RESUMO
The frequency of human social and emotional disorders varies significantly between males and females. We have recently reported that oxytocin receptor interneurons (OxtrINs) modulate female sociosexual behavior. Here, we show that, in male mice, OxtrINs regulate anxiety-related behaviors. We demonstrate that corticotropin-releasing-hormone-binding protein (CRHBP), an antagonist of the stress hormone CRH, is specifically expressed in OxtrINs. Production of CRHBP blocks the CRH-induced potentiation of postsynaptic layer 2/3 pyramidal cell activity of male, but not female, mice, thus producing an anxiolytic effect. Our data identify OxtrINs as critical for modulation of social and emotional behaviors in both females and males and reveal a molecular mechanism that acts on local medial prefrontal cortex (mPFC) circuits to coordinate responses to OXT and CRH. They suggest that additional studies of the impact of the OXT/OXTR and CRHBP/CRH pathways in males and females will be important in development of gender-specific therapies.
Assuntos
Ansiedade/psicologia , Proteínas de Transporte/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Interneurônios/metabolismo , Ocitocina/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Ocitocina/metabolismo , Caracteres Sexuais , Animais , Ansiedade/metabolismo , Comportamento Animal , Feminino , Potenciação de Longa Duração , Masculino , Redes e Vias Metabólicas , Camundongos , Fatores SexuaisRESUMO
Diabetes is far more prevalent in smokers than non-smokers, but the underlying mechanisms of vulnerability are unknown. Here we show that the diabetes-associated gene Tcf7l2 is densely expressed in the medial habenula (mHb) region of the rodent brain, where it regulates the function of nicotinic acetylcholine receptors. Inhibition of TCF7L2 signalling in the mHb increases nicotine intake in mice and rats. Nicotine increases levels of blood glucose by TCF7L2-dependent stimulation of the mHb. Virus-tracing experiments identify a polysynaptic connection from the mHb to the pancreas, and wild-type rats with a history of nicotine consumption show increased circulating levels of glucagon and insulin, and diabetes-like dysregulation of blood glucose homeostasis. By contrast, mutant Tcf7l2 rats are resistant to these actions of nicotine. Our findings suggest that TCF7L2 regulates the stimulatory actions of nicotine on a habenula-pancreas axis that links the addictive properties of nicotine to its diabetes-promoting actions.
Assuntos
Transtornos do Metabolismo de Glucose/genética , Habenula/metabolismo , Transdução de Sinais , Tabagismo/complicações , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , AMP Cíclico/metabolismo , Glucose/metabolismo , Transtornos do Metabolismo de Glucose/metabolismo , Humanos , Camundongos , Mutagênese , Nicotina/metabolismo , Células PC12 , Pâncreas/metabolismo , Ratos , Receptores Nicotínicos/metabolismo , Tabagismo/genética , Tabagismo/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
Neuromodulatory substances can be released from distal afferents for communication between brain structures or produced locally to modulate neighboring circuit elements. Corticotropin-releasing hormone (CRH) from long-range neurons in the hypothalamus projecting to the medial prefrontal cortex (mPFC) has been shown to induce anxiety-like behaviors. However, the role of CRH produced in the mPFC has not been investigated. Here we demonstrate that a specific class of mPFC interneurons that express CRH (CrhINs) releases CRH upon high-frequency stimulation to enhance excitability of layer 2/3 pyramidal cells (L2/3 PCs) expressing the CRH receptors. When stimulated at low frequency, CrhINs release GABA resulting in the inhibition of oxytocin receptor-expressing interneurons (OxtrINs) and L2/3 PCs. Conditional deletion of CRH in mPFC CrhINs and chemogenetic activation of CrhINs have opposite effects on novelty exploration in male but not in female mice, and do not affect anxiety-related behaviors in either males or females. Our data reveal that CRH produced by local interneurons in the mPFC is required for sex-specific novelty exploration and suggest that our understanding of complex behaviors may require knowledge of local and remote neuromodulatory action.
Assuntos
Hormônio Liberador da Corticotropina , Córtex Pré-Frontal , Feminino , Masculino , Animais , Camundongos , Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina , Células Piramidais , InterneurôniosRESUMO
Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.
