Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABA_ARs). Although neurosteroids are accepted allosteric modulators of GABA_ARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABA_ARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABA_AR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABA_AR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABA_ARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.Neurosteroids are synthesized de novo in the brain from cholesterol, or steroid hormone precursors. Raising neurosteroid levels in the CNS causes anxiolysis, sedation/hypnosis, anticonvulsant action, and anesthesia and reduces depressive-like behaviors (1–3). Accordingly, dysregulation of neurosteroid signaling is associated with premenstrual dysphoric disorder, panic disorder, depression, schizophrenia, and bipolar disorder. Neurosteroids exert the majority of their actions via potentiating the activity of γ-aminobutyric acid receptors (GABA_ARs), which mediate the majority of fast synaptic inhibition in the adult brain. Accordingly, at low nanomolar concentrations they potentiate GABA-dependent currents, whereas at micromolar concentrations they directly activate GABA_ARs (4–8).GABA_ARs are Cl⁻-preferring pentameric ligand-gated ion channels that assemble from eight families of subunits: α(1–6), β(1–3), γ(1–3), δ, ε, ө, π, and ρ(1–3) (9, 10). Receptor subtypes composed of α1–3βγ subunits largely mediate synaptic or phasic inhibition, whereas those constructed from α4–6β1–3, with or without γ/δ subunits, are principal determinants of tonic inhibition (11–13). Neurosteroids have been shown to bind GABA_ARs at an allosteric site distinct from that of GABA, benzodiazepines, or barbiturates (9, 14). Hosie et al. identified residues located within the transmembrane domain of GABA_AR α and β subunits that are critical for the direct activation (α1–6; Threonine 236, β1–3; Tyrosine 284) and allosteric potentiation (α1–6 Asparagine 407, and α1–6 Glutamine 246) of neurosteroids (15–17). Accordingly, mutation of glutamine 241 (Q241) within the α1–6 subunits prevents allosteric potentiation of GABA_AR composed of αβγ and αβδ subunits by neurosteroids (15, 16).In addition to modulating channel gating, neurosteroids exert potent effects on the expression levels of GABA_ARs (1, 18–20). Moreover, in the hippocampus, prolonged exposure to physiological concentrations of neurosteroids has been shown to enhance the tonic conductance mediated by extrasynaptic GABA_ARs containing the α4/δ subunits, while having little effect on the phasic conductance mediated by synaptic GABA_ARs (6, 21). However, the molecular mechanisms by which neurosteroids regulate GABA_AR expression levels remain unknown.Here, we reveal that neurosteroids act to increase the PKC-dependent phosphorylation of serine 443 (S443) within the intracellular domain of the α4 subunit. This process leads to increased insertion of α4 subunit-containing GABA_ARs into the plasma membrane and a selective enhancement of tonic inhibition. Thus, our experiments reveal a previously unidentified molecular mechanism by which neurosteroids exert sustained effects on GABAergic inhibition by selectively increasing α4-containing GABA_ARs in the membrane and therefore potentiate tonic inhibition.     

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors

GABA_A-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABA_A receptors, and requires a newly described interaction with GABARAP, a GABA_A receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.Interneurons that release γ-aminobutyric acid (GABA) are a major source of inhibitory signaling in vertebrate nervous systems, and play important roles in cognition, mood, and behavior (1, 2). Many of these inhibitory interneurons release GABA, which binds to ionotropic ligand-gated GABA_A receptors located at GABAergic synapses and at extrasynaptic sites, and these GABA_A receptors are sites of action for benzodiazepine and barbiturates (3). GABA_A receptors are dynamic, with continuous exchange between synaptic and extrasynaptic sites in the plane of the membrane, as well as endocytic trafficking between the cell surface and intracellular compartments (3–6). GABA_A receptor cell surface expression is believed to be required for formation of GABAergic synapses based on studies with heterogeneously-expressed GABA_A receptors (7). However, the role of GABA_A receptors in preserving GABAergic synapses has not yet been described in a native neuronal environment.GABAergic synapses localize to both the axon initial segment (AIS) as well as somatodendritic sites of target neurons (2, 8, 9). In the cerebellum, basket and stellar interneurons project specific axon terminals to the AISs of Purkinje cells, forming GABAergic “pinceau” synapses (10). Formation of these pinceau synapses depends on a steep gradient of the cell adhesion molecule neurofascin, which is enriched at the AIS (11, 12). Both GABAergic pinceau synapses and the neurofascin gradient are missing in mice with cerebellar knockout out of the membrane adaptor ankyrin-G (11, 13). Ankyrin-G coordinates multiple proteins at AISs including voltage-gated sodium channels (VGSC), KCNQ2/3 channels, 186-kDa neurofascin, and beta-4 spectrin (14). A role of ankyrin-G in stabilizing GABAergic synapses outside of the the AIS of cerebellar neurons has not been explored.Assembly of AISs as well as their GABAergic synapses requires giant ankyrin-G, which contains a 7.8-kb alternatively spliced nervous system-specific exon found only in vertebrates (14). In addition to ANK repeats and a beta-spectrin-binding domain, giant ankyrin-G (480-kDa ankyrin-G) contains 2,600 residues configured as an extended fibrous polypeptide (14–17). Giant ankyrin-G has been assumed to be confined to AISs and nodes of Ranvier and a general role for ankyrin-G in GABAergic synapse stability at other cellular sites has not been entertained (14, 15, 18).Here we report that giant ankyrin-G is present in extrasynaptic microdomains on the somatodendritic surfaces of hippocampal and cortical neurons, and describe a giant ankyrin-G–based mechanism required for cell surface expression of GABA_A receptors and for maintaining somatodendritic GABAergic synapses. We find that somatodendritic giant ankyrin-G inhibits GABA_A receptor endocytosis through an interaction with the GABA_A receptor-associated protein (GABARAP). This previously unidentified role for giant ankyrin-G provides a newly resolved step in the formation of GABA_A-receptor-mediated circuitry in the cerebral cortex as well as a rationale for recent linkage of human mutations in the giant ankyrin exon with autism and severe cognitive dysfunction (19).     

