RESUMO
BACKGROUND: The ventral tegmental area (VTA) is a dopaminergic brain area that is critical in the development and maintenance of addiction. During withdrawal from chronic ethanol exposure, the response of VTA neurons to GABA (gamma-aminobutyric acid) is reduced through an epigenetically regulated mechanism. In the current study, a whole-genome transcriptomic approach was used to investigate the underlying molecular mechanism of GABA hyposensitivity in the VTA during withdrawal after chronic ethanol exposure. METHODS: We performed RNA sequencing of the VTA of Sprague Dawley male rats withdrawn for 24 hours from a chronic ethanol diet as well as sequencing of the VTA of control rats fed the Lieber-DeCarli diet. RNA sequencing data were analyzed using weighted gene coexpression network analysis to identify modules that contained coexpressed genes. Validation was performed with quantitative polymerase chain reaction, gas chromatography-mass spectrometry, and electrophysiological assays. RESULTS: Pathway and network analysis of weighted gene coexpression network analysis module 1 revealed a significant downregulation of genes associated with the cholesterol synthesis pathway. Consistent with this association, VTA cholesterol levels were significantly decreased during withdrawal. Chromatin immunoprecipitation indicated a decrease in levels of acetylated H3K27 at the transcriptional control regions of these genes. Electrophysiological studies in VTA slices demonstrated that GABA hyposensitivity during withdrawal was normalized by addition of exogenous cholesterol. In addition, inhibition of cholesterol synthesis produced GABA hyposensitivity, which was reversed by adding exogenous cholesterol to VTA slices. CONCLUSIONS: These results suggest that decreased expression of cholesterol synthesis genes may regulate GABA hyposensitivity of VTA neurons during alcohol withdrawal. Increasing cholesterol levels in the brain may be a novel avenue for therapeutic intervention to reverse detrimental effects of chronic alcohol exposure.
Assuntos
Alcoolismo , Síndrome de Abstinência a Substâncias , Ratos , Masculino , Animais , Ácido gama-Aminobutírico/metabolismo , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/metabolismo , Área Tegmentar Ventral , Alcoolismo/metabolismo , Ratos Sprague-Dawley , Etanol/farmacologiaRESUMO
Excessive alcohol intake, e.g., binge drinking, is a serious and mounting public health problem in the United States and throughout the world. Hence the need for novel insights into the underlying neurobiology that may help improve prevention and therapeutic strategies. Therefore, our group employed a darkness-induced alcohol intake protocol to define the reward deficiency domains of alcohol and other substance use disorders in terms of reward pathways' reduced dopamine signaling and its restoration via specifically-designed therapeutic compounds. It has been determined that KCNK13 and RASGRF2 genes, respectively, code for potassium two pore domain channel subfamily K member 13 and Ras-specific guanine nucleotide-releasing factor 2, and both genes have important dopamine-related functions pertaining to alcohol binge drinking. We present a hypothesis that identification of KCNK13 and RASGRF2 genes' risk polymorphism, coupled with genetic addiction risk score (GARS)-guided precision pro-dopamine regulation, will mitigate binge alcohol drinking. Accordingly, we review published reports on the benefits of this unique approach and provide data on favorable outcomes for both binge-drinking animals and drunk drivers, including reductions in alcohol intake and prevention of relapse to drinking behavior. Since driving under the influence of alcohol often leads to incarceration rather than rehabilitation, there is converging evidence to support the utilization of GARS with or without KCNK13 and RASGRF2 risk polymorphism in the legal arena, whereby the argument that "determinism" overrides the "free will" account may be a plausible defense strategy. Obviously, this type of research is tantamount to helping resolve a major problem related to polydrug abuse.
RESUMO
BACKGROUND: Alcohol excites neurons of the ventral tegmental area (VTA) and the release of dopamine from these neurons is a key event in ethanol (EtOH)-induced reward and reinforcement. Many mechanisms have been proposed to explain EtOH's actions on neurons of the VTA, but antagonists generally do not eliminate the EtOH-induced excitation of VTA neurons. We have previously demonstrated that the ion channel KCNK13 plays an important role in the EtOH-related excitation of mouse VTA neurons. Here, we elaborate on that finding and further assess the importance of KCNK13 in rats. METHODS: Rats (Sprague-Dawley and Fisher 344) were used in these studies. In addition to single-unit electrophysiology in brain slices, we used quantitative PCR and immunohistochemistry to discern the effects of EtOH and the brain slice preparation method on the expression levels of the Kcnk13 gene and KCNK13 protein. RESULTS: Immunohistochemistry demonstrated that the levels of KCNK13 were significantly reduced during procedures normally used to prepare brain slices for electrophysiology, with a reduction of about 75% in KCNK13 protein at the time that electrophysiological recordings would normally be made. Extracellular recordings demonstrated that EtOH-induced excitation of VTA neurons was reduced after knockdown of Kcnk13 using a small interfering RNA (siRNA) delivered via the recording micropipette. Real-time PCR demonstrated that the expression of Kcnk13 was altered in a time-dependent manner after alcohol withdrawal. CONCLUSIONS: KCNK13 plays an important role in EtOH-induced stimulation of rat VTA neurons and is dynamically regulated by cell damage and EtOH exposure, and during withdrawal. KCNK13 is a novel alcohol-sensitive protein, and further investigation of this channel may offer new avenues for the development of agents useful in altering the rewarding effect of alcohol.
