RESUMO
Fear behaviors are regulated by adaptive mechanisms that dampen their expression in the absence of danger. By studying circuits and the molecular mechanisms underlying this adaptive response, we show that cholinergic neurons of the medial habenula reduce fear memory expression through GABAB presynaptic excitation. Ablating these neurons or inactivating their GABAB receptors impairs fear extinction in mice, whereas activating the neurons or their axonal GABAB receptors reduces conditioned fear. Although considered exclusively inhibitory, here, GABAB mediates excitation by amplifying presynaptic Ca(2+) entry through Cav2.3 channels and potentiating co-release of glutamate, acetylcholine, and neurokinin B to excite interpeduncular neurons. Activating the receptors for these neurotransmitters or enhancing neurotransmission with a phosphodiesterase inhibitor reduces fear responses of both wild-type and GABAB mutant mice. We identify the role of an extra-amygdalar circuit and presynaptic GABAB receptors in fear control, suggesting that boosting neurotransmission in this pathway might ameliorate some fear disorders.
Assuntos
Neurônios Colinérgicos/metabolismo , Medo/fisiologia , Habenula/fisiologia , Memória/fisiologia , Receptores de GABA-B/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais , Neurotransmissores/metabolismo , Transmissão SinápticaRESUMO
Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.
Assuntos
Comunicação Autócrina , Neurônios/metabolismo , Dor/metabolismo , Receptores de GABA-B/metabolismo , Canais de Cátion TRPV/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Retroalimentação , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.
Assuntos
Camundongos Knockout , Transdução de Sinais , Sinaptotagminas , Animais , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Camundongos , Humanos , Neurônios/metabolismo , Transmissão Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/genética , Complexo de Golgi/metabolismo , Ligação Proteica , Células HEK293RESUMO
GABAB receptors are obligatory heterodimers responsible for prolonged neuronal inhibition in the central nervous system. The two receptor subunits are encoded by GABBR1 and GABBR2. Variants in GABBR2 have been associated with a Rett-like phenotype (MIM: 617903), epileptic encephalopathy (MIM: 617904), and milder forms of developmental delay with absence epilepsy. To date, however, no phenotypes associated with pathogenic variants of GABBR1 have been established. Through GeneMatcher, we have ascertained four individuals who each have a monoallelic GABBR1 de novo non-synonymous variant; these individuals exhibit motor and/or language delay, ranging from mild to severe, and in one case, epilepsy. Further phenotypic features include varying degrees of intellectual disability, learning difficulties, autism, ADHD, ODD, sleep disorders, and muscular hypotonia. We functionally characterized the four de novo GABBR1 variants, p.Glu368Asp, p.Ala397Val, p.Ala535Thr, and p.Gly673Asp, in transfected HEK293 cells. GABA fails to efficiently activate the variant receptors, most likely leading to an increase in the excitation/inhibition balance in the central nervous system. Variant p.Gly673Asp in transmembrane domain 3 (TMD3) renders the receptor completely inactive, consistent with failure of the receptor to reach the cell surface. p.Glu368Asp is located near the orthosteric binding site and reduces GABA potency and efficacy at the receptor. GABA exhibits normal potency but decreased efficacy at the p.Ala397Val and p.Ala535Thr variants. Functional characterization of GABBR1-related variants provides a rationale for understanding the severity of disease phenotypes and points to possible therapeutic strategies.