Tobacco smoking interferes with GABAA receptor neuroadaptations during prolonged alcohol withdrawal

Understanding the effects of tobacco smoking on neuroadaptations in GABA_A receptor levels over alcohol withdrawal will provide critical insights for the treatment of comorbid alcohol and nicotine dependence. We conducted parallel studies in human subjects and nonhuman primates to investigate the differential effects of tobacco smoking and nicotine on changes in GABA_A receptor availability during acute and prolonged alcohol withdrawal. We report that alcohol withdrawal with or without concurrent tobacco smoking/nicotine consumption resulted in significant and robust elevations in GABA_A receptor levels over the first week of withdrawal. Over prolonged withdrawal, GABA_A receptors returned to control levels in alcohol-dependent nonsmokers, but alcohol-dependent smokers had significant and sustained elevations in GABA_A receptors that were associated with craving for alcohol and cigarettes. In nonhuman primates, GABA_A receptor levels normalized by 1 mo of abstinence in both groups—that is, those that consumed alcohol alone or the combination of alcohol and nicotine. These data suggest that constituents in tobacco smoke other than nicotine block the recovery of GABA_A receptor systems during sustained alcohol abstinence, contributing to alcohol relapse and the perpetuation of smoking.Alcohol dependence and tobacco smoking are highly comorbid (1). Alcohol-dependent smokers who quit drinking but continue smoking may have a reduced severity of alcohol withdrawal and relapse risk (2) compared with alcohol-dependent smokers who stop smoking and drinking at the same time (3–5). This has led to some complacency in the field about treating the addiction to nicotine in alcohol-dependent smokers, and few treatment settings provide any systematic tobacco treatment (6). However, a large part of the morbidity and mortality from alcohol dependence can be attributed to concurrent tobacco smoking (7), and a large number of alcohol-dependent individuals in treatment express a desire to quit smoking (8). Understanding the involvement of tobacco smoking in the neuroadaptations and behavioral changes that occur during alcohol withdrawal will provide critical insights to direct treatment strategies.Given the multiple molecular targets for alcohol in the brain and numerous constituents of tobacco smoke, it is likely that the neurobiology of this comorbidity is complex. However, the γ-aminobutyric acid (GABA) system may be an important point of convergence of the effects of tobacco smoke and alcohol in the brain. For example, nicotine reinforcement has been critically linked to activation of GABA neurons (9), and alcohol appears to both directly stimulate extrasynaptic GABA_A receptors with relatively high affinity (10) and to indirectly stimulate the release of GABA and neurosteroids (11), such as allopregnanolone, that also stimulate extrasynaptic GABA_A receptors (12, 13). Alcohol and neurosteroids can act at synaptic GABA_A receptors, but the affinity is low, the response is variable, and the dose of alcohol that would facilitate signaling at these synaptic receptors would induce a coma in humans (14, 15).Studies in both animals and humans have yielded a tentative model about the convergence of the codependency produced by smoking and alcohol consumption, as has been reviewed (16). In the absence of smoking, chronic alcohol administration produces an adaptive down-regulation of synaptic GABA_A receptor function by altering GABA_A receptor subunit composition and subtly shifting subpopulations of receptors from a relative predominance of low-affinity high Cl– conductance type to greater numbers of a high-affinity low Cl– conductance subtype, characteristic of extrasynaptic GABA_A receptors (15). In early recovery, there is a transitional phase, during which deficits in GABA_A receptor signaling are thought to contribute to withdrawal-related cortical hyperexcitability and low-affinity high-conductance receptors are recruited to reestablish the cortical balance of excitation and inhibition. The recruitment of the additional GABA_A receptors was demonstrated by a transient increase in ligand binding over the first week of alcohol withdrawal (17). In this same cross-sectional study (17), a subset of smokers did not show similar time-dependent alterations during early recovery. Moreover, GABA_A receptor availability was positively correlated with alcohol withdrawal symptoms in nonsmokers but not smokers, suggesting that smoking may have suppressed withdrawal symptoms by preventing alcohol-related neuroadaptations in GABA_A receptors.The goal of the current study was to systematically examine the effect of tobacco smoking on alcohol withdrawal-related neuroadaptations in GABA_A levels. The first study was designed to extend the previous cross-sectional findings to determine differences in GABA_A receptor levels in alcohol-dependent smokers versus nonsmokers at multiple times during early withdrawal and during extended abstinence. Additionally, tobacco smoke consists of over 4,000 chemicals. Many of these chemicals may play a role in influencing alcohol-related withdrawal symptoms; however, nicotine, the primary addictive chemical in tobacco smoke, has been the most widely studied tobacco constituent and has been associated with GABA system regulation (9, 18). Thus, a second parallel study was conducted in nonhuman primates that were randomized to self-administer alcohol with or without concurrent access to a nicotine solution rather than tobacco smoke to determine the role of nicotine per se on alcohol withdrawal-related neuroadaptations.     

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome

Haploinsufficiency of the voltage-gated sodium channel Na_V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na_V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na_V1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.Dravet syndrome (DS), also referred to as “severe myoclonic epilepsy in infancy,” is a rare genetic epileptic encephalopathy characterized by frequent intractable seizures, severe cognitive deficits, and premature death (1–3). DS is caused by loss-of-function mutations in SCN1A, the gene encoding type I voltage-gated sodium channel Na_V1.1, which usually arise de novo in the affected individuals (4–7). Like DS patients, mice with heterozygous loss-of-function mutations in Scn1a exhibit ataxia, sleep disorder, cognitive deficit, autistic-like behavior, and premature death (8–14). Like DS patients, DS mice first become susceptible to seizures caused by elevation of body temperature and subsequently experience spontaneous myoclonic and generalized tonic-clonic seizures (11). Global deletion of Na_V1.1 impairs Na⁺ currents and action potential (AP) firing in GABAergic-inhibitory interneurons (8–10), and specific deletion of Na_V1.1 in forebrain interneurons is sufficient to cause DS in mice (13, 15). These data suggest that the loss of interneuron excitability and resulting disinhibition of neural circuits cause DS, but the functional role of different subtypes of interneurons in the cerebral cortex in DS remains unknown.Neocortical GABAergic interneurons shape cortical output and display great diversity in morphology and function (16, 17). The expression of parvalbumin (PV) and somatostatin (SST) defines two large, nonoverlapping groups of interneurons (16, 18, 19). In layer V of the cerebral cortex, PV-expressing fast-spiking interneurons and SST-expressing Martinotti cells each account for ∼40% of interneurons, and these interneurons are the major inhibitory regulators of cortical network activity (17, 20). Layer V PV interneurons make synapses on the soma and proximal dendrites of pyramidal neurons (18, 19), where they mediate fast and powerful inhibition (21, 22). Selective heterozygous deletion of Scn1a in neocortical PV interneurons increases susceptibility to chemically induced seizures (23), spontaneous seizures, and premature death (24), indicating that this cell type may contribute to Scn1a deficits. However, selective deletion of Scn1a in neocortical PV interneurons failed to reproduce the effects of DS fully, suggesting the involvement of other subtypes of interneurons in this disease (23, 24). Layer V Martinotti cells have ascending axons that arborize in layer I and spread horizontally to neighboring cortical columns, making synapses on apical dendrites of pyramidal neurons (17, 25, 26). They generate frequency-dependent disynaptic inhibition (FDDI) that dampens excitability of neighboring layer V pyramidal cells (27–29), contributing to maintenance of the balance of excitation and inhibition in the neocortex. However, the functional roles of Martinotti cells and FDDI in DS are unknown.Because layer V forms the principal output pathway of the neocortex, reduction in inhibitory input to layer V pyramidal cells would have major functional consequences by increasing excitatory output from all cortical circuits. However, the effects of the DS mutation on interneurons and neural circuits that provide inhibitory input to layer V pyramidal cells have not been determined. Here we show that the intrinsic excitability of layer V fast-spiking PV interneurons and SST Martinotti cells and the FDDI mediated by Martinotti cells are reduced dramatically in DS mice, leading to an imbalance in the excitation/inhibition ratio. Our results suggest that loss of Na_V1.1 in these two major types of interneurons may contribute synergistically to increased cortical excitability, epileptogenesis, and cognitive deficits in DS.     

MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity

GABA_A receptors shape synaptic transmission by modulating Cl⁻ conductance across the cell membrane. Remarkably, animal toxins that specifically target GABA_A receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABA_A receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABA_A receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α⁺/β⁻ interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABA_A receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.Ionotropic γ-aminobutyric acid type A (GABA_A) receptors are found predominantly in the central nervous system, where they mediate inhibitory postsynaptic transmission by influencing Cl⁻ flux across the cell membrane (1–4). Imbalances between excitatory and inhibitory GABA_A receptor activity have been implicated in clinical phenotypes such as epilepsy, schizophrenia, and chronic pain (5, 6). As such, GABA_A receptors are targeted by various drugs including barbiturates, benzodiazepines, and anesthetics (1, 7).GABA_A receptors belong to the pentameric Cys-loop superfamily of ligand-gated ion channel receptors, which also encompasses the nicotinic acetylcholine (nAChRs), glycine (GlyR), and serotonin receptors (8). The numerous subunit isoforms (α1–6, β1–3, γ1–3, δ, ε, π, θ, and ρ1–3) that can make up a GABA_A receptor create multiple structural arrangements (9–11). In general, each subunit consists of four transmembrane domains, in which transmembrane domain 2 delineates the axially positioned Cl⁻ channel (12). Molecules can interact with various regions within one or more subunits, resulting in a complex pharmacologic landscape. For example, GABA, as well as the prototypic exogenous agonist muscimol (13), binds at the extracellular interface between a β and α subunit (β⁺/α⁻) (14, 15) whereas benzodiazepines require both the α and γ2 subunit to be pharmacologically active (16, 17). Conversely, anesthetics such as propofol most likely position themselves in transmembrane intersubunit pockets (18). So far, picrotoxin (PTX) is the only well-documented naturally occurring plant toxin that is known to block the GABA_A receptor pore and is experimentally used as a chemoconvulsant to induce epileptic seizures (19). In contrast, molecules isolated from plant extracts and snake and cone snail venoms have been used extensively to probe the structural and functional properties of nAChRs (20, 21).Here, we explore whether animal venoms contain toxins that primarily interact with GABA_A receptors. While examining the venom of Costa Rican coral snakes (22), we came across a major fraction that displayed evidence of GABA_A-related toxicity in mice. Within this fraction, we found micrurotoxin1 (MmTX1) and MmTX2, two equally potent peptides with a primary sequence belonging to the PATE-SLURP1-LYNX1-Ly-6/neurotoxin-like family (23–25). Extensive binding and competition studies revealed that GABA_A receptors are the primary target of these peptides, whereas nAChRs are unaffected. In contrast to PTX, which blocks the pore at micromolar concentrations, our data suggest that MmTX1 and MmTX2 modulate GABA_A receptor function at subnanomolar quantities by tightly binding to the α⁺/β⁻ subunit interface, a novel benzodiazepine-like binding site with promising therapeutic potential (26). Electrophysiologic experiments with recombinantly and synthetically produced MmTX1 and MmTX2 on hippocampal neurons, HEK 293 cells, and Xenopus oocytes expressing common receptor compositions indicate that these toxins allosterically increase GABA_A receptor sensitivity to agonist, thereby reshaping channel opening as well as desensitization. Overall, our results demonstrate that potent and selective GABA_A-receptor modulating toxins can be found in snake venom and reveal the exciting prospect of discovering new tools to study these receptors.     

Dystroglycan mediates homeostatic synaptic plasticity at GABAergic synapses

Dystroglycan (DG), a cell adhesion molecule well known to be essential for skeletal muscle integrity and formation of neuromuscular synapses, is also present at inhibitory synapses in the central nervous system. Mutations that affect DG function not only result in muscular dystrophies, but also in severe cognitive deficits and epilepsy. Here we demonstrate a role of DG during activity-dependent homeostatic regulation of hippocampal inhibitory synapses. Prolonged elevation of neuronal activity up-regulates DG expression and glycosylation, and its localization to inhibitory synapses. Inhibition of protein synthesis prevents the activity-dependent increase in synaptic DG and GABA_A receptors (GABA_ARs), as well as the homeostatic scaling up of GABAergic synaptic transmission. RNAi-mediated knockdown of DG blocks homeostatic scaling up of inhibitory synaptic strength, as does knockdown of like-acetylglucosaminyltransferase (LARGE)—a glycosyltransferase critical for DG function. In contrast, DG is not required for the bicuculline-induced scaling down of excitatory synaptic strength or the tetrodotoxin-induced scaling down of inhibitory synaptic strength. The DG ligand agrin increases GABAergic synaptic strength in a DG-dependent manner that mimics homeostatic scaling up induced by increased activity, indicating that activation of this pathway alone is sufficient to regulate GABA_AR trafficking. These data demonstrate that DG is regulated in a physiologically relevant manner in neurons and that DG and its glycosylation are essential for homeostatic plasticity at inhibitory synapses.Muscular dystrophies are often associated with mild to severe cognitive deficits, epilepsy, and other neurological deficits (1–3). This is particularly evident in muscular dystrophies caused by mutations that affect glycosylation of the membrane glycoprotein α-dystroglycan (α-DG) (4). α-DG docks with transmembrane β-DG to form the functional core of the dystrophin-associated glycoprotein complex (DGC) that links adhesive proteins in the extracellular matrix to dystrophin (5). α-DG is heavily glycosylated and interacts via its carbohydrate side chains with laminin and laminin G-like domains in a variety of proteins including agrin, perlecan, slit, neurexin, and pikachurin (6–10). Key carbohydrate residues are added onto α-DG by several glycosyltransferases, most notably like-acetylglucosaminyltransferase (LARGE) (11). LARGE is necessary for functional glycosylation of α-DG (12), and is mutated in muscular dystrophies associated with severe cognitive deficits (4).DG was first identified in the nervous system (13), where it is important during development for neuroblast migration (14), axon guidance (7), and ribbon synapse formation (8). At neuromuscular synapses, DG is required for the stabilization of acetylcholine receptors in the postsynaptic density and contributes to the accumulation of acetylcholinesterase (10, 15). However, the function of DG at central synapses remains essentially unknown. In the mature central nervous system (CNS), neuronal DGC components are exclusively colocalized with GABA_A receptors (GABA_ARs) in multiple brain regions (16–18), raising the possibility for a role in GABA_AR regulation. However, DG is dispensable for GABAergic synapse formation in hippocampal cultures (17), although adult mice lacking full-length dystrophin show reduced clustering of GABA_ARs in the hippocampus and other brain regions (16, 19, 20). Because dystrophin localization at GABAergic synapses depends on DG (17), these findings suggest that DG may regulate the plasticity of mature GABAergic synapses. Homeostatic synaptic plasticity is widely thought to be essential for brain function and involves the reciprocal regulation of glutamatergic and GABAergic synapses to stabilize neuronal activity (21). Chronic elevation of neuronal activity is associated with an increase in synaptic GABA_ARs (22, 23), but the mechanistic details are incompletely understood.Here, we assess the roles of DG and α-DG glycosylation in regulating the expression of homeostatic synaptic plasticity at GABAergic synapses. We find that in mature hippocampal cultures, prolonged elevation of neuronal activity up-regulates DG expression and the coclustering of α-DG and GABA_ARs. Inhibition of protein synthesis or knockdown of DG blocks homeostatic scaling up of GABAergic synaptic strength. Knockdown of the selective α-DG glycosyltransferase LARGE also blocks homeostatic scaling up, suggesting a role for ligand binding. Furthermore, exogenous application of agrin—a ligand for glycosylated α-DG—is sufficient to scale up GABAergic synaptic strength in a DG-dependent fashion. These data identify a mechanism whereby expression of glycosylated α-DG is linked to neuronal activity level and is essential for homeostatic scaling up of GABAergic synaptic strength by regulating GABA_AR abundance at the synapse.     

ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity

ErbB4 signaling in the central nervous system is implicated in neuropsychiatric disorders and epilepsy. In cortical tissue, ErbB4 associates with excitatory synapses located on inhibitory interneurons. However, biochemical and histological data described herein demonstrate that the vast majority of ErbB4 is extrasynaptic and detergent-soluble. To explore the function of this receptor population, we used unbiased proteomics, in combination with electrophysiological, biochemical, and cell biological techniques, to identify a clinically relevant ErbB4-interacting protein, the GABA_A receptor α1 subunit (GABAR α1). We show that ErbB4 and GABAR α1 are robustly coexpressed in hippocampal interneurons, and that ErbB4-null mice have diminished cortical GABAR α1 expression. Moreover, we characterize a Neuregulin-mediated ErbB4 signaling modality, independent of receptor tyrosine kinase activity, that couples ErbB4 to decreased postsynaptic GABAR currents on inhibitory interneurons. Consistent with an evolving understanding of GABAR trafficking, this pathway requires both clathrin-mediated endocytosis and protein kinase C to reduce GABAR inhibitory currents, surface GABAR α1 expression, and colocalization with the inhibitory postsynaptic protein gephyrin. Our results reveal a function of ErbB4, independent of its tyrosine kinase activity, that modulates postsynaptic inhibitory control of hippocampal interneurons and may provide a novel pharmacological target in the treatment of neuropsychiatric disorders and epilepsy.ErbB4 signaling regulates neuronal excitability (1, 2) and synaptic plasticity (3, 4) in the adult brain, and has been implicated in psychiatric disorders (5, 6) and epilepsy (2, 7). In the neocortex and hippocampus of rodents, monkeys, and humans, ErbB4 expression is restricted to GABAergic interneurons, and its expression is particularly high in parvalbumin-positive fast-spiking (PV+) interneurons (8, 9). Of note, targeted ablation of ErbB4 specifically in PV+ interneurons recapitulates behavioral abnormalities of full ErbB4-null mice, highlighting the importance of ErbB4 signaling in this GABAergic interneuron subclass (10). Moreover, gamma oscillations, a type of high-frequency network activity that depends on synchronization of local circuits by PV+ interneurons, are augmented by Neuregulin (NRG)1 in vitro in an ErbB4-dependent manner (11).In the hippocampus, the GABA_A receptor α1 subunit (GABAR α1), which imparts rapid decay kinetics (12), is also selectively expressed in subsets of inhibitory interneurons, especially in PV+ neurons (13, 14). Furthermore, a mutation in GABRA1 has been linked to absence seizures (15), and heterozygous Gabra1-null mice show cortical absence epileptiform activity (16). Additionally, genome-wide linkage analyses have repeatedly identified a cluster of GABAR subunits, which includes GABRA1, as a schizophrenia (SCZ) susceptibility locus (17). Therefore, identifying mechanisms that acutely regulate α1-containing GABARs on interneurons is important to understanding their role in neuronal network activity and their association with SCZ and epilepsy.Numerous postmortem and functional imaging studies have implicated a selective loss of GABAergic interneuron function as a major deficit in SCZ (18). Interest has focused predominantly on PV+ basket and chandelier neurons in the dorsal lateral prefrontal cortex (DLPFC), because these interneurons target pyramidal neuron somata and axon initial segments to regulate excitatory–inhibitory balance and neuronal network activity important for numerous cognitive functions affected in SCZ (18, 19). However, there is mounting evidence for hippocampal dysfunction in SCZ (20), where PV+ interneurons (21) contribute to the altered gamma oscillations observed (18).Although a fraction of ErbB4 receptors is tightly associated with PSD-95 at glutamatergic synapses (3, 22), the majority of the receptors are extrasynaptic (see below). To explore this largely overlooked pool of ErbB4 (i.e., outside the glutamatergic synapse), we used unbiased proteomics of detergent-soluble ErbB4 isolated from synaptic plasma membranes. Using this approach, here we report a unique interaction between ErbB4 and GABAR α1. We show that NRG2, a homolog of NRG1 that is highly expressed in the adult brain (23, 24), increases the association of ErbB4 with α1-containing GABARs, causes internalization of these receptors, and reduces the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) on ErbB4+ interneurons. Unexpectedly, although the ErbB4 receptor is essential for reducing the mIPSCs in response to NRG2, its canonical receptor tyrosine kinase (RTK) activity is entirely dispensable. Our results are consistent with other studies suggesting a model for extrasynaptic GABAR trafficking (25–28), and they introduce NRG-ErbB4 signaling as a critical modulator of postsynaptic GABAR signaling in interneurons.     

GABAB autoreceptor-mediated cell type-specific reduction of inhibition in epileptic mice

