Neurodevelopmental and neuropsychiatric disorders such as autism and schizophrenia are devastating brain illnesses that are often associated with deficits in social behaviors. Thus, understanding the molecular mechanisms contributing to the etiology underlying these social defects will be critical in developing therapeutic strategies for these disorders. Here, we have identified a crucial role of carboxyl-terminus (C-tail) of Neuroligin 2 (NL2), a cell adhesion molecule highly enriched at inhibitory synapses, in social behaviors. Indeed, we have found that genetic deletion of NL2 C-tail in mice (StopKI mice) significantly reduces GABAergic synaptic density and inhibitory synaptic transmission in hippocampal CA1 neurons. Importantly, both male and female StopKI mice also manifest elevated obsessive-compulsive disorder-like phenotypes. In addition, we have observed impaired social cognition behaviors in these mice that have not been previously observed in NL2 knock-out mice. These data reveal an unappreciated role of the NL2 C-tail in regulating social behaviors and highlight the importance of NL2 C-tail-mediated signaling in delineating molecular determinants for neurodevelopmental and neuropsychiatric disorders.

Abstract GABAA receptors (GABAARs) mediate most synaptic inhibition in the brain, but their cell-autonomous role in regulating glutamatergic transmission remains poorly understood. By targeting GABAAR β1–3 subunit alleles (GABRB1-3) at once, we genetically eliminated GABAARs in individual hippocampal CA1 pyramidal neurons. We find that single-cell silencing of GABAergic transmission does not alter AMPAR-mediated synaptic transmission, but leads to a reduction in NMDAR-mediated synaptic transmission, loss of long-term potentiation (LTP), and impaired spatial memory. Genetic rescue experiments reveal that NMDAR-mediated whole-cell currents and synaptic transmission depend on specific GABAAR subtypes and are tightly regulated by neuronal excitability. Pharmacologically restoring NMDAR function in β123-CRISPR mice rescues both LTP and spatial memory deficits induced by the loss of GABAARs in CA1 neurons. Our data uncover a previously unknown regulation of synaptic NMDAR functions by GABAARs at the single-cell level and provide insight into excitation and inhibition balance between GABAARs and NMDARs in the brain.

Neuroligins (NLGNs) are a family of postsynaptic adhesion molecules that bind to their presynaptic partners, neurexins, facilitating the formation and maintenance of synapses. In humans, there are five genes encoding NLGNs (NLGN1-3, NLGN4X, and NLGN4Y), with NLGN1-3 having highly conserved counterparts in rodents, allowing these genes to be studied with high confidence of translational validity in mouse models. Human NLGN4X and 4Y were often assumed to serve similar functions because they share a 97% sequence homology, whereas mouse NLGN4-like is quite divergent. Many NLGN-mediated synaptic effects are modulated through post-translation modifications, which exert temporal and spatial control. In this report, we characterize a conserved phosphorylation site, serine 712, on NLGN4X and 4Y. Despite serine 712 being located in a highly conserved region between NLGN4X and 4Y, we observed kinase specificity. PKA exclusively phosphorylates NLGN4X S712, whereas Cdk5 phosphorylates S712 on both NLGN4X and 4Y. NLGN4X S712 phosphorylation regulated spine density, with phosphorylation reducing mature mushroom spines and unphosphorylated S712 increasing spines and enhancing miniature excitatory postsynaptic current frequency.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients’ SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3–/–) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

Current treatments of anxiety and depressive disorders are plagued by considerable side effects and limited efficacies, underscoring the need for additional molecular targets that can be leveraged to improve medications. Here, we have identified a molecular cascade triggered by chronic stress that exacerbates anxiety- and depressive-like behaviors. Specifically, chronic stress enhances Src kinase activity and tyrosine phosphorylation of calmodulin, which diminishes MyosinVa (MyoVa) interaction with Neuroligin2 (NL2), resulting in decreased inhibitory transmission and heightened anxiety-like behaviors. Importantly, pharmacological inhibition of Src reinstates inhibitory synaptic deficits and effectively reverses heightened anxiety-like behaviors in chronically stressed mice, a process requiring the MyoVa–NL2 interaction. These data demonstrate the reversibility of anxiety- and depressive-like phenotypes at both molecular and behavioral levels and uncover a therapeutic target for anxiety and depressive disorders.

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABAA receptor (GABAAR) auxiliary subunit. Functionally, TMEM132B promotes GABAAR expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABAAR interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABAAR-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABAAR auxiliary subunit, identify the TMEM132B-GABAAR complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors.

