RESUMO
A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.
Assuntos
Encéfalo/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de GABA-A/deficiência , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Marcadores de Afinidade , Animais , Azidas , Benzodiazepinas/agonistas , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/efeitos dos fármacos , Feminino , Agonistas GABAérgicos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Muscimol , Mutação/genética , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ensaio Radioligante , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genética , Transmissão Sináptica/efeitos dos fármacos , Trítio , Ácido gama-Aminobutírico/farmacologiaRESUMO
We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.
Assuntos
Química Encefálica/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Convulsivantes/farmacologia , Receptores de GABA-A , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Convulsivantes/metabolismo , Agonistas GABAérgicos/metabolismo , Agonistas GABAérgicos/farmacologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , RNA Mensageiro/análise , Ensaio Radioligante , Receptores de GABA-A/química , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Radioisótopos de EnxofreRESUMO
GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.
Assuntos
Cerebelo/citologia , Cerebelo/metabolismo , Receptores de GABA-A/genética , Animais , Western Blotting , Membrana Celular/metabolismo , Diazepam/farmacologia , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Imuno-Histoquímica , Cinética , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Mutação , Testes de Precipitina , Ligação Proteica , Regulação para CimaRESUMO
BACKGROUND: The gamma-aminobutyric acid type A receptors (GABARs) are involved in mediating some of the behavioral effects of beverage alcohol (ethanol). However, the unique pharmacological and behavioral responses conferred by each of the various receptor subunits are not well understood. METHODS: To address the role of the GABAR delta subunit in mediating ethanol responses, gene knockout mice that lack this subunit were tested for a variety of ethanol-induced behavioral responses. RESULTS: Our results indicate that, compared with controls, delta-deficient mice (delta-/-) have (1) reduced ethanol consumption, (2) attenuated withdrawal from chronic ethanol exposure, and (3) reduced anticonvulsant (seizure-protective) effects of ethanol. These mice demonstrate a normal anxiolytic response to ethanol and a normal hypothermic response to ethanol, and they develop both chronic and acute tolerance. CONCLUSIONS: These results further establish the link between GABARs and specific behavioral responses to ethanol and begin to reveal the role of the delta subunit in these responses.
Assuntos
Comportamento Animal/efeitos dos fármacos , Etanol/farmacologia , Receptores de GABA-A/fisiologia , Consumo de Bebidas Alcoólicas/genética , Animais , Anticonvulsivantes/farmacologia , Tolerância a Medicamentos/genética , Etanol/farmacocinética , Feminino , Hipotermia/induzido quimicamente , Cinética , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidades Proteicas , Receptores de GABA-A/deficiência , Receptores de GABA-A/genética , Sono , Síndrome de Abstinência a Substâncias/genéticaRESUMO
gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.
Assuntos
Comportamento Animal/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Receptores de GABA-A/genética , Esteroides/farmacologia , Animais , Azidas/metabolismo , Azidas/farmacologia , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Hipnóticos e Sedativos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muscimol/metabolismo , Muscimol/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiologia , Transtornos do Sono-Vigília/genética , Esteroides/metabolismoRESUMO
The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Memória/fisiologia , Mutação/genética , Neurônios/patologia , Condutos Olfatórios/fisiopatologia , Sequência de Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Encéfalo/anormalidades , Encéfalo/patologia , Encéfalo/fisiopatologia , Divisão Celular/fisiologia , Sistema Nervoso Central/patologia , Anormalidades Congênitas/genética , Drosophila/crescimento & desenvolvimento , Transtornos da Memória/genética , Dados de Sequência Molecular , Mutação/fisiologia , Complexo de Reconhecimento de Origem , Pupa/fisiologia , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , Transgenes/fisiologiaRESUMO
Mutations in the latheo (lat) gene disrupt associative learning in Drosophila , but a role for LAT in regulating neuronal function has not been demonstrated. Here, we report that LAT plays a central role in regulating Ca2(+)- and activity-dependent synaptic plasticity. Immunological localization of the LAT protein indicates it is present at synaptic connections of the larval neuromuscular junction (NMJ) and is enriched in presynaptic boutons. Basal synaptic transmission amplitude at the lat mutant NMJ is elevated 3- to 4-fold, and Ca2+ dependence of transmission is significantly reduced. Multiple forms of synaptic facilitation and posttetanic potentiation (PTP) are strongly depressed or absent at the mutant synapse. Our results suggest that LAT is a novel presynaptic protein with a role in the Ca2(+)-dependent synaptic modulation mechanisms necessary for behavioral plasticity.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Drosophila/fisiologia , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Proteínas de Ligação a DNA/metabolismo , Drosophila/crescimento & desenvolvimento , Condutividade Elétrica , Estimulação Elétrica , Larva/fisiologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Neurotransmissores/antagonistas & inibidores , Terminações Pré-Sinápticas/metabolismo , Pupa/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Etanol/farmacologia , Gânglios Espinais/fisiologia , Variação Genética , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Animais , Ansiedade , Membrana Celular/metabolismo , Quimera , Cruzamentos Genéticos , Éxons , Feminino , Flunitrazepam/farmacocinética , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ensaio Radioligante , Receptores de GABA-A/deficiência , Receptores de GABA-A/genética , Deleção de Sequência , Sono/efeitos dos fármacos , Síndrome de Abstinência a Substâncias , Transcrição GênicaRESUMO
Genetically engineered animals (e.g., transgenics, gene knockouts, gene knockins) are being utilized with increasing frequency to investigate the mechanisms of action of alcohol and anesthetics. By creating and analyzing animals that harbor precise, preplanned changes in candidate genes, researchers are rapidly making progress toward uncovering how these drugs exert their effects on the central nervous system to bring about their behavioral effects. Since these sedative / hypnotic agents are likely to exert their effects by altering neurotransmission, the majority of investigations to date have focused on neurotransmitter receptors and modulators of neurotransmission such as kinases.
Assuntos
Anestésicos/farmacologia , Etanol/farmacologia , Engenharia Genética , Transmissão Sináptica/fisiologia , Intoxicação Alcoólica/genética , Intoxicação Alcoólica/fisiopatologia , Animais , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo I , Fosfotransferases/genética , Fosfotransferases/fisiologia , Ratos , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos alfa/fisiologia , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/fisiologia , Receptores de GABA-A/genética , Receptores de GABA-A/fisiologia , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/fisiologia , Receptores Opioides/genética , Receptores Opioides/fisiologia , Receptores de Serotonina/genética , Receptores de Serotonina/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
The Drosophila mutant turnip (tur) was isolated on the basis of its poor performance in an olfactory learning task, and also has a reduction in protein kinase C (PKC) activity. PKC has been found in the nervous systems of a wide range of organisms and appears to have an important role in learning and memory-related processes. Unfortunately, previous reports documenting the learning defect of tur lacked the controls required to assess the origins of the poor performance of the mutant. We have analyzed the effects of the tur mutation on both associative and nonassociative learning as well as on PKC activity. Additionally, the effects of the mutation on the task-relevant sensorimotor abilities of the flies were assessed. Although we were able to replicate previous behavioral and biochemical results obtained with tur, we discovered that the tur mutation also affected response to electric shock and caused a drastic reduction in the locomotor ability of the flies. Because locomotion is an essential component of the learning assays, this result makes it impossible to conclude that tur specifically affects learning and demonstrates the crucial importance of sensorimotor controls in conditioning experiments.