GABA_B receptors (GABA_BRs) mediate slow inhibitory effects on neuronal excitability and synaptic transmission in the brain. However, the GABA_BR agonist baclofen can also promote excitability and seizure generation in human patients and animals models. Here we show that baclofen has concentration-dependent effects on the hippocampal network in a mouse model of mesial temporal lobe epilepsy. Application of baclofen at a high dose (10 mg/kg i.p.) reduced the power of γ oscillations and the frequency of pathological discharges in the Cornu Ammonis area 3 (CA3) area of freely moving epileptic mice. Unexpectedly, at a lower dose (1 mg/kg), baclofen markedly increased γ activity accompanied by a higher incidence of pathological discharges. Intracellular recordings from CA3 pyramidal cells in vitro further revealed that, although at a high concentration (10 µM), baclofen invariably resulted in hyperpolarization, at low concentrations (0.5 µM), the drug had divergent effects, producing depolarization and an increase in firing frequency in epileptic but not control mice. These excitatory effects were mediated by the selective muting of inhibitory cholecystokinin-positive basket cells (CCK⁺ BCs), through enhanced inhibition of GABA release via presynaptic GABA_BRs. We conclude that cell type–specific up-regulation of GABA_BR-mediated autoinhibition in CCK⁺ BCs promotes aberrant high frequency oscillations and hyperexcitability in hippocampal networks of chronic epileptic mice.Neuronal activity in the hippocampus shows oscillations in behavior-relevant frequency ranges including γ frequencies (30–80 Hz) (1). γ activity is prominent in the aroused brain and has been implicated in higher-level brain functions, such as sensory binding, perception (2), and storage and recall of information (3, 4). At the same time, γ frequency oscillations are also prevalent in epileptic patients and are most often observed at seizure onset during in depth EEG recordings (5). The GABAergic system plays a pivotal role in the generation of γ oscillations (6–8). However, it remains to be resolved how distinct GABAergic receptor subtypes, in particular GABA_B receptors (GABA_BRs), contribute to the generation and modulation of pathological network oscillatory activity.GABA_BRs mediate slow inhibitory effects and control synaptic transmission and the excitability of neurons in cortical networks. GABA_BRs are expressed both postsynaptically in somato-dendritic compartments and presynaptically in axon terminals, in excitatory principal cell and inhibitory interneurons (9–11). The effects of GABA_BR activation on the network are dominated by inhibition leading to an overall dampened population activity. However, if GABAergic interneurons are effected dominantly, as observed for example, during high-frequency stimulation, GABA_BR activation can produce disinhibition in principal cells (12, 13). Accordingly, the role of GABA_BRs in epilepsy and seizure generation remains ambiguous. GABA_BRs are expected to have an overall antiepileptic effect, and indeed, the receptor KO animals show an epileptic phenotype (14). However, there is also evidence that the receptor agonist baclofen can induce seizures in patients after intrathecal application (15, 16). The picture is further complicated by the fact that GABA_BR expression can be altered in both epileptic patients, e.g., in mesial temporal lobe epilepsy (mTLE) (17), and animal models (18). Thus, cell type–specific alterations in GABA_BR expression may change network excitability during the progression of mTLE.Using a chronic kainate (KA) model of mTLE, which reproduces major electrophysiological and histopathological characteristics of human mTLE (19, 20), we studied the role of GABA_BRs in altered hippocampal network activity. Our results suggest that enhanced and persistent GABA_BR activation in epileptic mice suppresses the inhibitory output from hippocampal interneurons, in particular cholecystokinin (CCK)-expressing basket cells (BCs) onto pyramidal cells (PCs). This reduction in the inhibitory output of interneurons, in turn, leads to disinhibition in hippocampal networks, enhances γ activity, and promotes the transition to pathological hyperexcitability.     

Heterodimeric coiled-coil interactions of human GABAB receptor

Metabotropic GABA_B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA_B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.The major inhibitory neurotransmitter in the CNS is GABA. Metabotropic GABA_B receptor is a G protein-coupled receptor (GPCR) that mediates slow synaptic inhibition (1, 2). It constitutes an important drug target for many neurological disorders, including epilepsy, spasticity, anxiety, and nociception (1, 2).Formation of a functional GABA_B receptor requires the heterodimeric assembly of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits (3–7). Both consist of an N-terminal extracellular domain, a seven-helix transmembrane domain, and a C-terminal intracellular domain. The intracellular domain of each subunit contains a stretch of coiled-coil sequence, and interaction between the coiled-coil helices is partly responsible for GABA_B receptor heterodimerization (5, 8).The intracellular region of GABA_B receptor hosts elements that control receptor trafficking (9). Specifically, GBR1 has a di-leucine internalization signal (EKSRLL) (9) and an endoplasmic reticulum (ER) retention signal (RSRR) (9–11) located within or near its coiled-coil domain (9). GBR1 is trapped within the ER when expressed alone (12) but can reach the cell surface upon association with GBR2 (9, 11). Mutation or removal of the ER retention signal in GBR1 results in plasma membrane expression of GBR1 (9–11). Furthermore, interaction between the coiled-coil domains of GBR1 and GBR2 masks this ER retention signal to facilitate the cell surface expression of both subunits (9–11). Although mutation of the di-leucine motif itself is not sufficient to release GBR1 from intracellular retention, it enhances cell surface expression of various GBR1 mutants that lack the ER retention signal (9).The coiled-coil domain of GBR1 associates with a number of intracellular proteins involved in trafficking, including the coat protein complex I (COPI) (13), the scaffolding protein 14-3-3 (13, 14), the GPCR interacting scaffolding protein GISP (15), and the guanidine exchange factor msec7-1 (16). In particular, COPI specifically recognizes the ER retention signal sequence of GBR1 and is involved in the intracellular retention of GBR1 (13). The msec7-1 protein increases the cell surface expression of GABA_B receptor by binding to the di-leucine internalization motif (16).Despite its important role in GABA_B receptor assembly and trafficking, the atomic details of the coiled-coil interaction between subunits are not known. In this study, we present the crystal structure of a GBR1/GBR2 coiled-coil heterodimer and identify specific contacts at the heterodimer interface that control the surface expression of GBR1.     

Differential vesicular sorting of AMPA and GABAA receptors

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABA_A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABA_A receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA_A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABA_A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.In the mammalian central nervous system, neurons receive excitatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neurotransmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA_A receptors, which are predominantly heteropentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a “plasma membrane sorting model,” different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a “vesicle sorting model,” different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the exocytic pathways of AMPA and GABA_A receptors have similar but also distinct properties (1, 2).Increasing evidence has suggested roles for the SNARE protein family in vesicular trafficking of AMPA and GABA_A receptors (4–17). SNAREs are a large family of membrane-associated proteins critical for many intracellular membrane trafficking events. The family is subdivided into v-SNAREs (synaptobrevin/VAMP, vesicle-associated membrane proteins) and t-SNAREs (syntaxins and SNAP25, synaptosomal-associated protein of 25 kDa) based on their localization on trafficking vesicles or target membranes, respectively. To mediate vesicle fusion with target membranes, SNARE proteins form a four-helix bundle (SNARE complex) consisting of two coiled-coil domains from SNAP25, one coiled-coil domain from syntaxin, and a coiled-coil domain from VAMPs (18). Formation of the helical bundle can be disrupted by neurotoxins, which specifically cleave different SNARE proteins (19). Each SNARE subfamily is composed of genes with high homology but different tissue specificity and subcellular localization. It remains to be determined whether individual SNAREs play specific roles in regulating the membrane trafficking of individual proteins.To address how AMPA and GABA_A receptors are sorted in the exocytic pathway and what molecules are involved in regulating exocytosis of these receptors, we specifically studied constitutive exocytosis of AMPA and GABA_A receptor subunits using total internal reflection fluorescence microscopy (TIRFM) in combination with immunocytochemistry, electrophysiology, and electron microscopy methods. Together, we revealed that AMPA and GABA_A receptors are initially sorted into different vesicles in the Golgi apparatus and delivered to different domains at the plasma membrane and are regulated by specific Rab proteins and SNARE complexes. These results reveal fundamental mechanisms underlying the sorting of excitatory and inhibitory neurotransmitter receptors in neurons and uncover the specific trafficking machinery involved in the constitutive exocytosis of each receptor type.     

