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Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and ß4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3ß4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and ß4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and ß4 subunits disappeared in α5ß4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that α3ß4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3ß4-containing nAChRs in the crucial pathway of nicotine dependence.
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Habénula , Núcleo Interpeduncular , Receptores Nicotínicos , Animales , Masculino , Ratones , Habénula/metabolismo , Núcleo Interpeduncular/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Sinapsis/metabolismoRESUMEN
Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxykynurenine (3-HK) protects from lethal bacterial infection. 3-HK, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages through a systemic mechanism that targets kainate-sensitive glutamate receptors. These findings reveal a new pathway by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with important implications for the coordination between the immune and nervous systems in pathological conditions.
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Bacterial infection involves a complex interaction between the pathogen and host where the outcome of infection is not solely determined by pathogen eradication. To identify small molecules that promote host survival by altering the host-pathogen dynamic, we conducted an in vivo chemical screen using zebrafish embryos and found that treatment with 3-hydroxy-kynurenine protects from lethal gram-negative bacterial infection. 3-hydroxy-kynurenine, a metabolite produced through host tryptophan metabolism, has no direct antibacterial activity but enhances host survival by restricting bacterial expansion in macrophages by targeting kainate-sensitive glutamate receptors. These findings reveal new mechanisms by which tryptophan metabolism and kainate-sensitive glutamate receptors function and interact to modulate immunity, with significant implications for the coordination between the immune and nervous systems in pathological conditions.
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Long-term potentiation (LTP), a well-characterized form of synaptic plasticity, is believed to underlie memory formation. Hebbian, postsynaptically expressed LTP requires TARPγ-8 phosphorylation for synaptic insertion of AMPA receptors (AMPARs). However, it is unknown whether TARP-mediated AMPAR insertion alone is sufficient to modify behavior. Here, we report the development of a chemogenetic tool, ExSYTE (Excitatory SYnaptic Transmission modulator by Engineered TARPγ-8), to mimic the cytoplasmic interaction of TARP with the plasma membrane in a doxycycline-dependent manner. We use this tool to examine the specific role of synaptic AMPAR potentiation in amygdala neurons that are activated by fear conditioning. Selective expression of active ExSYTE in these neurons potentiates AMPAR-mediated synaptic transmission in a doxycycline-dependent manner, occludes synaptically induced LTP, and mimics freezing triggered by cued fear conditioning. Thus, chemogenetic controlling of the TARP-membrane interaction is sufficient for LTP-like synaptic AMPAR insertion, which mimics fear conditioning.
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Doxiciclina , Potenciación a Largo Plazo , Potenciación a Largo Plazo/fisiología , Doxiciclina/farmacología , Sinapsis/metabolismo , Transmisión Sináptica , LípidosRESUMEN
The transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8) constitutes an auxiliary subunit of AMPA receptors, which mediates various brain functions including learning and memory. TARP γ-8 has emerged as a promising therapeutic target for central nervous system disorders. Despite considerable efforts, previously reported TARP γ-8 PET radioligands, such as [11C]TARP-1903 and [11C]TARP-1811 series, were plagued by limited brain uptake and/or high nonspecific binding in vivo. Herein, we developed two novel 11C-labeled probes, [11C]8 and [11C]15 (also named as [11C]TARP-2105), of which the latter exhibited a reasonable brain uptake as well as specific binding toward TARP γ-8 both in vitro and in vivo, as confirmed by blocking experiments with the commercially available TARP γ-8 inhibitor, JNJ-55511118 in the TARP γ-8-rich hippocampus. Overall, [11C]15 exhibited promising tracer characteristics and proved to be a lead positron-emission tomography ligand for the non-invasive quantification of TARP γ-8 in the mammalian brain.
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Canales de Calcio , Receptores AMPA , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Canales de Calcio/metabolismo , Hipocampo/metabolismo , Mamíferos/metabolismo , Tomografía de Emisión de Positrones , Receptores AMPA/metabolismoRESUMEN
Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.
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Lipofuscinosis Ceroideas Neuronales , Animales , Disulfuros/metabolismo , Lipoilación , Ratones , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Sinapsis/metabolismoRESUMEN
Mossy cells (MCs) of the dentate gyrus are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation, and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.SIGNIFICANCE STATEMENT Hilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.
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Fibras Musgosas del Hipocampo , Receptores de Ácido Kaínico , Animales , Ácido Glutámico , Ácido Kaínico , Ratones , Fibras Musgosas del Hipocampo/fisiología , Células Piramidales/fisiología , Receptores de Ácido Kaínico/metabolismo , Sinapsis/fisiologíaRESUMEN
Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABAA receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin-3 alpha, a putative trans-synaptic interactor with the native GABAA receptor complex, could recruit GABAA receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.
