RESUMO
The identification of proteins that are altered following nicotine/tobacco exposure can facilitate and positively impact the investigation of related diseases. In this report, we investigated the effects of chronic (-)-menthol exposure in 14 murine brain regions for changes in total ß2 subunit protein levels and changes in epibatidine binding levels using immunoblotting and radioligand binding assays. We identified the habenula as a region of interest due to the region's marked decreases in ß2 subunit and nAChR levels in response to chronic (-)-menthol alone. Thus, we further examined the habenula, a brain region associated with both the reward and withdrawal components of addiction, for additional protein level alterations using mass spectrometry. A total of 552 proteins with altered levels were identified after chronic (-)-menthol exposure. Enriched in the proteins with altered levels after (-)-menthol exposure were proteins associated with signaling, immune systems, RNA regulation, and protein transport. The continuation and expansion of the brain region-specific protein profiling in response to (-)-menthol will provide a better understanding of how this common flavorant in tobacco and e-liquid products may affect addiction and general health.
Assuntos
Habenula/efeitos dos fármacos , Habenula/metabolismo , Bombas de Infusão Implantáveis , Mentol/administração & dosagem , Proteogenômica/métodos , Receptores Nicotínicos/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nicotínicos/genéticaRESUMO
The identification of biomarkers that are altered following nicotine/tobacco exposure can facilitate the investigation of tobacco-related diseases. Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian central and peripheral nervous systems and the neuromuscular junction. Neuronal nAChR subunits (11) have been identified in mammals (α2-7, α9-10, ß2-4). We examined changes in ß2 nAChR subunit protein levels after chronic nicotine, (±)-menthol, or nicotine co-administered with (±)-menthol in nine murine brain regions. Our investigation of ß2 nAChR subunit level changes identified the hypothalamus as a novel region of interest for menthol exposure that demonstrated increased ß2 nAChR levels after (±)-menthol plus nicotine exposure compared to nicotine exposure alone. Using mass spectrometry, we further characterized changes in membrane protein abundance profiles in the hypothalamus to identify potential biomarkers of (±)-menthol plus nicotine exposure and proteins that may contribute to the elevated ß2 nAChR subunit levels. In the hypothalamus, 272 membrane proteins were identified with altered abundances after chronic nicotine plus menthol exposure with respect to chronic nicotine exposure without menthol. A comprehensive investigation of changes in nAChR and non-nAChR protein expression resulting from (±)-menthol plus nicotine in the brain may establish biomarkers to better understand the effects of these drugs on addiction and addiction-related diseases.
Assuntos
Nicotina , Receptores Nicotínicos , Animais , Encéfalo/metabolismo , Mentol , Camundongos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2α, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed α4, α6, and ß3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by â¼2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.
Assuntos
Potenciais de Ação/fisiologia , Neurônios Dopaminérgicos/fisiologia , Nicotiana/química , Nicotina/administração & dosagem , Fumaça , Resposta a Proteínas não Dobradas/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/administração & dosagem , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The glutamatergic subthalamic nucleus (STN) exerts control over motor output through nuclei of the basal ganglia. High-frequency electrical stimuli in the STN effectively alleviate motor symptoms in movement disorders, and cholinergic stimulation boosts this effect. To gain knowledge about the mechanisms of cholinergic modulation in the STN, we studied cellular and circuit aspects of nicotinic acetylcholine receptors (nAChRs) in mouse STN. We discovered two largely divergent microcircuits in the STN; these are regulated in part by either α4ß2 or α7 nAChRs. STN neurons containing α4ß2 nAChRs (α4ß2 neurons) received more glutamatergic inputs, and preferentially innervated GABAergic neurons in the substantia nigra pars reticulata. In contrast, STN neurons containing α7 nAChRs (α7 neurons) received more GABAergic inputs, and preferentially innervated dopaminergic neurons in the substantia nigra pars compacta. Interestingly, local electrical stimuli excited a majority (79%) of α4ß2 neurons but exerted strong inhibition in 58% of α7 neurons, indicating an additional diversity of STN neurons: responses to electrical stimulation. Chronic exposure to nicotine selectively affects α4ß2 nAChRs in STN: this treatment increased the number of α4ß2 neurons, upregulated α4-containing nAChR number and sensitivity, and enhanced the basal firing rate of α4ß2 neurons both ex vivo and in vivo. Thus, chronic nicotine enhances the function of the microcircuit involving α4ß2 nAChRs. This indicates chronic exposure to nicotinic agonist as a potential pharmacological intervention to alter selectively the balance between these two microcircuits, and may provide a means to inhibit substantia nigra dopaminergic neurons.
