Cationic peptides erase memories by removing synaptic AMPA receptors through endophilin-mediated endocytosis.

Administration of the Zeta Inhibitory Peptide (ZIP) interferes with memory maintenance and long-term potentiation (LTP). However, mice lacking its putative target, the protein kinase PKMζ, exhibit normal learning and memory as well as LTP, making ZIP's mechanism unclear. Here, we show that ZIP disrupts LTP by removing surface AMPA receptors through its cationic charge alone. This effect was fully blocked by drugs that block macropinocytosis and is dependent on endophilin A2 (endoA2)-mediated endocytosis. ZIP and other cationic peptides selectively removed newly inserted AMPAR nanoclusters, providing a mechanism by which these peptides erase memories without effects on basal synaptic function. Lastly, cationic peptides can be administered locally and/or systemically and can be combined with local microinjection of macropinocytosis inhibitors to modulate memories on local and brain-wide scales. Our findings have critical implications for an entire field of memory mechanisms and highlight a previously unappreciated mechanism by which memories can be lost.

Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses.

Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses.

Neurexin-3 subsynaptic densities are spatially distinct from Neurexin-1 and essential for excitatory synapse nanoscale organization in the hippocampus.

Proteins critical for synaptic transmission are non-uniformly distributed and assembled into regions of high density called subsynaptic densities (SSDs) that transsynaptically align in nanocolumns. Neurexin-1 and neurexin-3 are essential presynaptic adhesion molecules that non-redundantly control NMDAR- and AMPAR-mediated synaptic transmission, respectively, via transsynaptic interactions with distinct postsynaptic ligands. Despite their functional relevance, fundamental questions regarding the nanoscale properties of individual neurexins, their influence on the subsynaptic organization of excitatory synapses and the mechanisms controlling how individual neurexins engage in precise transsynaptic interactions are unknown. Using Double Helix 3D dSTORM and neurexin mouse models, we identify neurexin-3 as a critical presynaptic adhesion molecule that regulates excitatory synapse nano-organization in hippocampus. Furthermore, endogenous neurexin-1 and neurexin-3 form discrete and non-overlapping SSDs that are enriched opposite their postsynaptic ligands. Thus, the nanoscale organization of neurexin-1 and neurexin-3 may explain how individual neurexins signal in parallel to govern different synaptic properties.

LTP induction by structural rather than enzymatic functions of CaMKII.

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.

Ventral Subiculum Inputs to Nucleus Accumbens Medial Shell Preferentially Innervate D2R Medium Spiny Neurons and Contain Calcium Permeable AMPARs.

Ventral subiculum (vSUB) is the major output region of ventral hippocampus (vHIPP) and sends major projections to nucleus accumbens medial shell (NAcMS). Hyperactivity of the vSUB-NAcMS circuit is associated with substance use disorders and the modulation of vSUB activity alters drug seeking and drug reinstatement behavior in rodents. However, to the best of our knowledge, the cell type-specific connectivity and synaptic transmission properties of the vSUB-NAcMS circuit have never been directly examined. Instead, previous functional studies have focused on total ventral hippocampal (vHIPP) output to NAcMS without distinguishing vSUB from other subregions of vHIPP, including ventral CA1 (vCA1). Using ex vivo electrophysiology, we systematically characterized the vSUB-NAcMS circuit with cell type- and synapse-specific resolution in male and female mice and found that vSUB output to dopamine receptor type-1 (D1R) and type-2 (D2R) expressing medium spiny neurons (MSNs) displays a functional connectivity bias for D2R MSNs. Furthermore, we found that vSUB-D1R and vSUB-D2R MSN synapses contain calcium-permeable AMPA receptors in drug-naive mice. Finally, we find that, distinct from other glutamatergic inputs, cocaine exposure selectively induces plasticity at vSUB-D2R synapses. Importantly, we directly compared vSUB and vCA1 output to NAcMS and found that vSUB synapses are functionally distinct and that vCA1 output recapitulated the synaptic properties previously ascribed to vHIPP. Our work highlights the need to consider the contributions of individual subregions of vHIPP to substance use disorders and represents an important first step toward understanding how the vSUB-NAcMS circuit contributes to the etiologies that underlie substance use disorders.SIGNIFICANCE STATEMENT Inputs to nucleus accumbens (NAc) dopamine receptor type 1 (D1R) and D2R medium spiny neurons (MSNs) are critically involved in reward seeking behavior. Ventral subiculum (vSUB) provides robust synaptic input to nucleus accumbens medial shell (NAcMS) and activity of this circuit is linked to substance use disorders. Despite the importance of the vSUB to nucleus accumbens circuit, the functional connectivity and synaptic transmission properties have not been tested. Here, we systematically interrogated these properties and found that basal connectivity and drug-induced plasticity are biased for D2R medium spiny neurons. Overall, we demonstrate that this circuit is distinct from synaptic inputs from other brain regions, which helps to explain how vSUB dysfunction contributes to the etiologies that underlie substance use disorders.