Assuntos
Habenula , Pneumopatias , Receptores Nicotínicos , Camundongos , Animais , Nicotina/farmacologia , Nicotina/metabolismo , Habenula/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Receptores Nicotínicos/metabolismo , Neurônios Colinérgicos/metabolismo , Pneumopatias/metabolismoRESUMO
Over the last decade, the understanding of the habenula has rapidly advanced from being an understudied brain area with the Latin name 'habena" meaning "little rein", to being considered a "major rein" in the control of key monoaminergic brain centers. This ancient brain structure is a strategic node in the information flow from fronto-limbic brain areas to brainstem nuclei. As such, it plays a crucial role in regulating emotional, motivational, and cognitive behaviors and has been implicated in several neuropsychiatric disorders, including depression and addiction. This review will summarize recent findings on the medial (MHb) and lateral (LHb) habenula, their topographical projections, cell types, and functions. Additionally, we will discuss contemporary efforts that have uncovered novel molecular pathways and synaptic mechanisms with a focus on MHb-Interpeduncular nucleus (IPN) synapses. Finally, we will explore the potential interplay between the habenula's cholinergic and non-cholinergic components in coordinating related emotional and motivational behaviors, raising the possibility that these two pathways work together to provide balanced roles in reward prediction and aversion, rather than functioning independently.
Assuntos
Habenula , Núcleo Interpeduncular , Motivação , Habenula/metabolismo , Núcleo Interpeduncular/metabolismo , EmoçõesRESUMO
The habenula, an ancient small brain area in the epithalamus, densely expresses nicotinic acetylcholine receptors and is critical for nicotine intake and aversion. As such, identification of strategies to manipulate habenular activity may yield approaches to treat nicotine addiction. Here we show that GPR151, an orphan G-protein-coupled receptor (GPCR) highly enriched in the habenula of humans and rodents, is expressed at presynaptic membranes and synaptic vesicles and associates with synaptic components controlling vesicle release and ion transport. Deletion of Gpr151 inhibits evoked neurotransmission but enhances spontaneous miniature synaptic currents and eliminates short-term plasticity induced by nicotine. We find that GPR151 couples to the G-alpha inhibitory protein Gαo1 to reduce cyclic adenosine monophosphate (cAMP) levels in mice and in GPR151-expressing cell lines that are amenable to ligand screens. Gpr151- knockout (KO) mice show diminished behavioral responses to nicotine and self-administer greater quantities of the drug, phenotypes rescued by viral reexpression of Gpr151 in the habenula. These data identify GPR151 as a critical modulator of habenular function that controls nicotine addiction vulnerability.
Assuntos
Habenula/fisiologia , Plasticidade Neuronal/fisiologia , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Animais , Células CHO , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Habenula/metabolismo , Humanos , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Receptores Acoplados a Proteínas G/genética , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster, coding for the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. ß4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that ß4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. ß4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, ß4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and ß4KO self-administered more than WT mice, whereas ß4-overexpressing mice avoided nicotine injections. Viral expression of ß4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of ß4KO mice revealed dose- and region-dependent differences: ß4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas ß4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional ß4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of ß4*nAChRs in the MHb-IPN. These data indicate that ß4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENT Human genetic studies have provided strong evidence for a relationship between variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and nicotine addiction. Yet, little is known about the role of ß4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of ß4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the ß4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the ß4 subunit into either the MHb or IPN restored a "stop" signal on nicotine self-administration. These results suggest that ß4*nAChRs provide a promising novel drug target for smoking cessation.
