RESUMO
The discovery of genetic variants in the cholinergic receptor nicotinic CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula-interpeduncular axis as a critical relay circuit in the control of nicotine dependence. Although clear roles for α3, ß4, and α5 receptors in nicotine aversion and withdrawal have been established, the cellular and molecular mechanisms that participate in signaling nicotine use and contribute to relapse have not been identified. Here, using translating ribosome affinity purification (TRAP) profiling, electrophysiology, and behavior, we demonstrate that cholinergic neurons, but not peptidergic neurons, of the medial habenula (MHb) display spontaneous tonic firing of 2-10 Hz generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels and that infusion of the HCN pacemaker antagonist ZD7288 in the habenula precipitates somatic and affective signs of withdrawal. Further, we show that a strong, α3ß4-dependent increase in firing frequency is observed in these pacemaker neurons upon acute exposure to nicotine. No change in the basal or nicotine-induced firing was observed in cholinergic MHb neurons from mice chronically treated with nicotine. We observe, however, that, during withdrawal, reexposure to nicotine doubles the frequency of pacemaking activity in these neurons. These findings demonstrate that the pacemaking mechanism of cholinergic MHb neurons controls withdrawal, suggesting that the heightened nicotine sensitivity of these neurons during withdrawal may contribute to smoking relapse.
Assuntos
Relógios Biológicos/efeitos dos fármacos , Neurônios Colinérgicos , Habenula , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Síndrome de Abstinência a Substâncias , Animais , Cardiotônicos/farmacologia , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Habenula/metabolismo , Habenula/patologia , Habenula/fisiopatologia , Humanos , Camundongos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Pirimidinas/farmacologia , Fumar/metabolismo , Fumar/patologia , Fumar/fisiopatologia , Abandono do Hábito de Fumar , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na(+)-H(+) exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3ß4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation.
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Receptores Nicotínicos/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Canais Iônicos Sensíveis a Ácido , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Encéfalo/fisiologia , Linhagem Celular , Guanidinas/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Pirazóis/farmacologia , Canais de Sódio/deficiência , Canais de Sódio/genética , Canais de Sódio/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Transmissão Sináptica/efeitos dos fármacos , XenopusRESUMO
The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3ß4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that ß4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of ß4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that ß4A90I and ß4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant ß4D447Y, significantly increased nicotine-evoked current amplitudes, while ß4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express ß4 or ß4 variants in the MHb. Immunoprecipitation studies confirmed that ß4 lentiviral-mediated expression leads to specific upregulation of α3ß4 but not ß2 nAChRs in the Mhb. Mice injected with the ß4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the ß4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the ß4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular ß4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.
RESUMO
Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.
Assuntos
Córtex Cerebral/metabolismo , Inibição Neural/fisiologia , Sinapses/metabolismo , Animais , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Receptores de GABA-A/metabolismo , Sinapses/ultraestrutura , XenopusRESUMO
Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3ß4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the ß4 subunit is rate limiting for receptor activity, and that current increase by ß4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a ß4-specific residue (S435), mapping to the intracellular vestibule of the α3ß4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the medial habenula (MHb). Thus, this study both provides insights into α3ß4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.