RESUMO
Synaptic inhibition plays a crucial role in regulating neuronal excitability, which is the foundation of nervous system function. This inhibition is largely mediated by the neurotransmitters GABA and glycine that activate Cl--permeable ion channels, which means that the strength of inhibition depends on the Cl- gradient across the membrane. In neurons, the Cl- gradient is primarily mediated by two secondarily active cation-chloride cotransporters (CCCs), NKCC1 and KCC2. CCC-mediated regulation of the neuronal Cl- gradient is critical for healthy brain function, as dysregulation of CCCs has emerged as a key mechanism underlying neurological disorders including epilepsy, neuropathic pain, and autism spectrum disorder. This review begins with an overview of neuronal chloride transporters before explaining the dependent relationship between these CCCs, Cl- regulation, and inhibitory synaptic transmission. We then discuss the evidence for how CCCs can be regulated, including by activity and their protein interactions, which underlie inhibitory synaptic plasticity. For readers who may be interested in conducting experiments on CCCs and neuronal excitability, we have included a section on techniques for estimating and recording intracellular Cl-, including their advantages and limitations. Although the focus of this review is on neurons, we also examine how Cl- is regulated in glial cells, which in turn regulate neuronal excitability through the tight relationship between this nonneuronal cell type and synapses. Finally, we discuss the relatively extensive and growing literature on how CCC-mediated neuronal excitability contributes to neurological disorders.
Assuntos
Transtorno do Espectro Autista , Doenças do Sistema Nervoso , Simportadores , Humanos , Cloretos/metabolismo , Simportadores/metabolismo , Neurônios/metabolismo , Doenças do Sistema Nervoso/metabolismo , Proteínas de Membrana TransportadorasRESUMO
Kainate receptors (KARs) are glutamate-type receptors that mediate both canonical ionotropic currents and non-canonical metabotropic signalling. While KARs are expressed widely throughout the brain, synaptic KAR currents have only been recorded at a limited set of synapses, and the KAR currents that have been recorded are relatively small and slow, which has led to the question, what is the functional significance of KARs? While the KAR current itself is relatively modest, its impact on inhibition in the hippocampus can be profound. In the CA1 region of the hippocampus, presynaptic KAR activation bidirectionally regulates γ-aminobutyric acid (GABA) release in a manner that depends on the glutamate concentration; lower levels of glutamate facilitate GABA release via an ionotropic pathway, while higher levels of glutamate depress GABA release via a metabotropic pathway. Postsynaptic interneuron KAR activation increases spike frequency through an ionotropic current, which in turn can strengthen inhibition. In the CA3 region, postsynaptic KAR activation in pyramidal neurons also strengthens inhibition, but in this case through a metabotropic pathway which regulates the neuronal chloride gradient and hyperpolarizes the reversal potential for GABA (EGABA ). Taken together, the evidence for KAR-mediated regulation of the strength of inhibition via pre- and postsynaptic mechanisms provides compelling evidence that KARs are ideally positioned to regulate excitation-inhibition balance - through sensing the excitatory tone and concomitantly tuning the strength of inhibition.
Assuntos
Hipocampo , Receptores de Ácido Caínico , Hipocampo/metabolismo , Interneurônios/metabolismo , Células Piramidais/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismoRESUMO
Amyotrophic lateral sclerosis is a fatal disease resulting from motor neuron degeneration in the cortex and spinal cord. Cortical hyperexcitability is a hallmark feature of amyotrophic lateral sclerosis and is accompanied by decreased intracortical inhibition. Using electrophysiological patch-clamp recordings, we revealed parvalbumin interneurons to be hypoactive in the late pre-symptomatic SOD1*G93A mouse model of amyotrophic lateral sclerosis. We discovered that using adeno-associated virus-mediated delivery of chemogenetic technology targeted to increase the activity of the interneurons within layer 5 of the primary motor cortex, we were able to rescue intracortical inhibition and reduce pyramidal neuron hyperexcitability. Increasing the activity of interneurons in the layer 5 of the primary motor cortex was effective in delaying the onset of amyotrophic lateral sclerosis-associated motor deficits, slowing symptom progression, preserving neuronal populations, and increasing the lifespan of SOD1*G93A mice. Taken together, this study provides novel insights into the pathogenesis and treatment of amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Interneurônios/fisiologia , Córtex Motor/fisiologia , Inibição Neural/fisiologia , Adenoviridae , Animais , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Destreza Motora/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Superóxido Dismutase-1/genética , TransfecçãoRESUMO
Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an â¼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.
Assuntos
Membrana Celular/metabolismo , Hipocampo/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Receptores de Ácido Caínico/metabolismo , Simportadores/metabolismo , Animais , Membrana Celular/genética , Células Cultivadas , Hipocampo/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Receptores de Ácido Caínico/genética , Simportadores/genética , Cotransportadores de K e Cl- , Receptor de GluK2 CainatoRESUMO
Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Análise de Célula Única , TemozolomidaRESUMO
KCC2 is a neuron-specific K(+)-Cl(-) cotransporter that is essential for Cl(-) homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl(-) extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.
Assuntos
Cloretos/metabolismo , Hipocampo/citologia , Proteínas de Membrana/deficiência , Neurônios/metabolismo , Simportadores/metabolismo , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/citologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Simportadores/química , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG-binding protein 2 (MeCP2) gene, resulting in severe deficits in learning and memory. Alterations in synaptic plasticity have been reported in RTT, however most electrophysiological studies have been performed in male mice only, despite the fact that RTT is primarily found in females. In addition, most studies have focused on excitation, despite the emerging evidence for the important role of inhibition in learning and memory. Here, we performed an electrophysiological characterization in the CA1 region of the hippocampus in both males and females of RTT mouse models with a focus on neurogliaform (NGF) interneurons, given that they are the most abundant dendrite-targeting interneuron subtype in the hippocampus. We found that theta-burst stimulation (TBS) failed to induce long-term potentiation (LTP) in either pyramidal neurons or NGF interneurons in male or female RTT mice, with no apparent changes in short-term plasticity (STP). This failure to induce LTP was accompanied by excitation/inhibition (E/I) imbalances and altered excitability, in a sex- and cell-type specific manner. Specifically, NGF interneurons of male RTT mice displayed increased intrinsic excitability, a depolarized resting membrane potential, and decreased E/I balance, while in female RTT mice, the resting membrane potential was depolarized. Understanding the role of NGF interneurons in RTT animal models is crucial for developing targeted treatments to improve cognition in individuals with this disorder.
Assuntos
Síndrome de Rett , Masculino , Feminino , Camundongos , Animais , Síndrome de Rett/genética , Potenciação de Longa Duração , Proteína 2 de Ligação a Metil-CpG/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal/genética , Modelos Animais de DoençasRESUMO
KCC2 is the neuron-specific member of the of K(+)-Cl(-) cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl(-) from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl(-), which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K(+)-Cl(-) cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl(-) through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl(-) regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K(+)-Cl(-) cotransport in neurons.
Assuntos
Simportadores/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Transporte Biológico Ativo/fisiologia , Tamanho Celular , Cloretos/metabolismo , DNA/genética , DNA/isolamento & purificação , Feminino , Hipocampo/citologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Transporte de Íons , Microscopia Confocal , Oócitos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Xenopus , Cotransportadores de K e Cl-RESUMO
During development, neural circuit formation requires the stabilization of active γ-aminobutyric acidmediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calciumcalmodulinadenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.
Assuntos
Adenosina/metabolismo , Hipocampo/crescimento & desenvolvimento , Neurônios/fisiologia , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Antagonistas do Receptor A2 de Adenosina , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cognição , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipocampo/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso , Fosforilação , Receptor A2A de Adenosina/genética , Receptores de GABA-A/metabolismoRESUMO
Finely tuned excitation-inhibition balance is essential for proper brain function, and loss of balance resulting from reduced synaptic inhibition is associated with neurological disorders. Savardi and colleagues have discovered a novel inhibitor of a cation-chloride transporter that is required for synaptic inhibition, and which restores behaviors associated with Down syndrome (DS) and autism spectrum disorder (ASD).
RESUMO
The K+-Cl- co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl-]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAA receptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl- as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl--sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl-]i to GABAAR activity.
Assuntos
Cloretos/metabolismo , Receptores de GABA-A/metabolismo , Simportadores/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Endocitose , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Transdução de Sinais , Simportadores/genética , Transmissão Sináptica , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Cotransportadores de K e Cl-RESUMO
KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.
Assuntos
Cloretos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de Ácido Caínico/metabolismo , Simportadores/metabolismo , Animais , Feminino , Hipocampo/citologia , Homeostase , Masculino , Camundongos Endogâmicos C57BL , Neurônios/citologia , Cotransportadores de K e Cl-RESUMO
Memories are thought to be sparsely encoded in neuronal networks, but little is known about why a given neuron is recruited or allocated to a particular memory trace. Previous research shows that in the lateral amygdala (LA), neurons with increased CREB are selectively recruited to a fear memory trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability to bias neuronal recruitment, although this has not been shown experimentally. Here we use several methods to increase neuronal excitability and show this both biases recruitment into the memory trace and enhances memory formation. Moreover, artificial activation of these neurons alone is a sufficient retrieval cue for fear memory expression, showing that these neurons are critical components of the memory trace. These results indicate that neuronal memory allocation is based on relative neuronal excitability immediately before training.