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Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Ratones , Ratones Transgénicos , Densidad Postsináptica/metabolismo , Terminales Presinápticos/metabolismoRESUMEN
Although stressful events predispose individuals to psychiatric disorders, such as depression, not all people who undergo a stressful life experience become depressed, suggesting that gene-environment interactions (GxE) determine depression risk. The ventral hippocampus (vHPC) plays key roles in motivation, sociability, anhedonia, despair-like behaviors, anxiety, sleep, and feeding, pointing to the involvement of this brain region in depression. However, the molecular mechanisms underlying the cross talk between the vHPC and GxE in shaping behavioral susceptibility and resilience to chronic stress remain elusive. Here, we show that Ca2+/calmodulin-dependent protein kinase IIß (CaMKIIß) activity in the vHPC is differentially modulated in GxE mouse models of depression susceptibility and resilience, and that CaMKIIß-mediated TARPγ-8 phosphorylation enhances the expression of AMPA receptor subunit GluA1 in the postsynaptic sites to enable stress resilience. We present previously missing molecular mechanisms underlying chronic stress-elicited behavioral changes, providing strategies for preventing and treating stress-related psychiatric disorders.
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Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary AMPA receptor subunits that play a key role in receptor trafficking and in modulating receptor gating. The ability of TARPs to slow both deactivation and desensitization is isoform specific. However, TARP isoform-specific modulation of receptor properties remains uncharacterized. Here, we compare the isoform-specific effects of γ-2, γ-3, γ-4, and γ-8 TARPs on recovery from desensitization and responses to pairs of brief applications of glutamate. All four isoforms were able to reduce receptor-mediated paired-pulse depression and significantly speed recovery from desensitization in an isoform-specific manner. In the presence of TARPs, recovery time courses were observed to contain two components, fast and slow. The proportion of fast and slow components was determined by the TARP isoform. The time constant of recovery was also altered by the duration of glutamate application. When studies with TARP chimeras were performed, TARP extracellular loops were found to play a vital role in TARP modulation of recovery. Thus, isoform-specific differences in TARP modulation of recovery from desensitization influence receptor responses to repeated brief applications of glutamate, and these differences may impact frequency-dependent synaptic signaling in the mammalian central nervous system.SIGNIFICANCE STATEMENT AMPA receptors are major determinants of excitatory synaptic strength. The channel kinetics of AMPA receptors contribute to the kinetics of synaptic transmission. Transmembrane AMPA receptor regulatory proteins (TARPs) auxiliary subunits can modulate the decay kinetics of AMPA receptors. However, whether TARP isoforms specifically modulate receptor recovery is unclear. Here, we investigated the recovery kinetics of AMPA receptors by expressing various TARP isoforms and chimeras. We observed that the TARP isoforms and duration of glutamate application uniquely modulate time constants and the proportion of fast and slow components through a previously unidentified TARP domain. Given the impact of recovery kinetics on receptor responses to repetitive stimulation such as synaptic transmission, this work will be of great interest in the field of excitatory synaptic transmission research.
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Proteínas Nucleares/fisiología , Receptores AMPA/fisiología , Línea Celular , Espacio Extracelular/fisiología , Ácido Glutámico/farmacología , Humanos , Isomerismo , Cinética , Proteínas Mutantes Quiméricas , Proteínas Nucleares/química , Técnicas de Placa-Clamp , Receptores AMPA/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.
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Adenosina Trifosfato/farmacología , Conducta Animal/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animales , Calcio/metabolismo , Potenciales Evocados/efectos de los fármacos , Femenino , Genoma , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Sistemas de Lectura Abierta/genética , Dolor/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X3/deficiencia , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/metabolismoRESUMEN
Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA-based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel-dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Adenosina Trifosfato/metabolismo , Colesterol/metabolismo , Canales Iónicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Animales , Aniones/metabolismo , Células Cultivadas , Cerebelo/citología , Mutación con Ganancia de Función , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Concentración OsmolarRESUMEN
Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish.
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Conducta Animal , Sedación Consciente , Receptores de GABA-A/metabolismo , Receptores de Serotonina/metabolismo , Pez Cebra/metabolismo , Animales , Ligandos , Inhibición Neural , Neuronas/fisiología , Antagonistas de la Serotonina/química , Proteínas de Pez Cebra/metabolismoRESUMEN
The ionotropic GABA receptor (GABAAR) mediates fast inhibition in the brain. The GABAAR pore-forming (α, ß, and non-α/ß) subunits were isolated approximately 30 years ago and have since been the focus of extensive studies. As a result, many properties of GABAARs, including subunit assembly and channel and pharmacological properties, have been discovered. However, several of the underlying mechanisms such as the process for the synaptic localization of GABAARs remain unsolved. A reinvestigation of native GABAAR complexes in the brain and primary neurons identified two major molecular constituents, namely, the transmembrane GARLH/LHFPL protein family and the inhibitory synaptic protein neuroligin 2. This identification of the principal components of native receptor complexes may provide new mechanistic insight on receptor regulation.
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Receptores de GABA-A/metabolismo , Membrana CelularRESUMEN
a-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are implicated in the pathology of neurological diseases such as epilepsy and schizophrenia. As pan antagonists for this target are often accompanied with undesired effects at high doses, one of the recent drug discovery approaches has shifted to subtype-selective AMPA receptor (AMPAR) antagonists, specifically, via modulating transmembrane AMPAR regulatory proteins (TARPs). The quantification of AMPARs by positron emission tomography (PET) would help obtain insights into disease conditions in the living brain and advance the translational development of AMPAR antagonists. Herein we report the design, synthesis and preclinical evaluation of a series of TARP γ-8 antagonists, amenable for radiolabeling, for the development of subtype-selective AMPAR PET imaging agents. Based on the pharmacology evaluation, molecular docking studies and physiochemical properties, we have identified several promising lead compounds 3, 17-19 and 21 for in vivo PET studies. All candidate compounds were labeled with [11C]COCl2 in high radiochemical yields (13-31% RCY) and high molar activities (35-196 GBq/µmol). While tracers 30 ([11C]17) &32 ([11C]21) crossed the blood-brain barrier and showed heterogeneous distribution in PET studies, consistent with TARP γ-8 expression, high nonspecific binding prevented further evaluation. To our delight, tracer 31 ([11C]3) showed good in vitro specific binding and characteristic high uptake in the hippocampus in rat brain tissues, which provides the guideline for further development of a new generation subtype selective TARP γ-8 dependent AMPAR tracers.
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Imagen Molecular/métodos , Sondas Moleculares/farmacología , Tomografía de Emisión de Positrones/métodos , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Radioisótopos de Carbono , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This corrects the article DOI: 10.1038/nrd.2018.14.
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There are significant challenges in identifying receptor-specific functional interactors in vivo. In this issue of Neuron, Ge et al. (2018) identify a novel GABAA receptor (GABAAR)-interacting protein, Clptm1, that regulates forward trafficking of GABAARs and inhibitory transmission.
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Proteómica , Receptores de GABA-A , Neuronas , Transporte de Proteínas , Ácido gamma-AminobutíricoRESUMEN
A large proportion of biomedical research and the development of therapeutics is focused on a small fraction of the human genome. In a strategic effort to map the knowledge gaps around proteins encoded by the human genome and to promote the exploration of currently understudied, but potentially druggable, proteins, the US National Institutes of Health launched the Illuminating the Druggable Genome (IDG) initiative in 2014. In this article, we discuss how the systematic collection and processing of a wide array of genomic, proteomic, chemical and disease-related resource data by the IDG Knowledge Management Center have enabled the development of evidence-based criteria for tracking the target development level (TDL) of human proteins, which indicates a substantial knowledge deficit for approximately one out of three proteins in the human proteome. We then present spotlights on the TDL categories as well as key drug target classes, including G protein-coupled receptors, protein kinases and ion channels, which illustrate the nature of the unexplored opportunities for biomedical research and therapeutic development.
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Preservation of a balance between synaptic excitation and inhibition is critical for normal brain function. A number of homeostatic cellular mechanisms have been suggested to play a role in maintaining this balance, including long-term plasticity of GABAergic inhibitory synapses. Many previous studies have demonstrated a coupling of postsynaptic spiking with modification of perisomatic inhibition. Here, we demonstrate that activation of NMDA-type glutamate receptors leads to input-specific long-term potentiation of dendritic inhibition mediated by somatostatin-expressing interneurons. This form of plasticity is expressed postsynaptically and requires both CaMKIIα and the ß2 subunit of the GABA-A receptor. Importantly, this process may function to preserve dendritic inhibition, as genetic deletion of NMDAR signaling results in a selective weakening of dendritic inhibition. Overall, our results reveal a new mechanism for linking excitatory and inhibitory input in neuronal dendrites and provide novel insight into the homeostatic regulation of synaptic transmission in cortical circuits.
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Dendritas/fisiología , Potenciación a Largo Plazo/fisiología , Proteínas del Tejido Nervioso/fisiología , Inhibición Neural/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Piramidales/fisiología , Receptores de GABA-A/fisiologíaRESUMEN
GABAA receptor (GABAAR) pentamers are assembled from a pool of 19 subunits, and variety in subunit combinations diversifies GABAAR functions to tune brain activity. Pentamers with distinct subunit compositions localize differentially at synaptic and non-synaptic sites to mediate phasic and tonic inhibition, respectively. Despite multitudes of theoretical permutations, limited subunit combinations have been identified in the brain. Currently, no molecular model exists for combinatorial GABAAR assembly in vivo. Here, we reveal assembly rules of native GABAAR complexes that explain GABAAR subunit subcellular distributions using mice and Xenopus laevis oocytes. First, α subunits possess intrinsic signals to segregate into distinct pentamers. Second, γ2 is essential for GABAAR assembly with Neuroligin-2 (NL2) and GARLHs, which localize GABAARs at synapses. Third, δ suppresses α6 synaptic localization by preventing assembly with GARLHs/NL2. These findings establish the first molecular model for combinatorial GABAAR assembly in vivo and reveal an assembly pathway regulating GABAAR synaptic localization.