Assuntos
Epilepsia , Deficiência Intelectual , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Receptores de GABA-B , Humanos , Epilepsia/genética , Ácido gama-Aminobutírico/metabolismo , Células HEK293 , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Receptores de GABA-B/genéticaRESUMO
Kisspeptin and γ-amino butyric acid (GABA), synthesized in the central nervous system, are critical for reproduction. Both are also expressed in peripheral organs/tissues critical to metabolic control (liver/pancreas/adipose). Many kisspeptin neurons coexpress GABAB receptors (GABABR) and GABA controls kisspeptin expression and secretion. We developed a unique mouse lacking GABABR exclusively from kisspeptin cells/neurons (Kiss1-GABAB1KO) to evaluate the impact on metabolism/reproduction. We confirmed selective deletion of GABABR from Kiss1 cells in the anteroventral periventricular nucleus/periventricular nucleus continuum (AVPV/PeN; immunofluorescence and PCR) and arcuate nucleus (ARC), medial amygdala (MeA), pituitary, liver, and testes (PCR). Young Kiss1-GABAB1KO males were fertile, with normal LH and testosterone. Kiss1 expression was similar between genotypes in AVPV/PeN, ARC, MeA, bed nucleus of the stria terminalis (BNST), and peripheral organs (testis, liver, pituitary). Kiss1-GABAB1KO males presented higher fasted glycemia and insulin levels, an impaired response to a glucose overload, reduced insulin sensitivity, and marked insulin resistance. Interestingly, when Kiss1-GABAB1KO males got older (9 mo old) their body weight (BW) increased, in part due to an increase in white adipose tissue (WAT). Old Kiss1-GABAB1KO males showed higher fasted insulin, increased pancreatic insulin content, insulin resistance, and significantly decreased pancreatic kisspeptin levels. In sum, lack of GABABR specifically in Kiss1 cells severely impacts glucose homeostasis in male mice, reinforcing kisspeptin involvement in metabolic regulation. These alterations in glucose homeostasis worsened with aging. We highlight the impact of GABA through GABABR in the regulation of the pancreas kisspeptin system in contrast to liver kisspeptin that was not affected.NEW & NOTEWORTHY We developed a unique mouse lacking GABAB receptors specifically in Kiss1 cells to evaluate the impact on reproduction and metabolism. Knockout males showed a severe impact on glucose homeostasis, which worsened with aging. These results reinforce the proposed kisspeptin involvement in metabolic regulation and highlight the impact of GABA through GABABR in the regulation of the peripheral pancreas kisspeptin system.
Assuntos
Resistência à Insulina , Insulinas , Camundongos , Animais , Masculino , Kisspeptinas/genética , Kisspeptinas/metabolismo , Resistência à Insulina/genética , Estradiol/metabolismo , Camundongos Knockout , Reprodução/genética , Homeostase , Ácido gama-Aminobutírico/metabolismoRESUMO
The neurotransmitters glutamate and γ-aminobutyric acid (GABA) transmit synaptic signals by activating fast-acting ligand-gated ion channels and more slowly acting G-protein-coupled receptors (GPCRs). The GPCRs for these neurotransmitters, metabotropic glutamate (mGlu) and GABAB receptors, are atypical GPCRs with a large extracellular domain and a mandatory dimeric structure. Recent studies have revealed how these receptors are activated through multiple allosteric interactions between subunit domains. It emerges that the molecular complexity of these receptors is further increased through association with trafficking, effector and regulatory proteins. The structure and composition of these receptors present opportunities for therapeutic intervention in mental health and neurological disorders.
Assuntos
Complexos Multiproteicos/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sítio Alostérico , Animais , Descoberta de Drogas , Humanos , Complexos Multiproteicos/química , Mapas de Interação de Proteínas , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Transdução de SinaisRESUMO
Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.
Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Cerebelo/patologia , Deleção de Genes , Hipocampo/patologia , Proteínas do Tecido Nervoso/deficiência , Receptores de GABA-B/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Cerebelo/metabolismo , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABAB receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K+-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABAB receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K+ currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABAB receptors and modulates physiologically induced K+ current responses in the hippocampus. SIGNIFICANCE STATEMENT: The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.
Assuntos
Canais de Potássio/genética , Canais de Potássio/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animais , Química Encefálica/genética , Células CHO , Cricetinae , Cricetulus , Fenômenos Eletrofisiológicos/genética , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Cinética , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores KIR/metabolismoRESUMO
Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.
Assuntos
Colecistocinina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/metabolismo , Canais de Potássio/metabolismo , Receptores de GABA-A/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Técnicas de Patch-Clamp , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
It has been demonstrated that GABAB receptors modulate nicotine (NIC) reward effect; nevertheless, the mechanism implicated is not well known. In this regard, we evaluated the involvement of GABAB receptors on the behavioral, neurochemical, biochemical and molecular alterations associated with the rewarding effects induced by NIC in mice, from a pharmacological and genetic approach. NIC-induced rewarding properties (0.5 mg/kg, subcutaneously, sc) were evaluated by conditioned place preference (CPP) paradigm. CPP has three phases: preconditioning, conditioning and postconditioning. GABAB receptor antagonist 2-hydroxysaclofen (0.25, 0.5 and 1 mg/kg; intraperitoneally, ip) or the GABAB receptor agonist baclofen (3 mg/kg; ip) was injected before NIC during the conditioning phase. GABAB1 knockout (GABAB1 KO) mice received NIC during the conditioning phase. Vehicle and wild-type controls were employed. Neurochemical (dopamine, serotonin and their metabolites), biochemical (nicotinic receptor α4ß2, α4ß2nAChRs) and molecular (c-Fos) alterations induced by NIC were analyzed after the postconditioning phase by high-performance liquid chromatography (HPLC), receptor-ligand binding assays and immunohistochemistry, respectively, in nucleus accumbens (Acb), prefrontal cortex (PFC) and ventral tegmental area (VTA). NIC induced rewarding effects in the CPP paradigm and increased dopamine levels in Acb and PFC, α4ß2nAChRs density in VTA and c-Fos expression in Acb shell (AcbSh), VTA and PFC. We showed that behavioral, neurochemical, biochemical and molecular alterations induced by NIC were prevented by baclofen. However, in 2-hydroxysaclofen pretreated and GABAB1 KO mice, these alterations were potentiated, suggesting that GABAB receptor activity is necessary to control alterations induced by NIC-induced rewarding effects. Therefore, the present findings provided important contributions to the mechanisms implicated in NIC-induced rewarding effects.
Assuntos
Encéfalo/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores de GABA-B/efeitos dos fármacos , Animais , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Encéfalo/metabolismo , Dopamina/metabolismo , Agonistas dos Receptores de GABA-B/farmacologia , Antagonistas de Receptores de GABA-B/farmacologia , Masculino , Camundongos , Camundongos Knockout , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de GABA-B/genética , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Recompensa , Serotonina/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
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 GABAB, but not GABA(A), receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA(B) receptor (GABA(B)R) blockade or genetic deletion of the GB(1a) receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA(B)Rs 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(V)2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB(1b)-containing receptors. Thus, GABA(B)Rs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA(B)R 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(B)Rs.
Assuntos
Hipocampo/fisiologia , Homeostase , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Animais , Células Cultivadas , Potenciais Evocados , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. We show that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. Our data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis.
Assuntos
Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Receptores de GABA-B/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Antagonistas de Receptores de GABA-B/farmacologia , Hipocampo/citologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Compostos Organofosforados/farmacologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-B/genética , Transdução de Sinais/fisiologiaRESUMO
GABA(B) receptors (GABA(B)Rs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the CNS. In the past 5 years, notable advances have been made in our understanding of the molecular composition of these receptors. GABA(B)Rs are now known to comprise principal and auxiliary subunits that influence receptor properties in distinct ways. The principal subunits regulate the surface expression and the axonal versus dendritic distribution of these receptors, whereas the auxiliary subunits determine agonist potency and the kinetics of the receptor response. This Review summarizes current knowledge on how the subunit composition of GABA(B)Rs affects the distribution of these receptors, neuronal processes and higher brain functions.
Assuntos
Neurônios/metabolismo , Receptores de GABA-B/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Dendritos/metabolismo , Dendritos/fisiologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Ácido Glutâmico/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Fosforilação , Filogenia , Terminações Pré-Sinápticas/fisiologia , Receptor Cross-Talk/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
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. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(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 GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression.
Assuntos
Depressão/metabolismo , Receptores de GABA-B/fisiologia , Anedonia , Animais , Comportamento Animal , Proliferação de Células , Corticosterona/metabolismo , Depressão/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Risco , Estresse Psicológico , NataçãoRESUMO
Although the dorsal raphe nucleus (DRN) has long been linked to neural control of aggression, little is known about the regulatory influences of the DRN when an animal engages in either adaptive species-typical aggressive behavior or escalated aggression. Therefore it is important to explore which neurotransmitter inputs into the DRN determine the escalation of aggression in male mice. Previously, we observed that microinjection of the GABAB receptor agonist baclofen into the DRN escalates aggressive behavior in male mice. Here, we used a serotonin (5-HT) neuron-specific GABAB receptor knock-out mouse to demonstrate that baclofen acts on nonserotonergic neurons to escalate aggression. Intra-DRN baclofen administration increased glutamate release, but did not alter GABA release, within the DRN. Microinjection of l-glutamate into the DRN escalated dose-dependently attack bites toward an intruder. In vivo microdialysis showed that glutamate release increased in the DRN during an aggressive encounter, and the level of glutamate was further increased when the animal was engaged in escalated aggressive behavior after social instigation. Finally, 5-HT release was increased within the DRN and also in the medial prefrontal cortex when animals were provoked by social instigation, and during escalated aggression after social instigation, but this increase in 5-HT release was not observed when animals were engaged in species-typical aggression. In summary, glutamate input into the DRN is enhanced during escalated aggression, which causes a phasic increase of 5-HT release from the DRN 5-HT neurons.
Assuntos
Agressão/fisiologia , Núcleo Dorsal da Rafe/fisiologia , Ácido Glutâmico/fisiologia , Agressão/efeitos dos fármacos , Animais , Baclofeno/administração & dosagem , Baclofeno/farmacologia , Núcleo Dorsal da Rafe/efeitos dos fármacos , Núcleo Dorsal da Rafe/metabolismo , Relação Dose-Resposta a Droga , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Camundongos , Camundongos Knockout , Microinjeções , Córtex Pré-Frontal/metabolismo , Receptores de GABA-B/genética , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Neurônios Serotoninérgicos/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Condutividade Elétrica , Agonistas dos Receptores de GABA-B , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Cinética , Camundongos , Neurônios/metabolismo , Oócitos/metabolismo , Potássio/metabolismo , Canais de Potássio/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Transdução de Sinais , XenopusRESUMO
G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.
Assuntos
Receptores de GABA-B/química , Transdução de Sinais/fisiologia , Regulação Alostérica/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Multimerização Proteica/genética , Estabilidade Proteica , Receptores de GABA-B/deficiência , Receptores de GABA-B/genética , Transdução de Sinais/genéticaRESUMO
Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.
Assuntos
Dendritos/metabolismo , Hipocampo/citologia , Interneurônios/metabolismo , Interneurônios/ultraestrutura , Parvalbuminas/metabolismo , Receptores de GABA-B/metabolismo , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/ultraestrutura , Colecistocinina/metabolismo , Simulação por Computador , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , GABAérgicos/farmacologia , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Modelos Neurológicos , Inibição Neural , Neuropeptídeo Y/metabolismo , Ácidos Nipecóticos/farmacologia , Ratos , Ratos Transgênicos , Ratos Wistar , Tiagabina , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca(2+)-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca(2+)-activated small-conductance type 2 (SK2)K(+) channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA(B(1a,2)) receptors [GABA(B(1a,2))Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA(B)Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA(B1)-GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA(B1a) isoform selectively inhibits release at efferent cholinergic synapses.
Assuntos
Células Ciliadas Auditivas/fisiologia , Potenciais Pós-Sinápticos Inibidores , Receptores de GABA-B/metabolismo , Sinapses/fisiologia , Acetilcolina/metabolismo , Animais , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Estimulação Elétrica , Células Ciliadas Auditivas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Eferentes/fisiologia , Receptores de GABA-B/genética , Sinapses/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
There is evidence suggesting that the GABA system in the arcuate nucleus, where orexigenic neuropeptide Y and agouti-related peptide as well as anorexigenic proopiomelanocortin (POMC) are expressed, plays an important role in energy balance. In this study, we generated POMC-specific GABAB receptor-deficient [knock-out (KO)] mice. Male KO mice on a high-fat diet (HFD) showed mild increases in body weight (BW) at the age of 9 weeks compared to wild-type (WT) mice, and the differences remained significant until 16 weeks old. However, there was no difference in BW in females between genotypes. While food intake was similar between genotypes, oxygen consumption was significantly decreased in the male KO mice. The insulin tolerance test revealed that the male KO mice were less insulin sensitive compared to WT mice at the age of 8 weeks, when there was no significant difference in BW between genotypes. Despite increased BW, POMC mRNA expression in the arcuate nucleus was significantly decreased in the KO mice compared to WT mice at the age of 16 weeks. Furthermore, the expression of TNFα as well as IL-6, proinflammatory markers in the hypothalamus, was significantly increased in the KO mice on a HFD compared to WT mice. This demonstrates that the deletion of GABAB receptors in POMC neurons in the male mice on a HFD results in obesity, insulin resistance, and hypothalamic inflammation. Furthermore, the decreased POMC expression in the obese KO mice suggests that the regulation of POMC expression through GABAB receptors is essential for proper energy balance.