Postsynaptic activity reverses the sign of the acetylcholine-induced long-term plasticity of GABAA inhibition

Acetylcholine (ACh) regulates forms of plasticity that control cognitive functions but the underlying mechanisms remain largely unknown. ACh controls the intrinsic excitability, as well as the synaptic excitation and inhibition of CA1 hippocampal pyramidal cells (PCs), cells known to participate in circuits involved in cognition and spatial navigation. However, how ACh regulates inhibition in function of postsynaptic activity has not been well studied. Here we show that in rat PCs, a brief pulse of ACh or a brief stimulation of cholinergic septal fibers combined with repeated depolarization induces strong long-term enhancement of GABA_A inhibition (GABA_A-LTP). Indeed, this enhanced inhibition is due to the increased activation of α₅βγ₂ subunit-containing GABA_A receptors by the GABA released. GABA_A-LTP requires the activation of M1-muscarinic receptors and an increase in cytosolic Ca²⁺. In the absence of PC depolarization ACh triggered a presynaptic depolarization-induced suppression of inhibition (DSI), revealing that postsynaptic activity gates the effects of ACh from presynaptic DSI to postsynaptic LTP. These results provide key insights into mechanisms potentially linked with cognitive functions, spatial navigation, and the homeostatic control of abnormal hyperexcitable states.Long-term potentiation (LTP) at excitatory synapses is thought to be the cellular substrate of learning of the brain. Less is known about LTP at inhibitory synapses, a vital process given that inhibition regulates network behavior and LTP at excitatory synapses (1–3). Cholinergic activity can influence intrinsic excitability, as well as both excitatory (4, 5) and inhibitory synaptic plasticity (6, 7). However, less is known about the postsynaptic cholinergic-mediated control of synaptic inhibition and specifically of its regulation by postsynaptic activity. The CA1 region of the hippocampus receives a significant cholinergic projection from the medial septal nuclei (8). These act primarily through acetylcholine (ACh) muscarinic receptors (mAChRs) on CA1 pyramidal cells (PCs) (9), as well as through mAChRs and nicotinic cholinergic receptors (nAChRs) on interneurons (10). In addition, the retrograde modulation of γ-aminobutyric acid (GABA)-mediated inhibition by endocannabinoids (eCBs) (11) and its regulation by ACh and postsynaptic activity have been analyzed (12).We analyzed the modifications induced in PCs in the CA1 of rat hippocampal slices by repeated postsynaptic depolarization, applied in combination with a single brief ACh pulse delivered to the apical dendritic shaft. The postsynaptic depolarization reproduced either the rhythmic bursting that typifies the hippocampal theta rhythm [i.e., theta burst stimulation (TBS)] or that of prolonged repeated depolarization. Indeed, these protocols induced a robust long-term enhancement of inhibition because of the increased activation of α₅βγ₂ subunit-containing GABA_A receptors (GABA_ARs) by the released GABA, with no involvement of GABA_BRs. We termed this long-term enhancement of inhibition GABA_A-LTP. GABA_A-LTP was also evoked by a physiological relevant stimulation of cholinergic septal fibers of the oriens/alveus (O/A), combined with repeated depolarization or TBS stimulation. This GABA_A-LTP required activation of the M1 subtype mAChRs (M1-mAChRs) and an increased cytosolic Ca²⁺. In the absence of postsynaptic depolarization, ACh generated a type 1 eCB receptor (CB₁R)-dependent depolarization-induced suppression of inhibition (DSI) (13), indicating that the effects of ACh on synaptic inhibition depend on the active or quiescent state of the postsynaptic PC. Therefore, ACh triggers a state-dependent gating that transfers the dominant effects of postsynaptic activity from presynaptic DSI to postsynaptic LTP. Such a relocation may be essential to regulate the network activity that may be linked to the information-processing capacity of the system in terms of spatial and cognitive functions (14) and of the homeostatic control of abnormal hyperexcitable states.     

Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission

In many brain regions, gephyrin and GABA_A receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.Chemical synaptic transmission between neurons requires the tight packing of ionotropic neurotransmitter receptors in the postsynaptic plasma membrane. Core components of many inhibitory GABAergic postsynapses are the cell adhesion protein neuroligin 2 (NL2), the scaffolding protein gephyrin, the guanine nucleotide exchange factor (GEF) collybistin (Cb), and GABA_A receptors (GABA_ARs) (1, 2). The assembly of such GABAergic postsynapses is triggered by the interaction of NL2 with the src homology 3 (SH3) domain of Cb. This leads to the activation of Cb, which is otherwise autoinhibited by intramolecular interactions of its SH3 domain with the Dbl homology (DH) and pleckstrin homology (PH) domains, followed by membrane recruitment of Cb and synaptic accumulation of gephyrin and GABA_ARs (3). However, this NL2/Cb/gephyrin/GABA_AR interaction cascade cannot account for the formation of all GABAergic synapses, because in the hippocampus of NL2 KO mice gephyrin and GABA_AR clusters are lost only from perisomatic regions of CA1 pyramidal neurons (3). In contrast, deletion of Cb leads to a loss of gephyrin from both perisomatic and dendritic postsynapses (4). Thus, the formation of a substantial subset of GABAergic postsynapses must be regulated by Cb-interacting proteins other than NL2.Another class of Cb interaction partners with a potential role in gephyrin clustering are small Rho-like GTPases. They regulate many fundamental cellular processes, including actin cytoskeleton rearrangements (5), and the actin cytoskeleton plays an important role in the formation of inhibitory postsynapses, particularly at early stages of synapse formation (6, 7). The small GTPase Cdc42 is an established Cb substrate (8–10), and a recent analysis of 12 Rho-like GTPases identified Cdc42 as the only family member that can be activated in vitro by the human Cb ortholog hPem2 (11). However, Cdc42 expression is not required for gephyrin and GABA_AR clustering at postsynapses, indicating that Cb may regulate cytoskeleton remodeling by activating other Rho-like GTPases (10). The small GTPase most closely related to Cdc42 is TC10. Its sequence [67.4% amino acid identity (12)] and structure (13) are similar to those of Cdc42, it shares common cellular functions and effectors with Cdc42 (14), and profilin, an actin and gephyrin binding protein (15, 16), is an effector of TC10 (14). In contrast to Cdc42, which is ubiquitously expressed in the mammalian brain, the expression of TC10 is limited to specific areas, including the CA1 region of the hippocampus (17), where the most prominent reduction in gephyrin and GABA_AR clustering is observed in Cb KO mice (4). Here we provide evidence for an effector-type binding of GTP-TC10 to the PH domain of Cb that results in Cb activation, triggers synaptic gephyrin clustering, and enhances GABAergic neurotransmission.     