Assuntos
Condicionamento Operante/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Nicotina/administração & dosagem , Receptores Nicotínicos/metabolismo , Recompensa , Autocontrole , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Aprendizagem por Discriminação/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Motivação/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Agonistas Nicotínicos/administração & dosagem , Receptores Nicotínicos/genética , AutoadministraçãoRESUMO
Repeated exposure to drugs of abuse can produce adaptive changes that lead to the establishment of dependence. It has been shown that allelic variation in the α5 nicotinic acetylcholine receptor (nAChR) gene CHRNA5 is associated with higher risk of tobacco dependence. In the brain, α5-containing nAChRs are expressed at very high levels in the interpeduncular nucleus (IPN). Here we identified two nonoverlapping α5 + cell populations (α5- Amigo1 and α5- Epyc ) in mouse IPN that respond differentially to nicotine. Chronic nicotine treatment altered the translational profile of more than 1,000 genes in α5- Amigo1 neurons, including neuronal nitric oxide synthase (Nos1) and somatostatin (Sst). In contrast, expression of few genes was altered in the α5- Epyc population. We show that both nitric oxide and SST suppress optically evoked neurotransmitter release from the terminals of habenular (Hb) neurons in IPN. Moreover, in vivo silencing of neurotransmitter release from the α5- Amigo1 but not from the α5- Epyc population eliminates nicotine reward, measured using place preference. This loss of nicotine reward was mimicked by shRNA-mediated knockdown of Nos1 in the IPN. These findings reveal a proaddiction adaptive response to chronic nicotine in which nitric oxide and SST are released by a specific α5+ neuronal population to provide retrograde inhibition of the Hb-IPN circuit and thereby enhance the motivational properties of nicotine.
Assuntos
Núcleo Interpeduncular/efeitos dos fármacos , Nicotina/farmacologia , Óxido Nítrico Sintase Tipo I/genética , Receptores Nicotínicos/genética , Somatostatina/genética , Tabagismo/genética , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Habenula/efeitos dos fármacos , Habenula/metabolismo , Habenula/patologia , Núcleo Interpeduncular/metabolismo , Núcleo Interpeduncular/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurotransmissores/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Nicotínicos/metabolismo , Recompensa , Somatostatina/metabolismo , Técnicas Estereotáxicas , Transmissão Sináptica , Tabagismo/metabolismo , Tabagismo/patologiaRESUMO
Behavioral flexibility and impulse control are necessary for successful execution of adaptive behavior. They are impaired in patients with damage to the prefrontal cortex (PFC) and in some clinically important conditions, such as obsessive-compulsive disorder. Although the medial prefrontal cortex (mPFC) has been investigated as a critical structure for behavioral flexibility and impulse control, the contribution of the underlying pyramidal neuron cell types in the mPFC remained to be understood. Here we show that interneuron-mediated local inactivation of pyramidal neurons in the mPFC of male and female mice induces both premature responses and choice bias, and establish that these impulsive and compulsive responses are modulated independently. Cell-type-specific photoinhibition of pyramidal deep layer corticostriatal or corticothalamic neurons reduces behavioral flexibility without inducing premature responses. Together, our data confirm the role of corticostriatal neurons in behavioral flexibility and demonstrate that flexible behaviors are also modulated by direct projections from deep layer corticothalamic neurons in the mPFC to midline thalamic nuclei.SIGNIFICANCE STATEMENT Behavioral flexibility and impulse control are indispensable for animals to adapt to changes in the environment and often affected in patients with PFC damage and obsessive-compulsive disorder. We used a probabilistic reversal task to dissect the underlying neural circuitry in the mPFC. Through characterization of the three major pyramidal cell types in the mPFC with optogenetic silencing, we demonstrated that corticostriatal and corticothalamic but not corticocortical pyramidal neurons are temporally recruited for behavioral flexibility. Together, our findings confirm the role of corticostriatal projections in cognitive flexibility and identify corticothalamic neurons as equally important for behavioral flexibility.
Assuntos
Comportamento Animal/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Algoritmos , Animais , Comportamento de Escolha , Comportamento Compulsivo/psicologia , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Feminino , Comportamento Impulsivo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/citologia , Vias Neurais/fisiologia , Optogenética , Células Piramidais/fisiologia , Tempo de Reação , Tálamo/citologia , Tálamo/fisiologiaRESUMO
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
A large number of studies have demonstrated that the nucleus accumbens (NAC) is a critical site in the neuronal circuits controlling reward responses, motivation, and mood, but the neuronal cell type(s) underlying these processes are not yet known. Identification of the neuronal cell types that regulate depression-like states will guide us in understanding the biological basis of mood and its regulation by diseases like major depressive disorder. Taking advantage of recent findings demonstrating that the serotonin receptor chaperone, p11, is an important molecular regulator of depression-like states, here we identify cholinergic interneurons (CINs) as a primary site of action for p11 in the NAC. Depression-like behavior is observed in mice after decrease of p11 levels in NAC CINs. This phenotype is recapitulated by silencing neuronal transmission in these cells, demonstrating that accumbal cholinergic neuronal activity regulates depression-like behaviors and suggesting that accumbal CIN activity is crucial for the regulation of mood and motivation.