Assuntos
Rede Nervosa/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Núcleo Subtalâmico/efeitos dos fármacos , Animais , Colinérgicos/farmacologia , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/farmacologia , Sinapses/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacosRESUMO
Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express alpha6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive alpha6(L9'S)* receptors. alpha6(L9'S) mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by alpha6alpha4* pentamers, as alpha6(L9'S) mice lacking alpha4 subunits displayed essentially normal behavior. In alpha6(L9'S) mice, receptor numbers are normal, but loss of alpha4 subunits leads to fewer and less sensitive alpha6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires alpha4 subunits, implicating alpha6alpha4beta2* nAChRs in alpha6(L9'S) mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by alpha6(L9'S) nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in alpha6(L9'S), but not WT or alpha4KO/alpha6(L9'S), mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from alpha6(L9'S) presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate alpha6alpha4beta2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Locomoção/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Estimulantes Ganglionares/farmacologia , Hipercinese/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Nicotina/farmacologia , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Sinaptossomos/metabolismoRESUMO
These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional alpha4beta2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and alpha4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-beta-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3-1000 mum ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional alpha4* nAChRs were not found on the neuronal somata; however, nicotine acts via alpha4beta2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these alpha4beta2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of alpha4beta2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease.
Assuntos
Nicotina/administração & dosagem , Receptores Nicotínicos/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Animais , Dopamina/metabolismo , Esquema de Medicação , Potenciais Evocados , GABAérgicos/administração & dosagem , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Substância Negra/citologia , Regulação para CimaRESUMO
Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.
Assuntos
Tolerância a Medicamentos/fisiologia , Potenciação de Longa Duração/fisiologia , Mesencéfalo/metabolismo , Nicotina/administração & dosagem , Via Perfurante/metabolismo , Receptores Nicotínicos/biossíntese , Animais , Relação Dose-Resposta a Droga , Potenciação de Longa Duração/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Via Perfurante/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha4, alpha6, beta2, and beta3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing alpha6 and beta3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta3 subunits were fully functional compared with receptors with untagged beta3 subunits. We find that beta3- and alpha6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha4 and beta2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of beta3 and alpha6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha4- and alpha6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of alpha6- and beta3-containing receptors using TIRF.
Assuntos
Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Frações Subcelulares/metabolismo , Transferência Ressonante de Energia de Fluorescência , Transporte Proteico , Receptores Nicotínicos/químicaRESUMO
Nicotinic receptors containing the alpha 4 subunit (alpha 4* nAChRs) have high sensitivity and are widely expressed in the central nervous system, yet their contributions to behavioral tolerance, a hallmark of nicotine dependence, are unclear. To evaluate the contribution of alpha 4* and non-alpha 4 nAChRs in the development of tolerance to hypothermia and locomotor suppression, alpha 4 knockout (KO), hypersensitive Leu9'Ala alpha 4 knock-in, and wild-type (WT) mice received daily nicotine injections, and their behaviors were compared. Repeated selective activation of alpha 4* nAChRs in Leu9'Ala mice produced profound tolerance to hypothermia over 7 days, whereas no tolerance was observed in alpha 4 KO animals. The summed time course and temperature response (after appropriate normalizations) from these two mutant mouse strains resembled the time course of WT tolerance. In addition, daily selective activation of alpha 4* nAChRs elicited locomotor activation in Leu9'Ala mice, but nicotine suppressed activity in alpha 4 KO mice and this did not change with daily drug exposure. Again, appropriately combined responses from the two mutant strains resembled the biphasic nicotine-induced activity in WT animals. Thus, by analyzing nicotinic responses in two complementary mouse lines, one lacking alpha 4* nAChRs, the other expressing hypersensitive alpha 4* nAChRs, one can accurately separate non-alpha 4 nAChR responses from alpha 4 nAChR responses, and one can also account for WT tolerance to both hypothermia and locomotor suppression. Our study suggests a new paradigm for bridging the gap between genetic manipulation of a single receptor and whole animal behavioral studies and shows that activation of alpha 4* nAChRs is both necessary and sufficient for the expression of tolerance.
