RESUMO
Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%âa 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.
Assuntos
Aminas , Cumarínicos , Processos Fotoquímicos , Cumarínicos/química , Aminas/química , Estrutura Molecular , FotóliseRESUMO
Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.
Assuntos
Nicotina/farmacologia , Processos Fotoquímicos , Receptores Nicotínicos/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cálcio , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Antagonism of nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb) or interpeduncular nucleus (IPN) triggers withdrawal-like behaviors in mice chronically exposed to nicotine, implying that nicotine dependence involves the sensitization of nicotinic signaling. Identification of receptor and/or neurophysiological mechanisms underlying this sensitization is important, as it could promote novel therapeutic strategies to reduce tobacco use. Using an approach involving photoactivatable nicotine, we previously demonstrated that chronic nicotine (cNIC) potently enhances nAChR function in dendrites of MHb neurons. However, whether cNIC modulates downstream components of the habenulo-interpeduncular (Hb-IP) circuit is unknown. In this study, cNIC-mediated changes to Hb-IP nAChR function were examined in mouse (male and female) brain slices using molecular, electrophysiological, and optical techniques. cNIC enhanced action potential firing and modified spike waveform characteristics in MHb neurons. Nicotine uncaging revealed nAChR functional enhancement by cNIC on proximal axonal membranes. Similarly, nAChR-driven glutamate release from MHb axons was enhanced by cNIC. In IPN, the target structure of MHb axons, neuronal morphology, and nAChR expression is complex, with stronger nAChR function in the rostral subnucleus [rostral IPN (IPR)]. As in MHb, cNIC induced strong upregulation of nAChR function in IPN neurons. This, coupled with cNIC-enhanced nicotine-stimulated glutamate release, was associated with stronger depolarization responses to brief (1 ms) nicotine uncaging adjacent to IPR neurons. Together, these results indicate that chronic exposure to nicotine dramatically alters nicotinic cholinergic signaling and cell excitability in Hb-IP circuits, a key pathway involved in nicotine dependence.SIGNIFICANCE STATEMENT This study uncovers several neuropharmacological alterations following chronic exposure to nicotine in a key brain circuit involved in nicotine dependence. These results suggest that smokers or regular users of electronic nicotine delivery systems (i.e., "e-cigarettes") likely undergo sensitization of cholinergic circuitry in the Hb-IP system. Reducing the activity of Hb-IP nAChRs, either volitionally during smoking cessation or inadvertently via receptor desensitization during nicotine intake, may be a key trigger of withdrawal in nicotine dependence. Escalation of nicotine intake in smokers, or tolerance, may involve stimulation of these sensitized cholinergic pathways. Smoking cessation therapeutics are only marginally effective, and by identifying cellular/receptor mechanisms of nicotine dependence, our results take a step toward improved therapeutic approaches for this disorder.
Assuntos
Habenula/efeitos dos fármacos , Núcleo Interpeduncular/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Nicotina/farmacologia , Animais , Feminino , Habenula/metabolismo , Núcleo Interpeduncular/metabolismo , Masculino , Camundongos , Vias Neurais/metabolismo , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Tabagismo/metabolismoRESUMO
Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse ß2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.
Assuntos
Encéfalo/fisiologia , Neurônios Colinérgicos/fisiologia , Edição de Genes/métodos , Vetores Genéticos/genética , Receptores Nicotínicos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Proteína 9 Associada à CRISPR/biossíntese , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/fisiologia , Feminino , Vetores Genéticos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de ÓrgãosRESUMO
Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and ß4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, ß2, ß3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 µm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.
Assuntos
Expressão Gênica/genética , Habenula/citologia , Habenula/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Colina O-Acetiltransferase/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Habenula/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nicotina/farmacologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Nicotinic acetylcholine receptors (nAChRs) are the molecular target of nicotine. nAChRs in the medial habenula (MHb) have recently been shown to play a role in nicotine dependence, but it is not clear which nAChR subtypes or MHb neuron types are most important. To identify MHb nAChRs and/or cell types that play a role in nicotine dependence, we studied these receptors and cells with brain slice electrophysiology using both acute and chronic nicotine application. Cells in the ventroinferior (MHbVI) and ventrolateral MHb (MHbVL) subregions expressed functional nAChRs with different pharmacology. Further, application of nicotine to cells in these subregions led to different action potential firing patterns. The latter result was correlated with a differing ability of nicotine to induce nAChR desensitization. Chronic nicotine caused functional upregulation of nAChRs selectively in MHbVI cells, but did not change nAChR function in MHbVL. Importantly, firing responses were also differentially altered in these subregions following chronic nicotine. MHbVI neurons treated chronically with nicotine exhibited enhanced basal pacemaker firing but a blunted nicotine-induced firing response. MHbVL neurons did not change their firing properties in response to chronic nicotine. Together, these results suggest that acute and chronic nicotine differentially affect nAChR function and output of cells in MHb subregions. Because the MHb extensively innervates the interpeduncular nucleus, an area critical for both affective and somatic signs of withdrawal, these results could reflect some of the neurophysiological changes thought to occur in the MHb to the interpeduncular nucleus circuit in human smokers.
Assuntos
Habenula/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/biossíntese , Tabagismo/metabolismo , Animais , Feminino , Habenula/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Nicotina/administração & dosagem , Técnicas de Cultura de Órgãos , GravidezRESUMO
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated, cation-selective ion channels expressed throughout the brain. Although these channels have been investigated for several decades, it is still challenging 1) to identify the important nAChR subunits in cholinergic transmission and nicotine dependence and 2) to develop nAChR subtype-specific ligands. To overcome these challenges, we and others have studied mice expressing mutant, gain-of-function nAChR subunits. In this review, we discuss this research approach and the results it has yielded to date. Gain-of-function mutations, including those in nAChR subunits, provide an approach that is complementary to loss-of-function studies such as gene knockouts; the former allows one to answer questions of sufficiency and the latter addresses questions of necessity. Mutant mice expressing gain-of-function nAChR subunits are commonly produced using traditional gene targeting in embryonic stem cells, but novel approaches such as bacterial artificial chromosome transgenesis have yielded important insights as well. α7 nAChRs were the first nAChRs to be targeted with a gain-of-function mutation, followed by a pair of α4 nAChR gain-of-function mutant mice. These α4 nAChR gain-of-function mice (α4 L9'S mice, followed by α4 L9'A mice) provided an important system to probe α4 nAChR function in vivo, particularly in the dopamine reward system. α6 nAChR gain-of-function mice provided the first robust system allowing specific manipulation of this receptor subtype. Other targeted mutations in various nAChR subunits have also been produced and have yielded important insights into nicotinic cholinergic biology. As nAChR research advances and more details associated with nAChR expression and function emerge, we expect that existing and new mouse lines expressing gain-of-function nAChR subunits will continue to provide new insights.
Assuntos
Mutação , Receptores Nicotínicos/fisiologia , Tabagismo/fisiopatologia , Animais , Dopamina/fisiologia , Camundongos , Neurônios/fisiologia , Subunidades Proteicas/fisiologiaRESUMO
α6ß2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6ß2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6ß2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6ß2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels.
Assuntos
Dopamina/metabolismo , Receptores Nicotínicos/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Dopamina/biossíntese , Inibidores da Captação de Dopamina/farmacologia , Fenômenos Eletrofisiológicos , Espaço Extracelular/metabolismo , Mecamilamina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neostriado/metabolismo , Neurotransmissores/metabolismo , Antagonistas Nicotínicos/farmacologia , Núcleo Accumbens/metabolismo , Técnicas de Patch-Clamp , Piperazinas/farmacologia , Reação em Cadeia da Polimerase , Receptores Nicotínicos/genética , RecompensaRESUMO
Signaling through nicotinic acetylcholine receptors (nAChRs) plays a role in cocaine reward and reinforcement, suggesting that the cholinergic system could be manipulated with therapeutics to modulate aspects of cocaine use disorder (CUD). We examined the interaction between nAChRs and cocaine reinforcement by expressing a hypersensitive ß2 nAChR subunit (ß2Leu9'Ser) in the ventral tegmental area of male Sprague Dawley rats. Compared to control rats, ß2Leu9'Ser rats acquired (fixed ratio) intravenous cocaine self-administration faster and with greater likelihood. By contrast, ß2Leu9'Ser rats were approximately equivalent to controls in their intake of cocaine on a progressive ratio schedule of reinforcement, suggesting differential effects of cholinergic signaling depending on experimental parameters. Like progressive ratio cocaine SA, ß2Leu9'Ser rats and controls did not differ significantly in food SA assays, including acquisition on a fixed ratio schedule or in progressive ratio sessions. These results highlight the specific role of high-affinity, heteropentameric ß2* (ß2-containing) nAChRs in acquisition of cocaine SA, suggesting that mesolimbic acetylcholine signaling is active during this process.