Long-term potentiation of glycinergic synapses triggered by interleukin 1β

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABA_A receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.Glycine mediates fast synaptic inhibition throughout the spinal cord, brainstem, and midbrain, controlling normal motor behavior and rhythm generation, somatosensory processing, auditory and retinal signaling, and coordination of reflex responses (1). Strychnine-sensitive glycine receptors (GlyRs) are pentameric ligand-gated chloride channels of the Cys-loop receptor family that together with GABA_A receptors (GABA_ARs) dynamically interact with the synaptic scaffold protein gephyrin to form inhibitory synapses (1, 2). In the dorsal horn of the spinal cord, glycinergic synapses are essential for nociceptive and tactile sensory processing both during adaptive and pathological pain states (3–7). However, compared with glutamatergic and GABAergic synapses, little is known about the regulation of their synaptic strength. Several studies have examined glycine receptor trafficking in cultured neurons and in heterologous expression systems (8, 9). Intracellular Ca²⁺ appears important in the stabilization of GlyRs at synapses in culture (10), and elevation of intracellular Ca²⁺ can also potently increase glycine receptor single channel openings in cultured cells and in heterologous systems (11). However, the modulation of glycinergic synaptic strength in native tissue remains relatively unexplored.Following peripheral injury or inflammation, changes in tactile perception develop, including hyperalgesia (exaggerated pain upon noxious stimulation), allodynia (pain in response to innocuous stimuli), and secondary hyperalgesia (pain spreading beyond the confines of the injured region). Inhibitory interneurons of the spinal dorsal horn have been proposed to gate the flow of innocuous and nociceptive sensory information from the periphery to higher brain centers (12), and supportive evidence for this idea is growing (13–17). Loss of GABAergic/glycinergic inhibition contributes to enhanced transmission of nociceptive signals through the dorsal horn circuit during pain states, resulting in hyperalgesia and allodynia (3, 18–20). For example, polysynaptic A-fiber inputs onto neurokinin 1 receptor (NK1R)-expressing projection neurons become apparent only when GABA_AR and GlyRs are pharmacologically blocked, indicating that under conditions of disinhibition, nonnoxious mechanical stimuli can drive nociceptive-specific projection pathways and elicit allodynia (21). The majority of neurons tested in the dorsal horn receive glycinergic synapses, including lamina I projection neurons, both excitatory and inhibitory interneurons of lamina II (22, 23), and inhibitory glycinergic neurons (24). Given the diversity of afferent targets, it is likely that glycinergic synapses are differentially modulated in a cell type- and subregion-specific manner. For example, during chronic inflammation, prostaglandin E₂ selectively depresses glycinergic synaptic inputs onto nonglycinergic neurons (24). Similarly, peripheral nerve injury suppresses glycinergic inhibition of a specific excitatory interneuron class [protein kinase C (PKC)γ⁺ neurons receiving Aβ fiber inputs], allowing excitatory afferents carrying nonnociceptive tactile information to activate ascending projections of nociceptive pathways that are normally under strong inhibitory control (23).Both hyperalgesia and allodynia occur within minutes of peripheral inflammation, but the mechanisms underlying these rapid perceptual alterations are poorly understood. The proinflammatory cytokine, IL-1β, is a potent hyperalgesic agent (25–27), contributing both to peripheral and central sensitization after tissue damage (28–31). Following tissue trauma, nerve injury, or inflammation, IL-1β levels are up-regulated in the spinal cord itself (29, 32, 33), and delivery of IL-1β intrathecally increases the activity of superficial dorsal horn neurons that transmit pain signals to the brain (34, 35). Intrathecal delivery of an IL-1 receptor antagonist blocks allodynia in rodent models of inflammatory pain (36, 37). A recent study also found that IL-1β application rapidly potentiated primary afferent (glutamatergic) synapses in dorsal horn slices, through unidentified signaling molecules released from glial cells (38). Here we report that IL-1β rapidly elicits a postsynaptic form of long-term potentiation (LTP) at glycinergic synapses on lamina II inhibitory neurons (GlyR LTP), and that the same glycinergic synapses are potentiated after peripheral inflammation.     

Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation

Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABA_A receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phospho-mutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKII-dependent phosphorylation site on gephyrin as critically important for this process.Several activity-dependent plasticity and homeostatic mechanisms (1, 2) contribute to regulate synaptic strength at excitatory synapses. Similar mechanisms are also expected to finely tune the level of inhibition in response to activity in individual neurons, but the mechanisms remain poorly understood. Different forms of plasticity at GABAergic synapses have been reported based on either presynaptic or postsynaptic mechanisms (3, 4). Similar to receptors at excitatory synapses, GABA_A receptors (GABA_ARs), which mediate the fast component of inhibitory transmission, display complex trafficking mechanisms that affect the surface localization and diffusion of receptors (5). The distribution and clustering of GABA_ARs at synapses is tightly regulated through interactions with the scaffolding protein gephyrin, one of the main structural constituent of inhibitory postsynaptic densities. Gephyrin forms multimeric complexes that allow the anchoring of GABA_ARs (6) via molecular mechanisms that include phosphorylation and interactions with the guanine-nucleotide exchange factor collybistin (7–12). In addition to changes in inhibitory strength, more recent in vivo experiments revealed that inhibitory synapses are also dynamic structures that can be formed and eliminated in response to sensory experience (13–15). The mechanisms implicated in the coordinated regulation of excitatory and inhibitory plasticity remain, however, poorly understood. We investigated here this issue by using repetitive confocal imaging of tagged gephyrin to monitor the dynamic behavior of perisomatic inhibitory synapses over periods of days. Our results show that induction of synaptic plasticity and neuronal activity induces the formation of newly formed inhibitory synapses through postsynaptic mechanisms involving the phosphorylation of gephyrin at a CaMKII-dependent site.     

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABA_B, but not GABA_A, receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA_B receptor (GABA_BR) blockade or genetic deletion of the GB_1a receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA_BRs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB_1a-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB_1a intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca_V2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB_1b-containing receptors. Thus, GABA_BRs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA_BR as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure''s persistence in the absence of functional GABA_BRs.Neural circuits achieve an ongoing balance between plasticity and stability to enable adaptations to constantly changing environments while maintaining neuronal activity within a stable regime. Hebbian-like plasticity, reflected by persistent changes in synaptic and intrinsic properties, is crucial for refinement of neural circuits and information storage; however, alone it is unlikely to account for the stable functioning of neural networks (1). In the last 2 decades, major progress has been made toward understanding the homeostatic negative feedback systems underlying restoration of a baseline neuronal function after prolonged activity perturbations (2–4). Homeostatic processes may counteract the instability by adjusting intrinsic neuronal excitability, inhibition-to-excitation balance, and synaptic strength via postsynaptic or presynaptic modifications (5, 6) through a profound molecular reorganization of synaptic proteins (7, 8). These stabilizing mechanisms have been collectively termed homeostatic plasticity. Homeostatic mechanisms enable invariant firing rates and patterns of neural networks composed from intrinsically unstable activity patterns of individual neurons (9).However, nervous systems are not always capable of maintaining constant output. Although some mutations, genetic knockouts, or pharmacologic perturbations induce a compensatory response that restores network firing properties around a predefined “set point” (10), the others remain uncompensated, or their compensation leads to pathological function (11). The inability of neural networks to compensate for a perturbation may result in epilepsy and various types of psychiatric disorders (12). Therefore, determining under which conditions activity-dependent regulation fails to compensate for a perturbation and identifying the key regulatory molecules of neuronal homeostasis is critical for understanding the function and malfunction of central neural circuits.In this work, we explored the mechanisms underlying the failure in stabilizing hippocampal network activity by combining long-term extracellular spike recordings by multielectrode arrays (MEAs), intracellular patch-clamp recordings of synaptic responses, imaging of synaptic vesicle exocytosis, and calcium dynamics, together with FRET-based analysis of intermolecular interactions at individual synapses. Our results demonstrate that metabotropic, G protein-coupled receptors for GABA, GABA_BRs, are essential for firing rate homeostasis in hippocampal networks. We explored the mechanisms by which GABA_BRs gate homeostatic synaptic plasticity. Our study raises the possibility that persistence of epileptic seizures in GABA_BR-deficient mice (13–15) is directly linked to impairments in a homeostatic control system.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