Assuntos
Anexina A2/metabolismo , Depressão/fisiopatologia , Interneurônios/metabolismo , Núcleo Accumbens/metabolismo , Proteínas S100/metabolismo , Acetilcolina/metabolismo , Animais , Antidepressivos/farmacologia , Comportamento Animal , Depressão/metabolismo , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Fenótipo , Receptores Colinérgicos/metabolismoRESUMO
In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(ß2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.
Assuntos
Células Quimiorreceptoras/metabolismo , Receptores Nicotínicos/metabolismo , Respiração , Traqueia/fisiologia , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Paladar , Traqueia/citologia , Traqueia/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Determining the localization of intracerebral implants in rodent brain stands as a critical final step in most physiological and behaviroral studies, especially when targeting deep brain nuclei. Conventional histological approaches, reliant on manual estimation through sectioning and slice examination, are error-prone, potentially complicating data interpretation. Leveraging recent advances in tissue-clearing techniques and light-sheet fluorescence microscopy, we introduce a method enabling virtual brain slicing in any orientation, offering precise implant localization without the limitations of traditional tissue sectioning. To illustrate the method's utility, we present findings from the implantation of linear silicon probes into the midbrain interpeduncular nucleus (IPN) of anesthetized transgenic mice expressing chanelrhodopsin-2 and enhanced yellow fluorescent protein under the choline acetyltransferase (ChAT) promoter/enhancer regions (ChAT-Chr2-EYFP mice). Utilizing a fluorescent dye applied to the electrode surface, we visualized both the targeted area and the precise localization, enabling enhanced inter-subject comparisons. Three dimensional (3D) brain renderings, presented effortlessly in video format across various orientations, showcase the versatility of this approach.
RESUMO
At synaptic terminals, high voltage-activated Ca(v)2.1 and Ca(v)2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase-dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , ômega-Conotoxinas/farmacologia , Animais , Canais de Cálcio Tipo N/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Humanos , Integrases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dor/prevenção & controle , Ratos , Ratos Wistar , ômega-Conotoxinas/metabolismoRESUMO
Anxiety disorders are the most prevalent mental disorders in developed societies. Although roles for the prefrontal cortex, amygdala, hippocampus and mediodorsal thalamus in anxiety disorders are well documented, molecular mechanisms contributing to the functions of these structures are poorly understood. Here we report that deletion of Lynx2, a mammalian prototoxin gene that is expressed at high levels in anxiety associated brain areas, results in elevated anxiety-like behaviors. We show that LYNX2 can bind to and modulate neuronal nicotinic receptors, and that loss of Lynx2 alters the actions of nicotine on glutamatergic signaling in the prefrontal cortex. Our data identify Lynx2 as an important component of the molecular mechanisms that control anxiety, and suggest that altered glutamatergic signaling in the prefrontal cortex of Lynx2 mutant mice contributes to increased anxiety-related behaviors.
Assuntos
Ansiedade , Comportamento Animal , Glicoproteínas de Membrana/fisiologia , Neuropeptídeos/fisiologia , Animais , Transtornos de Ansiedade/etiologia , Ácido Glutâmico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Receptores Nicotínicos/metabolismo , Transmissão SinápticaRESUMO
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
Nicotinic acetylcholine receptors (nAChRs) affect a wide array of biological processes, including learning and memory, attention, and addiction. lynx1, the founding member of a family of mammalian prototoxins, modulates nAChR function in vitro by altering agonist sensitivity and desensitization kinetics. Here we demonstrate, through the generation of lynx1 null mutant mice, that lynx1 modulates nAChR signaling in vivo. Its loss decreases the EC(50) for nicotine by approximately 10-fold, decreases receptor desensitization, elevates intracellular calcium levels in response to nicotine, and enhances synaptic efficacy. lynx1 null mutant mice exhibit enhanced performance in specific tests of learning and memory. Consistent with reports that mutations resulting in hyperactivation of nAChRs can lead to neurodegeneration, aging lynx1 null mutant mice exhibit a vacuolating degeneration that is exacerbated by nicotine and ameliorated by null mutations in nAChRs. We conclude that lynx1 functions as an allosteric modulator of nAChR function in vivo, balancing neuronal activity and survival in the CNS.
Assuntos
Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fatores Etários , Animais , Aprendizagem por Associação/efeitos dos fármacos , Aprendizagem por Associação/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Mutantes , Mutação , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neuropeptídeos/efeitos dos fármacos , Neuropeptídeos/genética , Técnicas de Patch-Clamp , Receptores Nicotínicos/efeitos dos fármacosRESUMO
Vertebrate alpha-bungarotoxin-like molecules of the Ly-6 superfamily have been implicated as balancers of activity and survival in the adult nervous system. To determine whether a member of this family could be involved in the development of the avian ciliary ganglion, we identified 6 Gallus genes by their homology in structure to mouse lynx1 and lynx2. One of these genes, an ortholog of prostate stem cell antigen (psca), is barely detectable at embryonic day (E) 8, before neuronal cell loss in the ciliary ganglion, but increases >100-fold as the number of neurons begins to decline between E9 and E14. PSCA is highly expressed in chicken and mouse telencephalon and peripheral ganglia and correlates with expression of alpha7-containing nicotinic acetylcholine receptors (alpha7-nAChRs). Misexpressing PSCA before cell death in the ciliary ganglion blocks alpha7-nAChR activation by nicotine and rescues the choroid subpopulation from dying. Thus, PSCA, a molecule previously identified as a marker of prostate cancer, is a member of the Ly-6 neurotoxin-like family in the nervous system, and is likely to play a role as a modulator of alpha7 signaling-induced cell death during development.
Assuntos
Apoptose/genética , Proteínas Aviárias/metabolismo , Gânglios Parassimpáticos/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos/genética , Animais , Antígenos de Neoplasias , Proteínas Aviárias/genética , Sequência de Bases/genética , Galinhas , Proteínas Ligadas por GPI , Gânglios Parassimpáticos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurônios/citologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/metabolismo , Homologia de Sequência do Ácido Nucleico , Telencéfalo/embriologia , Telencéfalo/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 +/- 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
Assuntos
Conotoxinas/farmacologia , Nociceptores/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Southern Blotting , Cromossomos Artificiais Bacterianos/genética , Conotoxinas/química , DNA/biossíntese , DNA/genética , Eletrofisiologia , Feminino , Imuno-Histoquímica , Hibridização In Situ , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Camundongos , Camundongos Transgênicos , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/fisiologia , Oócitos/fisiologia , Dor/psicologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/inervação , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/genética , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Xenopus laevisRESUMO
The physiologies of cells depend on electrochemical signals carried by ion channels and receptors. Venomous animals produce an enormous variety of peptide toxins with high affinity for specific ion channels and receptors. The mammalian prototoxin lynx1 shares with alpha-bungarotoxin the ability to bind and modulate nicotinic receptors (nAChRs); however, lynx1 is tethered to the membrane via a GPI anchor. We show here that several classes of neurotoxins, including bungarotoxins and cobratoxins, retain their selective antagonistic properties when tethered to the membrane. Targeted elimination of nAChR function in zebrafish can be achieved with tethered alpha-bungarotoxin, silencing synaptic transmission without perturbing synapse formation. These studies harness the pharmacological properties of peptide toxins for use in genetic experiments. When combined with specific methods of cell and temporal expression, the extension of this approach to hundreds of naturally occurring peptide toxins opens a new landscape for cell-autonomous regulation of cellular physiology in vivo.