Assuntos
Cocaína , Receptores Nicotínicos , Ratos , Masculino , Animais , Ratos Sprague-Dawley , Cocaína/farmacologia , Receptores Nicotínicos/metabolismo , Transmissão Sináptica , Colinérgicos , AutoadministraçãoRESUMO
Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.
Assuntos
Neurônios/fisiologia , Receptores Nicotínicos/fisiologia , Colículos Superiores/fisiologia , Sinapses/metabolismo , Vias Visuais/fisiologia , Animais , Dopamina/metabolismo , Neurônios GABAérgicos/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Colículos Superiores/metabolismo , Vias Visuais/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine in rodents, suggesting that they may be key mediators of nicotine's action in humans. However, it is unknown which nAChR subtypes are sufficient to activate these neurons. To test the hypothesis that nAChRs containing α6 subunits are sufficient to activate VTA DA neurons, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9'S mice). In voltage-clamp recordings in brain slices from adult mice, 100 nM nicotine was sufficient to elicit inward currents in VTA DA neurons via α6ß2* nAChRs. In addition, we found that low concentrations of nicotine could act selectively through α6ß2* nAChRs to enhance the function of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors on the surface of these cells. In contrast, α6ß2* activation did not enhance N-methyl-D-aspartic acid receptor function. Finally, AMPA receptor (AMPAR) function was not similarly enhanced in brain slices from α6L9'S mice lacking α4 nAChR subunits, suggesting that α4α6ß2* nAChRs are important for enhancing AMPAR function in VTA DA neurons. Together, these data suggest that activation of α4α6ß2* nAChRs in VTA DA neurons is sufficient to support the initiation of cellular changes that play a role in addiction to nicotine. α4α6ß2* nAChRs may be a promising target for future smoking cessation pharmacotherapy.
Assuntos
Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores de AMPA/fisiologia , Receptores Nicotínicos/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Potenciais de Ação , Animais , Técnicas In Vitro , Ativação do Canal Iônico , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Subunidades Proteicas/agonistas , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologiaRESUMO
Mesolimbic nicotinic acetylcholine receptor (nAChRs) activation is necessary for nicotine reinforcement behavior, but it is unknown whether selective activation of nAChRs in the dopamine (DA) reward pathway is sufficient to support nicotine reinforcement. In this study, we tested the hypothesis that activation of ß2-containing (ß2*) nAChRs on VTA neurons is sufficient for intravenous nicotine self-administration (SA). We expressed ß2 nAChR subunits with enhanced sensitivity to nicotine (referred to as ß2Leu9'Ser) in the VTA of male Sprague Dawley (SD) rats, enabling very low concentrations of nicotine to selectively activate ß2* nAChRs on transduced neurons. Rats expressing ß2Leu9'Ser subunits acquired nicotine SA at 1.5 µg/kg/infusion, a dose too low to support acquisition in control rats. Saline substitution extinguished responding for 1.5 µg/kg/inf, verifying that this dose was reinforcing. ß2Leu9'Ser nAChRs also supported acquisition at the typical training dose in rats (30 µg/kg/inf) and reducing the dose to 1.5 µg/kg/inf caused a significant increase in the rate of nicotine SA. Viral expression of ß2Leu9'Ser subunits only in VTA DA neurons (via TH-Cre rats) also enabled acquisition of nicotine SA at 1.5 µg/kg/inf, and saline substitution significantly attenuated responding. Next, we examined electrically-evoked DA release in slices from ß2Leu9'Ser rats with a history of nicotine SA. Single-pulse evoked DA release and DA uptake rate were reduced in ß2Leu9'Ser NAc slices, but relative increases in DA following a train of stimuli were preserved. These results are the first to report that ß2* nAChR activation on VTA neurons is sufficient for nicotine reinforcement in rats.
Assuntos
Nicotina , Receptores Nicotínicos , Ratos , Masculino , Animais , Nicotina/farmacologia , Nicotina/metabolismo , Agonistas Nicotínicos/farmacologia , Área Tegmentar Ventral/metabolismo , Ratos Sprague-Dawley , Receptores Nicotínicos/metabolismo , Neurônios Dopaminérgicos/metabolismoRESUMO
The interpeduncular nucleus (IPN) plays a key role in nicotine dependence and is involved in regulation of fear responses, affective states, and novelty processing. IPN neurons express nicotinic acetylcholine receptors (nAChR) and receive strong cholinergic innervation from the ventral medial habenula. Dorsal medial habenula neurons are primarily peptidergic, releasing substance P (SP) mainly onto IPN neurons in the lateral subnucleus (IPL). IPL neurons are sensitive to SP, but it is not known if they are involved in cholinergic transmission like other IPN neurons. We examined nAChR subunit gene expression in IPL neurons, revealing that Chrna7 (α7 nAChR subunit) is expressed in a subset of GABAergic IPL neurons. In patch-clamp recordings from IPL neurons, ACh-evoked inward currents were attenuated by methyllycaconitine (α7 nAChR antagonist) and potentiated by NS1738 (α7 Type I positive allosteric modulator). We confirmed α7 functional expression in IPL neurons by also showing that ACh-evoked currents were potentiated by PNU-120596 (Type II positive allosteric modulator). Additional pharmacological experiments show that IPN neurons expressing α7 nAChRs also express α3ß4 nAChRs. Finally, we used 2-photon laser scanning microscopy and nicotine uncaging to directly examine the morphology of IPL neurons that express α7 nAChRs. These results highlight a novel aspect of α7 nAChR neurobiology, adding to the complexity of cholinergic modulation by nAChRs in the IPN.
Assuntos
Núcleo Interpeduncular , Receptores Nicotínicos , Colinérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Núcleo Interpeduncular/metabolismo , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Many tobacco smokers consume nicotine intermittently, but the underlying mechanisms and neurobiological changes associated with intermittent nicotine intake are unclear. Understanding intermittent nicotine intake is a high priority, as it could promote therapeutic strategies to attenuate tobacco consumption. We examined nicotine intake behavior and neurobiological changes in male rats that were trained to self-administer nicotine during brief (5 min) trials interspersed with longer (15 min) drug-free periods. Rats readily adapted to intermittent access (IntA) SA following acquisition on a continuous access (ContA) schedule. Probabilistic analysis of IntA nicotine SA suggested reduced nicotine loading behavior compared to ContA, and nicotine pharmacokinetic modeling revealed that rats taking nicotine intermittently may have increased intake to maintain blood levels of nicotine that are comparable to ContA SA. After IntA nicotine SA, rats exhibited an increase in unreinforced responses for nicotine-associated cues (incubation of craving) and specific alterations in the striatal proteome after 7 days without nicotine. IntA nicotine SA also induced nAChR functional upregulation in the interpeduncular nucleus (IPN), and it enhanced nicotine binding in the brain as determined via [11C]nicotine positron emission tomography. Reducing the saliency of the cue conditions during the 5 min access periods attenuated nicotine intake, but incubation of craving was preserved. Together, these results indicate that IntA conditions promote nicotine SA and nicotine seeking after a nicotine-free period.
Assuntos
Núcleo Interpeduncular , Nicotina , Animais , Comportamento Animal , Comportamento de Procura de Droga , Núcleo Interpeduncular/metabolismo , Masculino , Ratos , Recidiva , AutoadministraçãoRESUMO
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
The RGS7 (R7) family of RGS proteins bound to the divergent Gbeta subunit Gbeta5 is a crucial regulator of G protein-coupled receptor (GPCR) signaling in the visual and nervous systems. Here, we identify R7BP, a novel neuronally expressed protein that binds R7-Gbeta5 complexes and shuttles them between the plasma membrane and nucleus. Regional expression of R7BP, Gbeta5, and R7 isoforms in brain is highly coincident. R7BP is palmitoylated near its COOH terminus, which targets the protein to the plasma membrane. Depalmitoylation of R7BP translocates R7BP-R7-Gbeta5 complexes from the plasma membrane to the nucleus. Compared with nonpalmitoylated R7BP, palmitoylated R7BP greatly augments the ability of RGS7 to attenuate GPCR-mediated G protein-regulated inward rectifying potassium channel activation. Thus, by controlling plasma membrane nuclear-shuttling of R7BP-R7-Gbeta5 complexes, reversible palmitoylation of R7BP provides a novel mechanism that regulates GPCR signaling and potentially transduces signals directly from the plasma membrane to the nucleus.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Proteínas RGS/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Ácido Palmítico/metabolismo , Canais de Potássio/metabolismo , Proteínas RGS/genética , Proteínas RGS/isolamento & purificação , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Chronic nicotine upregulates nicotinic acetylcholine receptors (nAChRs) throughout the brain, and reducing their activity may promote somatic and affective states that lead to nicotine seeking. nAChRs are functionally upregulated in animal models using passive nicotine administration, but whether/how it occurs in response to volitional nicotine intake is unknown. The distinction is critical, as drug self-administration (SA) can induce neurotransmission and cellular excitability changes that passive drug administration does not. In this study, we probed the question of whether medial habenula (MHb) nAChRs are functionally augmented by nicotine SA. Male rats were implanted with an indwelling jugular catheter and trained to nose poke for nicotine infusions. A saline SA group controlled for non-specific responding and nicotine-associated visual cues. Using patch-clamp whole-cell recordings and local application of acetylcholine, we observed robust functional enhancement of nAChRs in MHb neurons from rats with a history of nicotine SA. To determine whether upregulated receptors are generally enhanced or directed to specific cellular compartments, we imaged neurons during recordings using two-photon laser scanning microscopy (2PLSM). nAChR activity at the cell soma and on proximal and distal dendrites was examined by local nicotine uncaging using a photoactivatable nicotine (PA-Nic) probe and focal laser flash photolysis. Results from this experiment revealed strong nAChR enhancement at all examined cellular locations. Our study demonstrates nAChR functional enhancement by nicotine SA, confirming that volitional nicotine intake sensitizes cholinergic systems in the brain. This may be a critical plasticity change supporting nicotine addiction.
Assuntos
Habenula , Receptores Nicotínicos , Tabagismo , Animais , Habenula/metabolismo , Masculino , Nicotina/farmacologia , Plásticos , Ratos , Receptores Nicotínicos/metabolismoRESUMO
Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.
Assuntos
Acetilcolina/metabolismo , Encéfalo/metabolismo , Lasers , Neurônios/fisiologia , Nicotina/metabolismo , Fotólise , Receptores Nicotínicos/metabolismo , Animais , Camundongos , Nicotina/efeitos da radiaçãoRESUMO
Ventral tegmental area (VTA) neurons receive glutamatergic and/or GABAergic input from other local neurons within the VTA. Nicotinic acetylcholine receptor (nAChR) activity is capable of modulating such intra-VTA transmission, but the mechanisms are unclear. Here, we isolated monosynaptic glutamate or GABA transmission from mouse medial VTA (mVTA) to lateral VTA (latVTA) using pharmacology and optogenetics, and we studied the ability of nicotine to modulate these modes of transmission. The action of nicotine on mVTA to latVTA glutamate transmission was bidirectional; nicotine enhanced glutamate release in half of the recorded latVTA cells and inhibited release in the other half. Nicotine-mediated reduction in glutamate release was reversed by blockade of GABAA receptors. This, coupled with expression data demonstrating coexpression of vesicular glutamate transporter 2 (VGluT2) and glutamate decarboxylase 2 (Gad2) in mVTA neurons, suggests that nicotine is able to stimulate GABA corelease from mVTA VGluT2+ neurons. Nicotine had an altogether different effect on mVTA to latVTA GABA release from Gad2+ cells; nicotine suppressed GABA release from mVTA Gad2+ terminals in nearly all cells tested. Together, these data uncover a complex system of local circuitry in the VTA that is modulated by nAChR activity. These actions of nicotine, which occurred at concentrations of nicotine found in the artificial CSF of cigarette smokers, may play a role in the adaptive response of the reward system to repeated nicotine exposure.
Assuntos
Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios GABAérgicos/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Optogenética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
The inhibitory activity of (±)-citalopram on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3ß4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in µM) hα3ß4 AChRs (5.1⯱â¯1.3) with higher potency than that for hα7 (18.8⯱â¯1.1) and hα4ß2 (19.1⯱â¯4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3ß4 with >2-fold higher affinity than that for hα4ß2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3ß4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3ß4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3ß4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.