Neurexins and their ligands at inhibitory synapses.

Since the discovery of neurexins (Nrxns) as essential and evolutionarily conserved synaptic adhesion molecules, focus has largely centered on their functional contributions to glutamatergic synapses. Recently, significant advances to our understanding of neurexin function at GABAergic synapses have revealed that neurexins can play pleiotropic roles in regulating inhibitory synapse maintenance and function in a brain-region and synapse-specific manner. GABAergic neurons are incredibly diverse, exhibiting distinct synaptic properties, sites of innervation, neuromodulation, and plasticity. Different classes of GABAergic neurons often express distinct repertoires of Nrxn isoforms that exhibit differential alternative exon usage. Further, Nrxn ligands can be differentially expressed and can display synapse-specific localization patterns, which may contribute to the formation of a complex trans-synaptic molecular code that establishes the properties of inhibitory synapse function and properties of local circuitry. In this review, we will discuss how Nrxns and their ligands sculpt synaptic inhibition in a brain-region, cell-type and synapse-specific manner.

Neurexin-3 defines synapse- and sex-dependent diversity of GABAergic inhibition in ventral subiculum.

Ventral subiculum (vSUB) is integral to the regulation of stress and reward; however, the intrinsic connectivity and synaptic properties of the inhibitory local circuit are poorly understood. Neurexin-3 (Nrxn3) is highly expressed in hippocampal inhibitory neurons, but its function at inhibitory synapses has remained elusive. Using slice electrophysiology, imaging, and single-cell RNA sequencing, we identify multiple roles for Nrxn3 at GABAergic parvalbumin (PV) interneuron synapses made onto vSUB regular-spiking (RS) and burst-spiking (BS) principal neurons. Surprisingly, we find that intrinsic connectivity of vSUB and synaptic function of Nrxn3 in vSUB are sexually dimorphic. We reveal that PVs make preferential contact with RS neurons in male mice, but BS neurons in female mice. Furthermore, we determine that despite comparable Nrxn3 isoform expression in male and female PV neurons, Nrxn3 knockout impairs synapse density, postsynaptic strength, and inhibitory postsynaptic current (IPSC) amplitude at PV-RS synapses in males, but enhances presynaptic release and IPSC amplitude in females.

Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.

Loss of nigral excitation of cholinergic interneurons contributes to parkinsonian motor impairments.

Progressive loss of dopamine inputs in Parkinson's disease leads to imbalances in coordinated signaling of dopamine and acetylcholine (ACh) in the striatum, which is thought to contribute to parkinsonian motor symptoms. As reciprocal interactions between dopamine inputs and cholinergic interneurons (ChIs) control striatal dopamine and ACh transmission, we examined how partial dopamine depletion in an early-stage mouse model for Parkinson's disease alters nigral regulation of cholinergic activity. We found region-specific alterations in how remaining dopamine inputs regulate cholinergic excitability that differ between the dorsomedial (DMS) and dorsolateral (DLS) striatum. Specifically, we found that dopamine depletion downregulates metabotropic glutamate receptors (mGluR1) on DLS ChIs at synapses where dopamine inputs co-release glutamate, abolishing the ability of dopamine inputs to drive burst firing. This loss underlies parkinsonian motor impairments, as viral rescue of mGluR1 signaling in DLS ChIs was sufficient to restore circuit function and attenuate motor deficits in early-stage parkinsonian mice.

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System.

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Modeling a Neurexin-3α Human Mutation in Mouse Neurons Identifies a Novel Role in the Regulation of Transsynaptic Signaling and Neurotransmitter Release at Excitatory Synapses.