Excitatory and inhibitory synapses do not overlap even when formed on one submicron-sized dendritic protrusion. How excitatory and inhibitory postsynaptic cytomatrices or densities (e/iPSDs) are segregated is not understood. Broadly, why membraneless organelles are naturally segregated in cellular subcompartments is unclear. Using biochemical reconstitutions in vitro and in cells, we demonstrate that ePSDs and iPSDs spontaneously segregate into distinct condensed molecular assemblies through phase separation. Tagging iPSD scaffold gephyrin with a PSD-95 intrabody (dissociation constant ~4 nM) leads to mistargeting of gephyrin to ePSD condensates. Unexpectedly, formation of iPSD condensates forces the intrabody-tagged gephyrin out of ePSD condensates. Thus, instead of diffusion-governed spontaneous mixing, demixing is a default process for biomolecules in condensates. Phase separation can generate biomolecular compartmentalization specificities that cannot occur in dilute solutions.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

>Sleep is a widely expressed behavior across the animal kingdom. In addition to the numerous health benefits that are associated with sleep, it is believed that sleep plays a pivotal role in mental processes such as learning and memory. Indeed, it has been demonstrated that learning and memory benefit from sleep, whereas sleep loss causes cognitive impairment(Rasch and Born, 2013). Changing the strength of synapses,

The crosstalk between the immune and the neuroendocrine system is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones release in response to environmental cues, such as self and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promotes peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. CD1d ligation on EC cells transduces a reverse signal and restrains potassium conductance, leading to calcium influx and 5-HT secretion. Together, we show that by coupling with iNKT cells, gut chemosensory cells perceive lipid antigens via CD1d to calibrate 5-HT release, modulating intestinal and systemic physiology.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.

Sleep is an essential process that consolidates memories by modulating synapses through poorly understood mechanisms. Here we report that GABAergic synapses in hippocampal CA1 pyramidal neurons undergo daily rhythmic alterations. Specifically, wake inhibits phasic inhibition whereas it promotes tonic inhibition compared to sleep. We further utilize a model of chemically induced inhibitory long-term potentiation (iLTP) to examine inhibitory plasticity. Intriguingly, while CA1 pyramidal neurons in both wake and sleep mice undergo iLTP, wake mice have a much higher magnitude. We also employ optogenetics and observe that inhibitory inputs from parvalbumin-, but not somatostatin-, expressing interneurons contribute to dynamic iLTP during sleep and wake. Finally, we demonstrate that synaptic insertion of α5-GABA A receptors underlies the wake-specific enhancement of iLTP at parvalbumin-synapses. These data reveal a previously unappreciated daily oscillation of inhibitory synaptic plasticity in hippocampal neurons and uncover a dynamic contribution of inhibitory synapses in memory mechanisms across sleep and wake.

GABA-A receptors (GABA A Rs) are crucial for development and function of the brain. Altered GABAergic transmission is hypothesized to be involved in neurodevelopmental disorders. Recently, we identified Shisa7 as a GABA A R auxiliary subunit that modulates GABA A R trafficking and GABAergic transmission. However, the underlying molecular mechanisms remain elusive. Here we generated a knock-in (KI) mouse line that is phospho-deficient at a phosphorylation site in Shisa7 (S405) and combined with electrophysiology, imaging and behavioral assays to illustrate the role of this site in GABAergic transmission and plasticity as well as behaviors. We found that expression of phospho-deficient mutants diminished α2-GABA A R trafficking in heterologous cells. Additionally, α1/α2/α5-GABA A R surface expression and GABAergic inhibition were decreased in hippocampal neurons in KI mice. Moreover, chemically induced inhibitory long-term potentiation was abolished in KI mice. Lastly, KI mice exhibited hyperactivity, increased grooming and impaired sleep homeostasis. Collectively, our study reveals a phosphorylation site critical for Shisa7-dependent GABA A Rs trafficking which contributes to behavioral endophenotypes displayed in neurodevelopmental disorders.

Sleep is an essential process that consolidates memories by modulating synapses through poorly understood mechanisms. Here, we report that GABAergic synapses in hippocampal CA1 pyramidal neurons undergo daily rhythmic alterations. Specifically, wake inhibits phasic inhibition, whereas it promotes tonic inhibition compared to sleep. We further utilize a model of chemically induced inhibitory long-term potentiation (iLTP) to examine inhibitory plasticity. Intriguingly, while CA1 pyramidal neurons in both wake and sleep mice undergo iLTP, wake mice have a much higher magnitude. We also employ optogenetics and observe that inhibitory inputs from parvalbumin-, but not somatostatin-, expressing interneurons contribute to dynamic iLTP during sleep and wake. Finally, we demonstrate that synaptic insertion of α5-GABAA receptors underlies the wake-specific enhancement of iLTP at parvalbumin-synapses, which is independent of time of the day. These data reveal a previously unappreciated daily oscillation of inhibitory LTP in hippocampal neurons and uncover a dynamic contribution of inhibitory synapses in memory mechanisms across sleep and wake.

GABA A receptors (GABA A Rs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABA A R-associated transmembrane proteins. We previously identified Shisa7 as a GABA A R auxiliary subunit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABA A R single-channel properties has yet to be determined. Here, we performed single-channel recordings of α2β3γ2L GABA A Rs cotransfected with Shisa7 in HEK293T cells and found that while Shisa7 does not change channel slope conductance, it reduced the frequency of receptor openings. Importantly, Shisa7 modulates GABA A R gating by decreasing the duration and open probability within bursts. Through kinetic analysis of individual dwell time components, activation modeling, and macroscopic simulations, we demonstrate that Shisa7 accelerates GABA A R deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABA A R gating and reveal for the first time that GABA A R single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission. SIGNIFICANCE STATEMENT Although GABA A receptor (GABA A R) single-channel properties are largely determined by pore-forming subunits, it remains unknown whether they are also controlled by GABA A R-associated transmembrane proteins. Here, we show that Shisa7, a recently identified GABA A R auxiliary subunit, modulates GABA A R activation by altering single-channel burst kinetics. These results reveal that Shisa7 primarily decreases the duration and open probability of receptor burst activity during gating, leading to accelerated GABA A R deactivation. These experiments are the first to assess the gating properties of GABA A Rs in the presence of an auxiliary subunit and provides a kinetic basis for how Shisa7 modifies temporal attributes of GABAergic transmission at the single-channel level.

The vast majority of fast inhibitory transmission in the brain is mediated by GABA acting on GABAA receptors (GABAARs), which provides inhibitory balance to excitatory drive and controls neuronal output. GABAARs are also effectively targeted by clinically important drugs for treatment in a number of neurological disorders. It has long been hypothesized that function and pharmacology of GABAARs are determined by the channel pore-forming subunits. However, recent studies have provided new dimensions in studying GABAARs due to several transmembrane proteins that interact with GABAARs and modulate their trafficking and function. In this review, we summarize recent findings on these novel GABAAR transmembrane regulators and highlight a potential avenue to develop new GABAAR psychopharmacology by targeting these receptor-associated membrane proteins.

SUMMARYTonic inhibition mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABAARs) play important roles in the regulation of various brain functions. However, the regulatory mechanisms for tonic inhibition remain largely unknown. Here we report distinct actions of GluN2A- and GluN2B-containing subtypes of NMDA receptors (NMDARs) on tonic inhibition in hippocampal neurons. Mechanistically, GluN2A- and GluN2B-containing NMDARs play differential roles in α5-GABAAR internalization. Additionally, GluN2A-, but not GluN2B-, containing receptors are required for the homeostatic potentiation of tonic inhibition. In an acute seizure model induced by kainic acid, tonic inhibition is decreased during acute seizures, while it is increased 24 h later, and these alterations are dependent on the distinct GluN2-containing NMDARs. Collectively, these data reveal a critical link between NMDARs and extrasynaptic GABAARs in both physiological and pathological conditions.

Sleep is a fundamental physiological process conserved across most species. As such, deficits in sleep can result in a myriad of psychological and physical health issues. However, the mechanisms underlying the induction of sleep are relatively unknown. Interestingly, general anesthetics cause unconsciousness by positively modulating GABA-A receptors (GABA A Rs). Based on this observation, it is hypothesized that GABA A Rs play a critical role in modulating circuits involved in sleep to promote unconsciousness. Recently, the lateral habenula (LHb) has been demonstrated to play a role in sleep physiology and sedation. Specifically, propofol has been shown to excite LHb neurons to promote sedation. However, the mechanism by which this occurs is unknown. Here, we utilize whole-cell voltage and current clamp recordings from LHb neurons obtained from 8-10 week old male mice to determine the physiological mechanisms for this phenomenon. We show that bath application of 1.5μM propofol is sufficient to increase LHb neuronal excitability involving synaptic transmission, but not through modulation of intrinsic properties. Additionally, although there is increased LHb neuronal excitability, GABA A Rs localized postsynaptically on LHb neurons are still responsive to propofol, as indicated by an increase in the decay time. Lastly, we find that propofol increases the synaptic drive onto LHb neurons involving enhanced presynaptic release of both glutamate and GABA. However, the greatest contributor to the potentiated synaptic drive is the increased release of glutamate which shifts the balance of synaptic transmission towards greater excitation. Taken together, this study is the first to identify the physiological basis for why LHb neurons are excited by propofol, rather than inhibited, and as a result promote sedation.

Tonic inhibition mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABAARs) critically regulates neuronal excitability and brain function. However, the mechanisms regulating tonic inhibition remain poorly understood. Here, we report that Shisa7 is critical for tonic inhibition regulation in hippocampal neurons. In juvenile Shisa7 knockout (KO) mice, α5-GABAAR-mediated tonic currents are significantly reduced. Mechanistically, Shisa7 is crucial for α5-GABAAR exocytosis. Additionally, Shisa7 regulation of tonic inhibition requires protein kinase A (PKA) that phosphorylates Shisa7 serine 405 (S405). Importantly, tonic inhibition undergoes activity-dependent regulation, and Shisa7 is required for homeostatic potentiation of tonic inhibition. Interestingly, in young adult Shisa7 KOs, basal tonic inhibition in hippocampal neurons is unaltered, largely due to the diminished α5-GABAAR component of tonic inhibition. However, at this stage, tonic inhibition oscillates during the daily sleep/wake cycle, a process requiring Shisa7. Together, these data demonstrate that intricate signaling mechanisms regulate tonic inhibition at different developmental stages and reveal a molecular link between sleep and tonic inhibition.