Assuntos
Adaptação Fisiológica/genética , Hipotermia/fisiopatologia , Locomoção/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nicotínicos/genéticaRESUMO
Understanding why the quit rate among smokers of menthol cigarettes is lower than non-menthol smokers requires identifying the neurons that are altered by nicotine, menthol, and acetylcholine. Dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediate the positive reinforcing effects of nicotine. Using mouse models, we show that menthol enhances nicotine-induced changes in nicotinic acetylcholine receptors (nAChRs) expressed on midbrain DA neurons. Menthol plus nicotine upregulates nAChR number and function on midbrain DA neurons more than nicotine alone. Menthol also enhances nicotine-induced changes in DA neuron excitability. In a conditioned place preference (CPP) assay, we observed that menthol plus nicotine produces greater reward-related behavior than nicotine alone. Our results connect changes in midbrain DA neurons to menthol-induced enhancements of nicotine reward-related behavior and may help explain how smokers of menthol cigarettes exhibit reduced cessation rates.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Mentol/administração & dosagem , Nicotina/administração & dosagem , Receptores Nicotínicos/fisiologia , Recompensa , Regulação para Cima/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Linhagem Celular , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Regulação para Cima/efeitos dos fármacosRESUMO
In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.
Assuntos
Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Mesencéfalo/citologia , Análise de Sequência de RNA , Tirosina 3-Mono-Oxigenase/genética , Animais , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Expressão Gênica , Camundongos , Neurônios/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Reprodutibilidade dos TestesRESUMO
GABA transporter subtype 1 (GAT1) knock-out (KO) mice display normal reproduction and life span but have reduced body weight (female, -10%; male, -20%) and higher body temperature fluctuations in the 0.2-1.5/h frequency range. Mouse GAT1 (mGAT1) KO mice exhibit motor disorders, including gait abnormality, constant 25-32 Hz tremor, which is aggravated by flunitrazepam, reduced rotarod performance, and reduced locomotor activity in the home cage. Open-field tests show delayed exploratory activity, reduced rearing, and reduced visits to the central area, with no change in the total distance traveled. The mGAT1 KO mice display no difference in acoustic startle response but exhibit a deficiency in prepulse inhibition. These open-field and prepulse inhibition results suggest that the mGAT1 KO mice display mild anxiety or nervousness. The compromised GABA uptake in mGAT1 KO mice results in an increased GABA(A) receptor-mediated tonic conductance in both cerebellar granule and Purkinje cells. The reduced rate of GABA clearance from the synaptic cleft is probably responsible for the slower decay of spontaneous IPSCs in cerebellar granule cells. There is little or no compensatory change in other proteins or structures related to GABA transmission in the mGAT1 KO mice, including GAT1-independent GABA uptake, number of GABAergic interneurons, and GABA(A)-, vesicular GABA transporter-, GAD65-, and GAT3-immunoreactive structures in cerebellum or hippocampus. Therefore, the excessive extracellular GABA present in mGAT1 KO mice results in behaviors that partially phenocopy the clinical side effects of tiagabine, suggesting that these side effects are inherent to a therapeutic strategy that targets the widely expressed GAT1 transporter system.
Assuntos
Ansiedade/genética , Ataxia/genética , Cerebelo/fisiopatologia , Proteínas da Membrana Plasmática de Transporte de GABA/deficiência , Tremor/genética , Animais , Comportamento Animal/fisiologia , Benzodiazepinas/uso terapêutico , Temperatura Corporal/genética , Peso Corporal/genética , Cerebelo/citologia , Estimulação Elétrica/métodos , Eletroencefalografia , Comportamento Exploratório/fisiologia , Antagonistas GABAérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Transtornos Neurológicos da Marcha/induzido quimicamente , Transtornos Neurológicos da Marcha/tratamento farmacológico , Transtornos Neurológicos da Marcha/genética , Glutamato Descarboxilase/metabolismo , Imuno-Histoquímica/métodos , Técnicas In Vitro , Inibição Psicológica , Isoenzimas/metabolismo , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Knockout , Atividade Motora/genética , Neurônios/fisiologia , Neurônios/efeitos da radiação , Pentilenotetrazol , Desempenho Psicomotor/fisiologia , Piridazinas/farmacologia , Receptores de GABA-A/metabolismo , Reflexo Acústico/genética , Teste de Desempenho do Rota-Rod/métodos , Convulsões/induzido quimicamente , Sinaptossomos/metabolismo , Tremor/tratamento farmacológico , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.
Assuntos
Fenótipo , Subunidades Proteicas/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Convulsões/genética , Transtornos do Sono-Vigília/genética , Animais , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Transgênicos , Mutação , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/toxicidade , Subunidades Proteicas/biossíntese , Convulsões/induzido quimicamente , Convulsões/metabolismo , Transtornos do Sono-Vigília/metabolismo , XenopusRESUMO
The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms, we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons; however, although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders.
Assuntos
Neurônios Colinérgicos/fisiologia , Locomoção/fisiologia , Mesencéfalo/fisiologia , Vias Neurais/fisiologia , Recompensa , Tegmento Mesencefálico/fisiologia , Animais , Parte Compacta da Substância Negra/fisiologia , Ratos , Área Tegmentar Ventral/fisiologiaRESUMO
Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into the global function of specific sets of neurons. We focus on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl alpha and beta) with a Sindbis virus and then activate these channels with IVM to produce inhibition via a Cl- conductance. We constructed a three-cistron Sindbis virus that expresses the alpha and beta subunits of a glutamate-gated chloride channel (GluCl) along with the green fluorescent protein (EGFP) marker. Expression of the C. elegans channel does not affect the normal spike activity or GABA/glutamate postsynaptic currents of cultured embryonic day 18 hippocampal neurons. At concentrations as low as 5 nm, IVM activates a Cl- current large enough to silence infected neurons effectively. This conductance reverses in 8 hr. These low concentrations of IVM do not potentiate GABA responses. Comparable results are observed with plasmid transfection of yellow fluorescent protein-tagged (EYFP) GluCl alpha and cyan fluorescent protein-tagged (ECFP) GluCl beta. The present study provides an in vitro model mimicking conditions that can be obtained in transgenic mice and in viral-mediated gene therapy. These experiments demonstrate the feasibility of using invertebrate ligand-activated Cl- channels as an approach to modulate excitability.
Assuntos
Canais de Cloreto/metabolismo , Ativação do Canal Iônico/fisiologia , Inibição Neural/fisiologia , Neurônios/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Caenorhabditis elegans/genética , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Canais de Cloreto/farmacologia , Cloretos/metabolismo , Estimulação Elétrica , Estudos de Viabilidade , Genes Reporter , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Ácido Glutâmico/metabolismo , Humanos , Ivermectina/farmacologia , Rim/citologia , Rim/metabolismo , Ligantes , Proteínas Luminescentes/genética , Inibição Neural/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Sindbis virus/genética , Transmissão Sináptica/efeitos dos fármacos , Transfecção/métodos , Ácido gama-Aminobutírico/metabolismoRESUMO
Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.
Assuntos
Mesencéfalo/metabolismo , Neurônios/metabolismo , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/análise , Células Clonais , Dendritos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Mesencéfalo/citologia , Camundongos , Microscopia de Fluorescência , Neurônios/química , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Subunidades Proteicas , Transporte Proteico , Receptores Nicotínicos/análise , Receptores Nicotínicos/fisiologia , Regulação para CimaRESUMO
Two series of knockin mouse strains have been constructed with point mutations that result in hypersensitive neuronal nicotinic acetylcholine receptors containing alpha 4- or alpha 7-subunits. The full expression of the stronger alleles produces neonatal excitotoxic lethality; however, mice with attenuated expression or milder alleles are viable, and display a range of hypersensitive responses to nicotine. To date, measurements have been made on nicotine-induced seizures, Straub tail, hypothermia, antinociception, electroencephalograms and cellular electrophysiological responses. These strains are helping to define the occurrence of these important receptor subtypes, and their role in the acute and chronic actions of nicotine. The hypersensitive strains may be useful for the development of nicotinic drug therapy.
Assuntos
Camundongos Mutantes/genética , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Camundongos , Modelos Animais , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Mutação Puntual , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiologia , Convulsões/induzido quimicamente , Convulsões/etiologia , Convulsões/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson's disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6ß2ß3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the ß3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4ß2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6ß2ß3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.
Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Regulação para Cima , Motivos de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Camundongos , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Receptores Nicotínicos/química , Receptores Nicotínicos/genéticaRESUMO
Several mutations in α4 or ß2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the ß2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (ß2VL). Previous studies indicated that the ß2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of ß2VL mice. No differences in ß2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the ß2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.
Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Nicotina/farmacologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/genética , Temperatura Corporal/fisiologia , Sensibilização do Sistema Nervoso Central/genética , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Dopamina/metabolismo , Distonia/induzido quimicamente , Distonia/genética , Distonia/fisiopatologia , Técnicas de Introdução de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Atividade Motora/fisiologia , Mutação de Sentido Incorreto/genética , Nicotina/administração & dosagem , Terminações Pré-Sinápticas/efeitos dos fármacos , Ensaio Radioligante/métodos , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Radioisótopos de Rubídio , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR.