RESUMO
Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.
Assuntos
Retroalimentação Fisiológica , Microglia/fisiologia , Inibição Neural , Neurônios/fisiologia , 5'-Nucleotidase/metabolismo , Potenciais de Ação , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Cálcio/metabolismo , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Inibição Neural/genética , Receptor A1 de Adenosina/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Fatores de TempoRESUMO
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
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
Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and ß2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including ß3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and ß4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.
Assuntos
Receptores Nicotínicos , Tabagismo , Encéfalo/metabolismo , Humanos , Nicotina , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , RecompensaRESUMO
Cigarette smoking has long been recognized as a risk factor for type 2 diabetes (T2D), although the precise causal mechanisms underlying this relationship remain poorly understood. Recent evidence suggests that nicotine, the primary reinforcing component in tobacco, may play a pivotal role in connecting cigarette smoking and T2D. Extensive research conducted in both humans and animals has demonstrated that nicotine can elevate blood glucose levels, disrupt glucose homeostasis, and induce insulin resistance. The review aims to elucidate the genetic variants of nicotinic acetylcholine receptors associated with diabetes risk and provide a comprehensive overview of the available data on the mechanisms through which nicotine influences blood glucose homeostasis and the development of diabetes. Here we emphasize the central and peripheral actions of nicotine on the release of glucoregulatory hormones, as well as its effects on glucose tolerance and insulin sensitivity. Notably, the central actions of nicotine within the brain, which encompass both insulin-dependent and independent mechanisms, are highlighted as potential targets for intervention strategies in diabetes management.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Receptores Nicotínicos , Animais , Humanos , Nicotina/efeitos adversos , Glicemia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptores Nicotínicos/genética , HomeostaseRESUMO
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
Allelic variation in CHRNA3, the gene encoding the α3 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco dependence and smoking-related diseases, but little is known about the role for α3-containing (α3*) nAChRs in regulating the addiction-related behavioral or physiological actions of nicotine. α3* nAChRs are densely expressed by medial habenula (mHb) neurons, which project almost exclusively to the interpeduncular nucleus (IPn) and are known to regulate nicotine avoidance behaviors. We found that Chrna3tm1.1Hwrt hypomorphic mice, which express constitutively low levels of α3* nAChRs, self-administer greater quantities of nicotine (0.4 mg kg-1 per infusion) than their wild-type littermates. Microinfusion of a lentivirus vector to express a short-hairpin RNA into the mHb or IPn to knock-down Chrna3 transcripts markedly increased nicotine self-administration behavior in rats (0.01-0.18 mg kg-1 per infusion). Using whole-cell recordings, we found that the α3ß4* nAChR-selective antagonist α-conotoxin AuIB almost completely abolished nicotine-evoked currents in mHb neurons. By contrast, the α3ß2* nAChR-selective antagonist α-conotoxin MII only partially attenuated these currents. Finally, micro-infusion of α-conotoxin AuIB (10 µm) but not α-conotoxin MII (10 µm) into the IPn in rats increased nicotine self-administration behavior. Together, these data suggest that α3ß4* nAChRs regulate the stimulatory effects of nicotine on the mHb-IPn circuit and thereby regulate nicotine avoidance behaviors. These findings provide mechanistic insights into how CHRNA3 risk alleles can increase the risk of tobacco dependence and smoking-related diseases in human smokers.SIGNIFICANCE STATEMENT Allelic variation in CHRNA3, which encodes the α3 nicotinic acetylcholine receptor (nAChR) subunit gene, increases risk of tobacco dependence but underlying mechanisms are unclear. We report that Chrna3 hypomorphic mice consume greater quantities of nicotine than wild-type mice and that knock-down of Chrna3 gene transcripts in the habenula or interpeduncular nucleus (IPn) increases nicotine intake in rats. α-Conotoxin AuIB, a potent antagonist of the α3ß4 nAChR subtype, reduced the stimulatory effects of nicotine on habenular neurons, and its infusion into the IPn increased nicotine intake in rats. These data suggest that α3ß4 nAChRs in the habenula-IPn circuit regulate the motivational properties of nicotine.
Assuntos
Habenula/metabolismo , Núcleo Interpeduncular/metabolismo , Receptores Nicotínicos/metabolismo , Tabagismo/metabolismo , Animais , Feminino , Predisposição Genética para Doença/genética , Variação Genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Receptores Nicotínicos/genética , Tabagismo/genéticaAssuntos
Abandono do Hábito de Fumar , Humanos , Fumar/efeitos adversos , Fumar Tabaco , Aumento de PesoRESUMO
The addiction-relevant molecular, cellular, and behavioral actions of nicotine are derived from its stimulatory effects on neuronal nicotinic acetylcholine receptors (nAChRs) in the central nervous system. nAChRs expressed by dopamine-containing neurons in the ventral midbrain, most notably in the ventral tegmental area (VTA), contribute to the reward-enhancing properties of nicotine that motivate the use of tobacco products. nAChRs are also expressed by neurons in brain circuits that regulate aversion. In particular, nAChRs expressed by neurons in the medial habenula (mHb) and the interpeduncular nucleus (IPn) to which the mHb almost exclusively projects regulate the "set-point" for nicotine aversion and control nicotine intake. Different nAChR subtypes are expressed in brain reward and aversion circuits and nicotine intake is titrated to maximally engage reward-enhancing nAChRs while minimizing the recruitment of aversion-promoting nAChRs. With repeated exposure to nicotine, reward- and aversion-related nAChRs and the brain circuits in which they are expressed undergo adaptations that influence whether tobacco use will transition from occasional to habitual. Genetic variation that influences the sensitivity of addiction-relevant brain circuits to the actions of nicotine also influence the propensity to develop habitual tobacco use. Here, we review some of the key advances in our understanding of the mechanisms by which nicotine acts on brain reward and aversion circuits and the adaptations that occur in these circuits that may drive addiction to nicotine-containing tobacco products.
Assuntos
Nicotina/farmacologia , Tabagismo/fisiopatologia , Tabagismo/psicologia , Adaptação Fisiológica , Animais , Dopamina/fisiologia , Humanos , Receptores Nicotínicos , RecompensaRESUMO
Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.
Assuntos
Cocaína/administração & dosagem , Locomoção/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Sinapses/metabolismo , Animais , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/patologia , Feminino , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Autoadministração , Síndrome de Abstinência a Substâncias/patologia , Sinapses/efeitos dos fármacos , Sinapses/patologiaRESUMO
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
Conserved across vertebrates, the habenular nuclei are a pair of small symmetrical structures in the epithalamus. The nuclei functionally link the forebrain and midbrain by receiving input from and projecting to several brain regions. Each habenular nucleus comprises two major asymmetrical subnuclei, the medial and lateral habenula. These subnuclei are associated with different physiological processes and disorders, such as depression, nicotine addiction, and encoding aversive stimuli or omitting expected rewarding stimuli. Elucidating the functions of the habenular nuclei at the molecular level requires knowledge of their neuropeptide complement. In this work, three mass spectrometry (MS) techniques-liquid chromatography (LC) coupled to Orbitrap tandem MS (MS/MS), LC coupled to Fourier transform (FT)-ion cyclotron resonance (ICR) MS/MS, and matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS-were used to uncover the neuropeptide profiles of the rodent medial and lateral habenula. With the assistance of tissue stabilization and bioinformatics, a total of 262 and 177 neuropeptides produced from 27 and 20 prohormones were detected and identified from the medial and lateral habenula regions, respectively. Among these neuropeptides, 136 were exclusively found in the medial habenula, and 51 were exclusively expressed in the lateral habenula. Additionally, novel sites of sulfation, a rare post-translational modification, on the secretogranin I prohormone are identified. The results demonstrate that these two small brain nuclei have a rich and differentiated peptide repertoire, with this information enabling a range of follow-up studies.
Assuntos
Habenula/química , Neuropeptídeos/análise , Proteômica/métodos , Animais , Cromogranina B/metabolismo , Epitálamo/química , Processamento de Proteína Pós-Traducional , Ratos , Sulfatos/metabolismoRESUMO
To truly reduce the rates of chronic kidney disease, a root cause of kidney damage, obesity, must be targeted. Weight loss is often unsustainable because imbalances in satiety regulators are frequently not addressed to ensure maintenance of weight loss. In a recent study, gastric bypass surgery rebalanced satiety signals through resensitization of the gut-brain axis in obesity. This research may lead to noninvasive strategies to reduce obesity and obesity-related kidney disease.
Assuntos
Derivação Gástrica , Obesidade , Encéfalo , Humanos , Redução de PesoRESUMO
Genetic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor subunit, increases vulnerability to tobacco addiction and lung cancer, but the underlying mechanisms are unknown. Here we report markedly increased nicotine intake in mice with a null mutation in Chrna5. This effect was 'rescued' in knockout mice by re-expressing α5 subunits in the medial habenula (MHb), and recapitulated in rats through α5 subunit knockdown in MHb. Remarkably, α5 subunit knockdown in MHb did not alter the rewarding effects of nicotine but abolished the inhibitory effects of higher nicotine doses on brain reward systems. The MHb extends projections almost exclusively to the interpeduncular nucleus (IPN). We found diminished IPN activation in response to nicotine in α5 knockout mice. Further, disruption of IPN signalling increased nicotine intake in rats. Our findings indicate that nicotine activates the habenulo-interpeduncular pathway through α5-containing nAChRs, triggering an inhibitory motivational signal that acts to limit nicotine intake.
Assuntos
Habenula/metabolismo , Nicotina/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais , Animais , Feminino , Habenula/efeitos dos fármacos , Habenula/fisiologia , Masculino , Camundongos , Camundongos Knockout , Nicotina/farmacocinética , Nicotina/farmacologia , Ratos , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , Recompensa , Transdução de Sinais/efeitos dos fármacos , Tabagismo/genética , Tabagismo/metabolismoRESUMO
Hypocretin (orexin) and dynorphin are neuropeptides with opposing actions on motivated behavior. Orexin is implicated in states of arousal and reward, whereas dynorphin is implicated in depressive-like states. We show that, despite their opposing actions, these peptides are packaged in the same synaptic vesicles within the hypothalamus. Disruption of orexin function blunts the rewarding effects of lateral hypothalamic (LH) stimulation, eliminates cocaine-induced impulsivity, and reduces cocaine self-administration. Concomitant disruption of dynorphin function reverses these behavioral changes. We also show that orexin and dynorphin have opposing actions on excitability of ventral tegmental area (VTA) dopamine neurons, a prominent target of orexin-containing neurons, and that intra-VTA orexin antagonism causes decreases in cocaine self-administration and LH self-stimulation that are reversed by dynorphin antagonism. Our findings identify a unique cellular process by which orexin can occlude the reward threshold-elevating effects of coreleased dynorphin and thereby act in a permissive fashion to facilitate reward.
Assuntos
Dinorfinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Recompensa , Área Tegmentar Ventral/metabolismo , Animais , Cocaína/administração & dosagem , Cocaína/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Dinorfinas/antagonistas & inibidores , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Comportamento Impulsivo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/antagonistas & inibidores , Receptores de Orexina/metabolismo , Orexinas , Autoadministração , Transmissão Sináptica/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
Neuronal nicotinic acetylcholine receptors containing α4, ß2, and sometimes other subunits (α4ß2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and ß2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is ß2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4ß2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4ß2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but ß2 and ß4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4ß2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.
Assuntos
Receptores Nicotínicos/metabolismo , Acetilcolina/química , Animais , Azetidinas/química , Sítios de Ligação , Citosina/química , DNA Complementar/metabolismo , Eletrofisiologia , Células HEK293 , Humanos , Mutação , Oócitos/citologia , Oócitos/metabolismo , Oxidiazóis/química , Ligação Proteica , Estrutura Terciária de Proteína , Piridinas/química , Relação Estrutura-Atividade , Xenopus laevisRESUMO
Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17ß-estradiol. Br-PBTC is much more potent than estrogens. Like 17ß-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4ß2)(α6ß2)ß3 but not (α6ß2)2ß3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.