Presynaptic α-neurexins are highly expressed and more frequently linked to neuropsychiatric and neurodevelopmental disorders than ß-neurexins. However, how extracellular sequences specific to α-neurexins enable synaptic transmission is poorly understood. We identified a mutation in an extracellular region of neurexin-3α (A687T), located in a region conserved among α-neurexins and throughout vertebrate evolution, in a patient diagnosed with profound intellectual disability and epilepsy. We systematically interrogated this mutation using a knockdown-replacement approach, and discovered that the A687T mutation enhanced presynaptic morphology and increased two critical presynaptic parameters: (1) presynaptic release probability, and (2) the size of the readily releasable pool exclusively at excitatory synapses in mixed sex primary mouse hippocampal cultures. Introduction of the mutation in vivo and subsequent analysis in ex vivo brain slices made from male and female mice revealed a significant increase in excitatory presynaptic neurotransmission that occluded presynaptic but not postsynaptic LTP. Mechanistically, neurexin-3αA687T enhanced binding to LRRTM2 without altering binding to postsynaptic neuroligin-1. Thus, neurexin-3αA687T unexpectedly produced the first neurexin presynaptic gain-of-function phenotype and revealed unanticipated novel insights into how α-neurexin extracellular sequences govern both transsynaptic adhesion and presynaptic neurotransmitter release.SIGNIFICANCE STATEMENT Despite decades of scientific scrutiny, how precise α-neurexin extracellular sequences control synapse function remains enigmatic. One largely unpursued avenue to identify the role of precise extracellular sequences is the interrogation of naturally occurring missense mutations. Here, we identified a neurexin-3α missense mutation in a compound heterozygous patient diagnosed with profound intellectual disability and epilepsy and systematically interrogated this mutation. Using in vitro and in vivo molecular replacement, electrophysiology, electron microscopy, and structure-function analyses, we reveal a novel role for neurexin-3α, unanticipated based on α-neurexin knock-out models, in controlling presynaptic morphology and neurotransmitter release at excitatory synapses. Our findings represent the first neurexin gain-of-function phenotype and provide new fundamentally important insight into the synaptic biology of α-neurexins.

Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent.

A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses.

AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing, we show that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated, but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1ß and neurexin-3ß SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses.

A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission.

Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.

AKAP150 Palmitoylation Regulates Synaptic Incorporation of Ca2+-Permeable AMPA Receptors to Control LTP.

Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs) containing GluA1 but lacking GluA2 subunits contribute to multiple forms of synaptic plasticity, including long-term potentiation (LTP), but mechanisms regulating CP-AMPARs are poorly understood. A-kinase anchoring protein (AKAP) 150 scaffolds kinases and phosphatases to regulate GluA1 phosphorylation and trafficking, and trafficking of AKAP150 itself is modulated by palmitoylation on two Cys residues. Here, we developed a palmitoylation-deficient knockin mouse to show that AKAP150 palmitoylation regulates CP-AMPAR incorporation at hippocampal synapses. Using biochemical, super-resolution imaging, and electrophysiological approaches, we found that palmitoylation promotes AKAP150 localization to recycling endosomes and the postsynaptic density (PSD) to limit CP-AMPAR basal synaptic incorporation. In addition, we found that AKAP150 palmitoylation is required for LTP induced by weaker stimulation that recruits CP-AMPARs to synapses but not stronger stimulation that recruits GluA2-containing AMPARs. Thus, AKAP150 palmitoylation controls its subcellular localization to maintain proper basal and activity-dependent regulation of synaptic AMPAR subunit composition.

Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons.

In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity.

Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1.

Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

ß-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling.

α- and ß-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although ß-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of ß-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The ß-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of ß-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of ß-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic ß-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for ß-neurexins in the endocannabinoid-dependent regulation of neural circuits.

Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses.

α- and ß-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available and targeting all α- and ß-neurexins produced by a particular gene is challenging. Using newly generated constitutive and conditional knockout mice that target all neurexin-3α and neurexin-3ß isoforms, we found that neurexin-3 was differentially required for distinct synaptic functions in different brain regions. Specifically, we found that, in cultured neurons and acute slices of the hippocampus, extracellular sequences of presynaptic neurexin-3 mediated trans-synaptic regulation of postsynaptic AMPA receptors. In cultured neurons and acute slices of the olfactory bulb, however, intracellular sequences of presynaptic neurexin-3 were selectively required for GABA release. Thus, our data indicate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms.