RESUMO
Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.
Assuntos
Encéfalo/metabolismo , Imagem Molecular/métodos , Receptores de Orexina/genética , Orexinas/análise , Proteínas Recombinantes/metabolismo , Animais , Comportamento Animal , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Receptores de Orexina/metabolismo , Orexinas/genética , Orexinas/farmacologia , Fótons , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Sono/fisiologiaRESUMO
The precise function of specialized GABAergic interneuron subtypes is required to provide appropriate synaptic inhibition for regulating principal neuron excitability and synchronization within brain circuits. Of these, parvalbumin-type (PV neuron) dysfunction is a feature of several sex-biased psychiatric and brain disorders, although, the underlying developmental mechanisms are unclear. While the transcriptional action of sex hormones generates sexual dimorphism during brain development, whether kinase signaling contributes to sex differences in PV neuron function remains unexplored. In the hippocampus, we report that gephyrin, the main inhibitory post-synaptic scaffolding protein, is phosphorylated at serine S268 and S270 in a developmentally-dependent manner in both males and females. When examining GphnS268A/S270A mice in which site-specific phosphorylation is constitutively blocked, we found that sex differences in PV neuron density in the hippocampal CA1 present in WT mice were abolished, coincident with a female-specific increase in PV neuron-derived terminals and increased inhibitory input onto principal cells. Electrophysiological analysis of CA1 PV neurons indicated that gephyrin phosphorylation is required for sexually dimorphic function. Moreover, while male and female WT mice showed no difference in hippocampus-dependent memory tasks, GphnS268A/S270A mice exhibited sex- and task-specific deficits, indicating that gephyrin phosphorylation is differentially required by males and females for convergent cognitive function. In fate mapping experiments, we uncovered that gephyrin phosphorylation at S268 and S270 establishes sex differences in putative PV neuron density during early postnatal development. Furthermore, patch-sequencing of putative PV neurons at postnatal day 4 revealed that gephyrin phosphorylation contributes to sex differences in the transcriptomic profile of developing interneurons. Therefore, these early shifts in male-female interneuron development may drive adult sex differences in PV neuron function and connectivity. Our results identify gephyrin phosphorylation as a new substrate organizing PV neuron development at the anatomical, functional, and transcriptional levels in a sex-dependent manner, thus implicating kinase signaling disruption as a new mechanism contributing to the sex-dependent etiology of brain disorders.
RESUMO
Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABAA receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho-proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABAARs for sleep through on-demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABAA receptor subtypes, α1- and α2-GABAARs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1-GABAARs emerges as central in sleep regulation, manifesting significant alterations in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake-dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV-eGFP-gephyrin or its phospho-null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep.
RESUMO
Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen-glucose deprivation as an in vitro model for ischemia, we show that BDNF-tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Ubiquitina-Proteína Ligases , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Membrana , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Tropomiosina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
Associative memory can be rendered malleable by a reminder. Blocking the ensuing reconsolidation process is suggested as a therapeutic target for unwanted aversive memories. Matrix metalloproteinase-9 (MMP-9) is required for structural synapse remodeling involved in memory consolidation. Inhibiting MMP-9 with doxycycline is suggested to attenuate human threat conditioning. Here, we investigated whether MMP-9 inhibition also interferes with threat memory reconsolidation. Male and female human participants (N = 78) learned the association between two visual conditioned stimuli (CS+) and a 50% chance of an unconditioned nociceptive stimulus (US), and between CS- and the absence of US. On day 7, one CS+ was reminded without reinforcement 3.5 h after ingesting either 200 mg of doxycycline or placebo. On day 14, retention of CS memory was assessed under extinction by fear-potentiated startle. Contrary to our expectations, we observed a greater CS+/CS- difference in participants who were reminded under doxycycline compared with placebo. Participants who were reminded under placebo showed extinction learning during the retention test, which was not observed in the doxycycline group. There was no difference between the reminded and the nonreminded CS+ in either group. In contrast, during relearning after the retention test, the CS+/CS- difference was more pronounced in the placebo group than in the doxycycline group. To summarize, a single dose of doxycycline before threat memory reminder appeared to have no specific impact on reconsolidation, but to globally impair extinction learning, and threat relearning, beyond drug clearance.SIGNIFICANCE STATEMENT Matrix metalloproteinase-9 inhibition appears to attenuate memory consolidation. It could also be a target for blocking reconsolidation. Here, we test this hypothesis in human threat conditioning. We find that doxycycline has no specific impact on a reminded cue, but confers a global reduction in extinction learning and threat learning beyond the clearance of the drug. This may point toward a more long-lasting impact of doxycycline treatment on memory plasticity.
Assuntos
Doxiciclina/farmacologia , Medo/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Memória/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Adulto , Extinção Psicológica/efeitos dos fármacos , Extinção Psicológica/fisiologia , Medo/fisiologia , Medo/psicologia , Feminino , Humanos , Masculino , Memória/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Distribuição Aleatória , Reflexo de Sobressalto/efeitos dos fármacos , Adulto JovemRESUMO
Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.
Assuntos
Estradiol/farmacologia , Inibição Neural/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Animais , Proteínas de Transporte/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Transmissão Sináptica/efeitos dos fármacosRESUMO
In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism.
Assuntos
Neurônios/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Sinapses/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese , Neurônios/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de GABA-A/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The neurotransmitters GABA and glycine mediate fast synaptic inhibition by activating ligand-gated chloride channels--namely, type A GABA (GABA(A)) and glycine receptors. Both types of receptors are anchored postsynaptically by gephyrin, which self-assembles into a scaffold and interacts with the cytoskeleton. Current research indicates that postsynaptic gephyrin clusters are dynamic assemblies that are held together and regulated by multiple protein-protein interactions. Moreover, post-translational modifications of gephyrin regulate the formation and plasticity of GABAergic synapses by altering the clustering properties of postsynaptic scaffolds and thereby the availability and function of receptors and other signalling molecules. Here, we discuss the formation and regulation of the gephyrin scaffold, its role in GABAergic and glycinergic synaptic function and the implications for the pathophysiology of brain disorders caused by abnormal inhibitory neurotransmission.
Assuntos
Proteínas de Transporte/metabolismo , Hipocampo/fisiologia , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Proteínas de Transporte/genética , Hipocampo/metabolismo , Proteínas de Membrana/genética , Neurônios/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Sinapses/metabolismoRESUMO
Inhibitory circuits are diverse, yet with a poorly understood cell biology. Functional characterization of distinct inhibitory neuron subtypes has not been sufficient to explain how GABAergic neurotransmission sculpts principal cell activity in a relevant fashion. Our Mini-Symposium brings together several emerging mechanisms that modulate GABAergic neurotransmission dynamically from either the presynaptic or the postsynaptic site. The first two talks discuss novel developmental and neuronal subtype-specific contributions to the excitatory/inhibitory balance and circuit maturation. The next three talks examine how interactions between cellular pathways, lateral diffusion of proteins between synapses, and chloride transporter function at excitatory and inhibitory synapses and facilitate inhibitory synapse adaptations. Finally, we address functional differences within GABAergic interneurons to highlight the importance of diverse, flexible, and versatile inputs that shape network function. Together, the selection of topics demonstrates how developmental and activity-dependent mechanisms coordinate inhibition in relation to the excitatory inputs and vice versa.
Assuntos
Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Humanos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Plasticidade NeuronalRESUMO
Gephyrin is a key scaffold protein mediating the anchoring of GABAA receptors at inhibitory synapses. Here, we exploited superresolution techniques combined with proximity-based clustering analysis and model simulations to investigate the single-molecule gephyrin reorganization during plasticity of inhibitory synapses in mouse hippocampal cultured neurons. This approach revealed that, during the expression of inhibitory LTP, the increase of gephyrin density at postsynaptic sites is associated with the promoted formation of gephyrin nanodomains. We demonstrate that the gephyrin rearrangement in nanodomains stabilizes the amplitude of postsynaptic currents, indicating that, in addition to the number of synaptic GABAA receptors, the nanoscale distribution of GABAA receptors in the postsynaptic area is a crucial determinant for the expression of inhibitory synaptic plasticity. In addition, the methodology implemented here clears the way to the application of the graph-based theory to single-molecule data for the description and quantification of the spatial organization of the synapse at the single-molecule level.SIGNIFICANCE STATEMENT The mechanisms of inhibitory synaptic plasticity are poorly understood, mainly because the size of the synapse is below the diffraction limit, thus reducing the effectiveness of conventional optical and imaging techniques. Here, we exploited superresolution approaches combined with clustering analysis to study at unprecedented resolution the distribution of the inhibitory scaffold protein gephyrin in response to protocols inducing LTP of inhibitory synaptic responses (iLTP). We found that, during the expression of iLTP, the increase of synaptic gephyrin is associated with the fragmentation of gephyrin in subsynaptic nanodomains. We demonstrate that such synaptic gephyrin nanodomains stabilize the amplitude of inhibitory postsynaptic responses, thus identifying the nanoscale gephyrin rearrangement as a key determinant for inhibitory synaptic plasticity.
