuracy correlated positively with the number of total and straight mitochondria per dlPFC bouton. In contrast, DR accuracy correlated inversely with the frequency of boutons containing donut-shaped mitochondria, which exhibited smaller active zone areas and fewer docked synaptic vesicles than those with straight or curved mitochondria. We then examined the effects of estrogen administration to test whether a treatment known to improve working memory influences mitochondrial morphology. Aged ovariectomized monkeys treated with vehicle displayed significant working memory impairment and a concomitant 44% increase in presynaptic donut-shaped mitochondria, both of which were reversed with cyclic estradiol treatment. Together, our data suggest that hormone replacement therapy may benefit cognitive aging, in part by promoting mitochondrial and synaptic health in the dlPFC.

Chronic changes in excitability and activity can induce homeostatic plasticity. These perturbations may be associated with neurological disorders, particularly those involving loss or dysfunction of GABA interneurons. In distal-less homeobox 1 (Dlx1(-/-)) mice with late-onset interneuron loss and reduced inhibition, we observed both excitatory synaptic silencing and decreased intrinsic neuronal excitability. These homeostatic changes do not fully restore normal circuit function, because synaptic silencing results in enhanced potential for long-term potentiation and abnormal gamma oscillations. Transplanting medial ganglionic eminence interneuron progenitors to introduce new GABAergic interneurons, we demonstrate restoration of hippocampal function. Specifically, miniature excitatory postsynaptic currents, input resistance, hippocampal long-term potentiation, and gamma oscillations are all normalized. Thus, in vivo homeostatic plasticity is a highly dynamic and bidirectional process that responds to changes in inhibition.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated frominduced pluripotent stemcells that were derived fromschizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts. [ABSTRACT FROM AUTHOR]

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca2+ uncaging experiments did not reveal changes in the intracellular Ca2+ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca2+ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca2+ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca2+ is explained by the simple mechanism of rapid Ca2+ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca2+ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca2+ channels. [ABSTRACT FROM AUTHOR]

Identifying molecular mediators of neural circuit development and/or function that contribute to circuit dysfunction when aberrantly reengaged in neurological disorders is of high importance. The role of the TWEAK/Fn14 pathway, which was recently reported to be a microglial/neuronal axis mediating synaptic refinement in experience-dependent visual development, has not been explored in synaptic function within the mature central nervous system. By combining electrophysiological and phosphoproteomic approaches, we show that TWEAK acutely dampens basal synaptic transmission and plasticity through neuronal Fn14 and impacts the phosphorylation state of pre- and postsynaptic proteins in adult mouse hippocampal slices. Importantly, this is relevant in two models featuring synaptic deficits. Blocking TWEAK/Fn14 signaling augments synaptic function in hippocampal slices from amyloid-beta-overexpressing mice. After stroke, genetic or pharmacological inhibition of TWEAK/Fn14 signaling augments basal synaptic transmission and normalizes plasticity. Our data support a glial/neuronal axis that critically modifies synaptic physiology and pathophysiology in different contexts in the mature brain and may be a therapeutic target for improving neurophysiological outcomes. [ABSTRACT FROM AUTHOR]

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin-2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABA A receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9-dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity., Competing Interests: Competing interests statement:The authors declare no competing interest.

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters (NCs) whose precise alignment across the cleft in a transsynaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses-those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses, presynaptic Munc13-1 and postsynaptic PSD-95 both form NCs that demonstrate alignment, underscoring synaptic nanostructure and the transsynaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell was also represented in Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of transsynaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses., Competing Interests: Competing interests statement:The authors declare no competing interest.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity., Competing Interests: Competing interests statement:The authors declare no competing interest.

AMPA receptors (AMPARs) mediate the majority of fast excitatory neurotransmission, and their density at postsynaptic sites determines synaptic strength. Ubiquitination is a posttranslational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167. Predominantly lysosomal, a subpopulation of RNF167 is located on the surface of cultured neurons. Using a RING mutant RNF167 or a specific shRNA to eliminate endogenous RNF167, we demonstrate that AMPAR surface expression increases in hippocampal neurons with disrupted RNF167 activity and that RNF167 is involved in activity-dependent ubiquitination of AMPARs. In addition, RNF167 regulates synaptic AMPAR currents, whereas synaptic NMDAR currents are unaffected. Therefore, our study identifies RNF167 as a selective regulator of AMPAR-mediated neurotransmission and expands our understanding of how ubiquitination dynamically regulates excitatory synapses.

The nanoscale co-organization of neurotransmitter receptors facing presynaptic release sites is a fundamental determinant of their coactivation and of synaptic physiology. At excitatory synapses, how endogenous AMPARs, NMDARs, and mGluRs are co-organized inside the synapse and their respective activation during glutamate release are still unclear. Combining single-molecule superresolution microscopy, electrophysiology, and modeling, we determined the average quantity of each glutamate receptor type, their nanoscale organization, and their respective activation. We observed that NMDARs form a unique cluster mainly at the center of the PSD, while AMPARs segregate in clusters surrounding the NMDARs. mGluR5 presents a different organization and is homogenously dispersed at the synaptic surface. From these results, we build a model predicting the synaptic transmission properties of a unitary synapse, allowing better understanding of synaptic physiology. [ABSTRACT FROM AUTHOR]

Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca2+ activation mechanism has been well-characterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilizes Syt1 oligomerization. Using a combination of fluorescence imaging and electrophysiology in neocortical synapses, we show that Syt1F349A is more efficient than wild-type Syt1 (Syt1WT) in triggering synchronous transmitter release but fails to clamp spontaneous and synaptotagmin 7 (Syt7)-mediated asynchronous release components both in rescue (Syt1-/- knockout background) and dominant-interference (Syt1+/+ background) conditions. Thus, we conclude that Ca2+-sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release. [ABSTRACT FROM AUTHOR]

Animal behavior is remarkably robust despite constant changes in neural activity. Homeostatic plasticity stabilizes central nervous system (CNS) function on time scales of hours to days. If and how CNS function is stabilized on more rapid time scales remains unknown. Here, we discovered that mossy fiber synapses in the mouse cerebellum homeostatically control synaptic efficacy within minutes after pharmacological glutamate receptor impairment. This rapid form of homeostatic plasticity is expressed presynaptically. We show that modulations of readily releasable vesicle pool size and release probability normalize synaptic strength in a hierarchical fashion upon acute pharmacological and prolonged genetic receptor perturbation. Presynaptic membrane capacitance measurements directly demonstrate regulation of vesicle pool size upon receptor impairment. Moreover, presynaptic voltage-clamp analysis revealed increased Ca2+-current density under specific experimental conditions. Thus, homeostatic modulation of presynaptic exocytosis through specific mechanisms stabilizes synaptic transmission in a CNS circuit on time scales ranging from minutes to months. Rapid presynaptic homeostatic plasticity may ensure stable neural circuit function in light of rapid activity-dependent plasticity. [ABSTRACT FROM AUTHOR]

Aging drives a progressive decline in cognition and decreases synapse numbers and synaptic function in the brain, thereby increasing the risk for neurodegenerative disease. Pioneering studies showed that introduction of blood from young mice into aged mice reversed age-associated cognitive impairments and increased synaptic connectivity in brain, suggesting that young blood contains specific factors that remediate age-associated decreases in brain function. However, whether such factors in blood from young animals act directly on neurons to enhance synaptic connectivity, or whether they act by an indirect mechanism remains unknown. Moreover, which factors in young blood mediate cognitive improvements in old mice is incompletely understood. Here, we show that serum extracted from the blood of young but not old mice, when applied to neurons transdifferentiated from human embryonic stem cells, directly increased dendritic arborization, augmented synapse numbers, doubled dendritic spine-like structures, and elevated synaptic Nmethyl- D-aspartate (NMDA) receptors, thereby increasing synaptic connectivity. Mass spectrometry revealed that thrombospondin-4 (THBS4) and SPARC-like protein 1 (SPARCL1) were enriched in serum from young mice. Strikingly, recombinant THBS4 and SPARCL1 both increased dendritic arborization and doubled synapse numbers in cultured neurons. In addition, SPARCL1 but not THBS4 tripled NMDA receptor-mediated synaptic responses. Thus, at least two proteins enriched in young blood, THBS4 and SPARCL1, directly act on neurons as synaptogenic factors. These proteins may represent rejuvenation factors that enhance synaptic connectivity by increasing dendritic arborization, synapse formation, and synaptic transmission. [ABSTRACT FROM AUTHOR]

Somatostatin-expressing interneurons (SOMIs) in the mouse dentate gyrus (DG) receive feedforward excitation from granule cell (GC) mossy fiber (MF) synapses and provide feedback lateral inhibition onto GC dendrites to support environment representation in the DG network. Although this microcircuitry has been implicated in memory formation, little is known about activity-dependent plastic changes at MF-SOMI synapses and their influence on behavior. Here, we report that the metabotropic glutamate receptor 1α (mGluR1α) is required for the induction of associative long-term potentiation (LTP) at MF-SOMI synapses. Pharmacological block of mGluR1α, but not mGluR5, prevented synaptic weight changes. LTP at MF-SOMI synapses was postsynaptically induced, required increased intracellular Ca 2+ , involved G-protein-mediated and Ca 2+ -dependent (extracellular signal-regulated kinase) ERK1/2 pathways, and the activation of NMDA receptors. Specific knockdown of mGluR1α in DG-SOMIs by small hairpin RNA expression prevented MF-SOMI LTP, reduced SOMI recruitment, and impaired object location memory. Thus, postsynaptic mGluR1α-mediated MF-plasticity at SOMI input synapses critically supports DG-dependent mnemonic functions., Competing Interests: Competing interests statement:The authors declare no competing interest.

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons. [ABSTRACT FROM AUTHOR]

Leucine-rich repeat transmembrane (LRRTM) proteins are synaptic cell adhesion molecules that influence synapse formation and function. They are genetically associated with neuropsychiatric disorders, and via their synaptic actions likely regulate the establishment and function of neural circuits in the mammalian brain. Here, we take advantage of the generation of a LRRTM1 and LRRTM2 double conditional knockout mouse (LRRTM1,2 cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of LRRTM1,2 in vivo in CA1 neurons using Cre recombinase-expressing lentiviruses dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins prevented rescue of LTP. In contrast to previous work using shRNA-mediated knockdown of LRRTM1,2, KO of these proteins at mature synapses also caused a decrease in AMPA receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP. [ABSTRACT FROM AUTHOR]

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ~10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/ depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons. [ABSTRACT FROM AUTHOR]

During a critical developmental period, cochlear inner hair cells (IHCs) exhibit sensory-independent activity, featuring action potentials in which Ca2+ ions play a fundamental role in driving both spiking and glutamate release onto synapses with afferent auditory neurons. This spontaneous activity is controlled by a cholinergic input to the IHC, activating a specialized nicotinic receptor with high Ca2+ permeability, and coupled to the activation of hyperpolarizing SK channels. The mechanisms underlying distinct excitatory and inhibitory Ca2+ roles within a small, compact IHC are unknown. Making use of Ca2+ imaging, afferent auditory bouton recordings, and electron microscopy, the present work shows that unusually high intracellular Ca2+ buffering and "subsynaptic" cisterns provide efficient compartmentalization and tight control of cholinergic Ca2+ signals. Thus, synaptic efferent Ca2+ spillover and cross-talk are prevented, and the cholinergic input preserves its inhibitory signature to ensure normal development of the auditory system. [ABSTRACT FROM AUTHOR]

Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+ channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+ channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+ buffer EGTA. These findings suggest that RBPs tether L-type Ca2+ channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits. [ABSTRACT FROM AUTHOR]

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. [ABSTRACT FROM AUTHOR]

Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications. [ABSTRACT FROM AUTHOR]