RESUMO
Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.
Assuntos
Hipogonadismo/enzimologia , Hipogonadismo/genética , Mutação , Sulfotransferases/genética , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Criança , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Genes de Helmintos , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Síndrome de Kallmann/enzimologia , Síndrome de Kallmann/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Sulfotransferases/química , Sulfotransferases/deficiência , Sulfotransferases/metabolismoRESUMO
GABA(A) receptors mediate both fast phasic inhibitory postsynaptic potentials and slower tonic extrasynaptic inhibition. Hyperpolarizing phasic GABAergic inhibition requires the activity of neuron-specific chloride-extruding potassium-chloride cotransporter KCC2 in adult CNS. However, the possible role of KCC2 in tonic GABAergic inhibition and the associated behaviors is unknown. Here, we have examined the role of KCC2 in phasic vs tonic GABA inhibition by measuring the behavioral effects of pharmacological agents that presumably enhance phasic vs tonic inhibition in mice that retain 15-20% of normal KCC2 protein levels. These KCC2-deficient mice show decreased sensitivity to diazepam-induced sedation and motor impairment consistent with the reported role for KCC2 in fast hyperpolarizing inhibition. In contrast, the mice exhibit normal responses to low-dose alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis; behaviors thought to be associated with tonic GABAergic inhibition. Electrophysiological recordings show that the tonic conductance is not affected. The results suggest that KCC2 activity is more critical for behaviors dependent on phasic than tonic GABAergic inhibition.
Assuntos
Anticonvulsivantes/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Diazepam/farmacologia , Etanol/farmacologia , Resposta de Imobilidade Tônica/fisiologia , Isoxazóis/farmacologia , Simportadores/deficiência , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/fisiologia , Hipocampo/efeitos da radiação , Resposta de Imobilidade Tônica/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos das Habilidades Motoras/induzido quimicamente , Transtornos das Habilidades Motoras/tratamento farmacológico , Neurônios/efeitos dos fármacos , Cotransportadores de K e Cl-RESUMO
GABA(A) receptor (GABA-AR)-mediated inhibition is critical for proper operation of neuronal networks. Synaptic inhibition either shifts the membrane potential farther away from the action potential firing threshold (hyperpolarizing inhibition) or via increase in the membrane conductance shunts the excitatory currents. However, the relative importance of these different forms of inhibition on the hippocampal function is unclear. To study the functional consequences of the absence of hyperpolarizing inhibition, we have used KCC2-deficient mice (KCC2hy/null) maintaining only 15-20% of the neuron-specific K-Cl-cotransporter. Gramicidin-perforated patch-clamp recordings in hippocampal CA1 pyramidal cells revealed that the reversal potential of the GABA-AR-mediated postsynaptic currents (E(GABA-A)) was approximately 20 mV more positive in KCC2hy/null mice than in wild-type (WT) animals. The basic glutamatergic transmission appeared unaltered in the KCC2hy/null mice, yet they displayed lowered threshold for stimulation-induced synchronous afterdischarges in the CA1 area. Also fatigue of field excitatory postsynaptic potentials/excitatory postsynaptic currents in response to repetitious stimulation was smaller in KCC2hy/null mice, indicating altered synaptic dynamics. Interestingly, this effect was present also under blockade of GABA-ARs and was dependent on the extracellular K+ concentration. Moreover, there were no differences in the levels of either long-term potentiation or long-term depression between the genotypes. The local hippocampal CA1 network can in several aspects maintain its functional viability even in the absence of hyperpolarizing inhibition in pyramidal cells. Our results underscore the central role of shunting type of inhibition in controlling the neuronal excitation/inhibition balance. Moreover, our data demonstrate a novel, unexpected role for the KCC2, namely the modulation of properties of glutamatergic transmission during repetitious afferent activity.
Assuntos
Hipocampo/citologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Receptores de GABA-A/fisiologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Inibição Neural/efeitos da radiação , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/efeitos da radiação , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Potássio/farmacologia , Células Piramidais/efeitos dos fármacos , Simportadores/deficiência , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Transmissão Sináptica/efeitos da radiação , Cotransportadores de K e Cl-RESUMO
Hyperpolarizing fast inhibitory neurotransmission by gamma-aminobutyric acid and glycine requires an efficient chloride extrusion mechanism in postsynaptic neurons. A major effector of this task in adult animals is the potassium-chloride co-transporter KCC2 that is selectively and abundantly expressed postsynaptically in most CNS neurons. Yet, the role of KCC2 in adult brain at the systems level is poorly known. Here, we characterize the behaviour of mice doubly heterozygous for KCC2 null and hypomorphic alleles that retain 15-20% of normal KCC2 protein levels in the brain. These hypomorphic KCC2-deficient mice were viable and fertile but weighed 15-20% less than wild-type littermates at 2 weeks old and thereafter. The mice displayed increased anxiety-like behaviour in several tests including elevated plus-maze and were more susceptible to pentylenetetrazole-induced seizures. Moreover, the mice were impaired in water maze learning and showed reduced sensitivity to tactile and noxious thermal stimuli in von Frey hairs, hot plate and tail flick tests. In contrast, the mice showed normal spontaneous locomotor activity in open field and Y-maze tests, and intact motor coordination in rotarod and beam tests. The results suggest that requirements for KCC2-dependent fast hyperpolarizing inhibition may differ among various functional systems of the CNS. As shunting inhibition is expected to be intact in KCC2-deficient neurons, these mice may provide a useful tool to study the specific functions and relative importance of hyperpolarizing fast synaptic inhibition in adult CNS that may have implications for human neuropsychiatric disorders, such as epilepsy, pain and anxiety.
Assuntos
Comportamento Animal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fenótipo , Simportadores/deficiência , Análise de Variância , Animais , Animais Recém-Nascidos , Anticonvulsivantes/uso terapêutico , Western Blotting/métodos , Peso Corporal/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Comportamento Exploratório/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora , Medição da Dor , Limiar da Dor/fisiologia , Pentilenotetrazol/toxicidade , Propofol/uso terapêutico , Desempenho Psicomotor/fisiologia , RNA Mensageiro/biossíntese , Tempo de Reação , Respiração/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Convulsões/tratamento farmacológico , Convulsões/etiologia , Convulsões/mortalidade , Convulsões/patologia , Comportamento Espacial/fisiologia , Coloração e Rotulagem/métodos , Simportadores/genética , Simportadores/fisiologia , Cotransportadores de K e Cl-RESUMO
Intracellular Cl- plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion-permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation-chloride cotransporters (CCCs), including the K-Cl cotransporters KCC1-4 and the Na-K-2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5-14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1-positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up-regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down-regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin-positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17-P0), there was a shift in NKCC1 expression to the neuron specific Class III beta-tubulin (betaIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.