GABAB(1) receptor subunit isoforms differentially regulate stress resilience

Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABA_B receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABA_B(2) subunit with either a GABA_B(1a) or a GABA_B(1b) subunit. Here we show that mice lacking the GABA_B(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABA_B(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABA_B(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABA_B(1b)^−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABA_B(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression.Although chronic and/or severe stress is a risk factor for the development of several different psychiatric disorders including depression and anxiety, many individuals remain resilient to such negative effects of stress. The mechanisms underlying this resilience are not yet fully understood, although it is thought to involve a complex interplay between several environmental factors such as social support and biological and genetic risk factors (1, 2). Currently, there is an impetus to determine the neural substrates underlying stress resilience and susceptibility, as these are poised to be key novel targets for the development of more effective treatments for depression and anxiety disorders.Accumulating evidence suggests that GABA_B receptors may be important therapeutic targets for the treatment of stress-related psychiatric disorders such as anxiety and depression (3–5). Functional GABA_B receptors are composed of heterodimers of GABA_B(1) and GABA_B(2) subunits (6). Interestingly, the GABA_B(1) subunit is expressed as different isoforms, and in the brain the predominant isoforms are GABA_B(1a) and GABA_B(1b) (6). Mice deficient in GABA_B(1a) and GABA_B(1b) exhibit differential cognitive and conditioned fear responses, indicating a potential role for these isoforms in psychiatric illness (7–10). Recent postmortem brain studies report alterations in the expression of GABA_B receptor subunits in depression (4, 11), and clinical studies suggest that neurophysiology deficits in GABA_B receptors may play a role in major depression (12) and the antidepressant response (13). In addition, mice lacking functional GABA_B receptors exhibit an antidepressant-like phenotype and increased anxiety (14, 15), and pharmacological blockade of these receptors induces antidepressant-like behavior (16–18). GABA_B receptor antagonists have also recently been shown to increase cell proliferation in the adult hippocampus (16), which is an important regulator of stress- and antidepressant-related neuroplasticity. However, the specific role of GABA_B receptor isoforms in stress sensitivity is unclear. Therefore, we assessed the susceptibility and resilience to stress during either early life (maternal separation) or adulthood (psychosocial stress) in GABA_B(1a)^−/− and GABA_B(1b)^−/− mice.     

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A_2AR-D₂R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D₂R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A_2AR agonists, but also A_2AR antagonists, decrease the affinity and intrinsic efficacy of D₂R agonists and the affinity of D₂R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A_2AR-D₂R heteromers as heterotetramers, constituted by A_2AR and D₂R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A_2AR antagonists behaved as A_2AR agonists and decreased D₂R function in the brain.Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A_2A receptor (A_2AR) agonists to decrease the affinity of dopamine D₂ receptor (D₂R) agonists in the A_2AR-D₂R heteromer (5). A_2AR-D₂R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6, 8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1–3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A_2AR-D₂R heteromer, where a decrease in D₂R agonist affinity cannot alone explain the ability of an A_2AR agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D₂R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8). Again, the A_2AR-D₂R heteromer provides a valuable example. A recent study has shown that different levels of intracellular Ca²⁺ exert different modulations of A_2AR-D₂R heteromer signaling (8). This depends on the ability of low and high Ca²⁺ to promote a selective interaction of the heteromer with different Ca²⁺-binding proteins, which differentially modulate allosteric interactions in the heteromer (8).It has been hypothesized that the allosteric interactions between A_2AR and D₂R agonists within the A_2AR-D₂R heteromer provide a mechanism responsible not only for the depressant effects of A_2AR agonists, but also for the psychostimulant effects of adenosine A_2AR antagonists and the nonselective adenosine receptor antagonist caffeine (9, 11, 12), with implications for several neuropsychiatric disorders (13). In fact, the same mechanism has provided the rationale for the use of A_2AR antagonists in patients with Parkinson’s disease (13, 14). The initial aim of the present study was to study in detail the ability of caffeine to counteract allosteric modulations between A_2AR and D₂R agonists (affinity and intrinsic efficacy) within the A_2AR-D₂R heteromer. Unexpectedly, when performing control radioligand-binding experiments, not only an A_2AR agonist, but also caffeine, significantly decreased D₂R agonist binding. However, when coadministered, the A_2AR agonist and caffeine co-counteracted their ability to modulate D₂R agonist binding. By exploring the molecular mechanisms behind these apparent inconsistencies, the present study provides new insight into the quaternary structure and function of A_2AR-D₂R heteromers.     

Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A_2A receptor (A_2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A_2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A_2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A_2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A_2AR deletion; and (iv) selective A_2AR deletion in forebrain neurons. Notably, A_2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A_2AR antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A_2AR in the control of chronic stress-induced modifications and suggest A_2AR as candidate targets to alleviate the consequences of chronic stress on brain function.Repeated stress elicits neurochemical and morphological changes that negatively affect brain functioning (1, 2). Thus, repeated stress is a trigger or a risk factor for neuropsychiatric disorders, namely depression, in both humans and animal models (2, 3). Given the absence of effective therapeutic tools, novel strategies to manage the impact of chronic stress are needed, and analyzing particular lifestyles can provide important leads. Notably, caffeine consumption increases in stressful conditions (4) and correlates inversely with the incidence of depression (5, 6) and the risk of suicide (7, 8). However, the molecular targets operated by caffeine to afford these beneficial effects have not been defined.Caffeine is the most widely consumed psychoactive drug. The only molecular targets for caffeine at nontoxic doses are the main adenosine receptors in the brain, namely the inhibitory A₁ receptors (A₁R) and the facilitatory A_2A receptors (A_2AR) (9). A_2AR blockade affords robust protection against noxious brain conditions (10), an effect that might result from the ability of neuronal A_2AR to control aberrant plasticity (11, 12) and synaptotoxicity (13–15) or from A_2AR’s impact on astrocytes (16) or microglia (17). The protection provided by A_2AR blockade prompts the hypothesis that A_2AR antagonism may underlie the beneficial effects of caffeine on chronic stress, in accordance with the role of synaptic (18, 19) or glial dysfunction (20) in mood disorders. Thus, A_2AR antagonists prolonged escape behavior in two screening tests for antidepressant activity (21–23) and prevented maternal separation-induced long-term cognitive impact (12). We combined pharmacological and tissue-selective A_2AR transgenic mice (24, 25) to test if neuronal A_2AR controlled the modifications caused by chronic unpredictable stress (CUS).