Assuntos
Proteínas de Transporte/metabolismo , Neurônios GABAérgicos/citologia , Depressão Sináptica de Longo Prazo/fisiologia , Proteínas de Membrana/metabolismo , Densidade Pós-Sináptica/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Algoritmos , Animais , Células Cultivadas , Simulação por Computador , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Hipocampo/citologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , N-Metilaspartato/farmacologia , Peptídeos/metabolismo , Polímeros , Densidade Pós-Sináptica/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Valina/análogos & derivados , Valina/farmacologiaRESUMO
In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post-synaptic proteins of GABAergic synapses are in part apposed to glutamatergic pre-synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre- and post-synaptic elements. We report here that the IQ motif and Sec7 domain-containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post-synaptic density (PSD) proteins. Over-expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co-expressing eGFP-gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP-gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP-gephyrin, endogenous GABAA receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over-expression. Conversely, GFP-PSD-95 clusters were unaffected by over-expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant-negative mutant over-expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca2+ concentrations. Taken together, our findings identify a post-synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre-synaptic terminals.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Sinapses/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Catálise , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Hipocampo/citologia , Processamento de Imagem Assistida por Computador , Densidade Pós-Sináptica , Gravidez , Terminações Pré-Sinápticas/metabolismo , Cultura Primária de Células , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/fisiologiaRESUMO
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 GABAA receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca(2+)/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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Inibição Neural , Sinapses/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/ultraestrutura , Hipocampo/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Inibição Neural/efeitos dos fármacos , Optogenética , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Piridazinas/farmacologia , Ratos , Receptores de GABA-A/metabolismo , Sinapses/efeitos dos fármacosRESUMO
Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity. SIGNIFICANCE STATEMENT: Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.
Assuntos
Colecistocinina/metabolismo , Distroglicanas/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Animais , Proteínas de Ligação ao Cálcio , Carbacol/farmacologia , Distroglicanas/genética , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Técnicas de Introdução de Genes , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agonistas Muscarínicos/farmacologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Alterations of neuronal activity due to changes in GABAA receptors (GABAA R) mediating tonic inhibition influence different hippocampal functions. Gabra5-null mice and α5 subunit((H105R)) knock-in mice exhibit signs of hippocampal dysfunction, but are capable of improved performance in several learning and memory tasks. Accordingly, alleviating abnormal GABAergic tonic inhibition in the hippocampal formation by selective α5-GABAA R modulators represents a possible therapeutic approach for several intellectual deficit disorders. Adult neurogenesis in the dentate gyrus is an important facet of hippocampal plasticity; it is regulated by tonic GABAergic transmission, as shown by deficits in proliferation, migration and dendritic development of adult-born neurons in Gabra4-null mice. Here, we investigated the contribution of α5-GABAA Rs to granule cell development, using retroviral vectors expressing eGFP for labeling precursor cells in the subgranular zone. Global α5-GABAA R knockout (α5-KO) mice showed no alterations in migration and morphological development of eGFP-positive granule cells. However, upregulation of α1 subunit-immunoreactivity was observed in the hippocampal formation and cerebral cortex. In contrast, partial gene inactivation in α5-heterozygous (α5-het) mice, as well as single-cell deletion of Gabra5 in newborn granule cells from α5-floxed mice, caused severe alterations of migration and dendrite development. In α5-het mice, retrovirally mediated overexpression of Cdk5 resulted in normal migration and dendritic branching, suggesting that Cdk5 cooperates with α5-GABAA Rs to regulate neuronal development. These results show that minor imbalance of α5-GABAA R-mediated transmission may have major consequences for neuronal plasticity; and call for caution upon chronic therapeutic use of negative allosteric modulators acting at these receptors.
Assuntos
Giro Denteado/citologia , Neurogênese , Neurônios/citologia , Receptores de GABA-A/metabolismo , Animais , Movimento Celular , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Espinhas Dendríticas/metabolismo , Giro Denteado/embriologia , Giro Denteado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Receptores de GABA-A/genéticaRESUMO
Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology.
Assuntos
Calpaína/metabolismo , Proteínas de Transporte/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Encéfalo/metabolismo , Eletrofisiologia/métodos , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Hipocampo/metabolismo , Humanos , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Plasmídeos/metabolismo , Ratos , Sinapses/metabolismoRESUMO
Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3ß (GSK3ß) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3ß inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca(2+)-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.
Assuntos
Proteínas de Transporte/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/citologia , Proteínas de Membrana/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Calpaína/metabolismo , Células Cultivadas , Eletrofisiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Imuno-Histoquímica , Cloreto de Lítio/farmacologia , Neurônios/metabolismo , Fosforilação , Ratos , Espectrometria de Massas em TandemRESUMO
Primary hippocampal cultures grown from genetically modified mice provide a simplified context to study molecular mechanisms underlying neuronal development, synaptogenesis, and synapse plasticity in vitro. Here, we describe a simple protocol for culturing hippocampal neurons from P0 to P2 mice and a strategy for inducing alterations in synaptic strength at inhibitory and excitatory synapses in vitro. We also describe approaches for immunofluorescent labeling, image acquisition, and quantification of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1.
Assuntos
Hipocampo , Neurônios , Animais , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Células Cultivadas , Técnicas de Cultura de Células/métodos , Sinapses/fisiologia , Sinapses/metabolismoRESUMO
Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1.
Assuntos
Dependovirus , Sinapses , Animais , Camundongos , Sinapses/metabolismo , Dependovirus/genética , Encéfalo/metabolismo , Neurônios/metabolismo , Técnicas de Transferência de Genes , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.