Assuntos
Etanol/farmacologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Gadolínio/farmacologia , Expressão Gênica/efeitos dos fármacos , Isoflurano/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/análise , Canais de Potássio de Domínios Poros em Tandem/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Área Tegmentar Ventral/química , Área Tegmentar Ventral/fisiologiaRESUMO
P2X4 receptors are found throughout the central nervous system, and studies have shown that these purinergic receptors are important regulators of alcohol intake. The ventral tegmental area (VTA) is an important region for the rewarding and reinforcing properties of alcohol, but the role of P2X4 receptors in this region is unknown. Using both immunohistochemical and electrophysiological methods, we examined the interaction between P2X4 receptors and alcohol on VTA neurons. Incubation of brain slices containing the VTA for 2 h with siRNA targeting P2X4 receptors resulted in about a 25% reduction in P2X4 immunoreactivity in tyrosine hydroxylase positive VTA neurons. In electrophysiological experiments, ATP (0.5-3 mM) produced a reduction in the spontaneous firing rate, and ethanol significantly reduced this inhibition. Exposure to siP2X4 for 2 h via the recording micropipette resulted in a suppression of the response of VTA neurons to ATP, but no significant reduction in the ethanol inhibition of the ATP response was observed after this P2X4 downregulation. These results support the idea that VTA neurons are inhibited by ATP, ethanol antagonizes this inhibition, and the ethanol-sensitive component of ATP inhibition is mediated by P2X4 receptors. This interaction of ethanol with P2X4 receptors may be an important regulator of the rewarding effects of ethanol, making P2X4 receptors an intriguing target for the development of agents to treat alcohol use disorders.
Assuntos
Etanol/farmacologia , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2X4/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Alcoolismo/tratamento farmacológico , Animais , Dopamina/fisiologia , Masculino , Recompensa , Ácido gama-Aminobutírico/farmacologiaRESUMO
Elevations in estrogen (17ß-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERß) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERß into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERß, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.SIGNIFICANCE STATEMENT Estrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances, such as alcohol. We investigated the mechanisms by which estrogen increases the response of neurons in the VTA to ethanol. We found that activation of the ERα increased the ethanol-induced excitation of VTA neurons. 17ß-Estradiol-mediated enhancement of ethanol-induced excitation required the metabotropic glutamate receptor mGluR1. We also demonstrated that ERs in the VTA regulate binge-like alcohol drinking by female, but not male, mice. The influence of ERs on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.
Assuntos
Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Receptor alfa de Estrogênio/metabolismo , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Consumo Excessivo de Bebidas Alcoólicas/psicologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Receptores de AMPA/metabolismo , Área Tegmentar Ventral/citologiaRESUMO
Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6â¯J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Consumo Excessivo de Bebidas Alcoólicas/patologia , Dopamina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio de Domínios Poros em Tandem/genética , RNA Mensageiro/metabolismo , Técnicas de Cultura de Tecidos , Área Tegmentar Ventral/metabolismo , Área Tegmentar Ventral/patologiaRESUMO
BACKGROUND: The ventral tegmental area (VTA) is important for alcohol-related reward and reinforcement. Mouse VTA neurons are hyposensitive to γ-aminobutyric acid (GABA) during ethanol (EtOH) withdrawal, and GABA responsiveness is normalized by in vitro treatment with histone deacetylase inhibitors (HDACi). The present study examined the effect of a systemically administered HDACi, suberanilohydroxamic acid (SAHA) on GABA sensitivity, and related molecular changes in VTA neurons during withdrawal after chronic EtOH intake in rats. METHODS: Sprague Dawley male adult rats were fed with Lieber-DeCarli diet (9% EtOH or control diet) for 16 days. Experimental groups included control diet-fed and EtOH diet-fed (0- or 24-hour withdrawal) rats treated with either SAHA or vehicle injection. Single-unit recordings were used to measure the response of VTA neurons to GABA. Immunohistochemistry was performed to examine levels of HDAC2, acetylated histone H3 lysine 9 (acH3K9), and GABAA receptor α1 and α5 subunits in the VTA; quantitative polymerase chain reaction was performed to examine the mRNA levels of HDAC2 and GABAA receptor subunits. RESULTS: VTA neurons from the withdrawal group exhibited GABA hyposensitivity. In vivo SAHA treatment 2 hours before sacrifice normalized the sensitivity of VTA neurons to GABA. EtOH withdrawal was associated with increased HDAC2 and decreased acH3K9 protein levels; SAHA treatment normalized acH3K9 levels. Interestingly, no significant change was observed in the mRNA levels of HDAC2. The mRNA levels, but not protein levels, of GABAA receptor α1 and α5 subunits were increased during withdrawal. CONCLUSIONS: Withdrawal from chronic EtOH exposure results in a decrease in GABA-mediated inhibition, and this GABA hyposensitivity is normalized by in vivo SAHA treatment. Disruption of signaling in the VTA produced by alteration of GABA neurotransmission could be 1 neuroadaptive physiological process leading to craving and relapse. These results suggest that HDACi pharmacotherapy with agents like SAHA might be an effective treatment for alcoholism.
Assuntos
GABAérgicos/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Área Tegmentar Ventral/efeitos dos fármacos , Vorinostat/uso terapêutico , Ácido gama-Aminobutírico/farmacologia , Animais , Dieta , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/biossíntese , Receptores de GABA-A/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
The ventral tegmental area (VTA) evaluates salience of environmental stimuli and provides dopaminergic innervation to many brain areas affected by acute and chronic ethanol exposure. While primarily associated with rewarding and reinforcing stimuli, recent evidence indicates a role for the VTA in aversion as well. Ethanol actions in the VTA may trigger neuroadaptation resulting in reduction of the aversive responses to alcohol and a relative increase in the rewarding responses. In searching for effective pharmacotherapies for the treatment of alcohol abuse and alcoholism, recognition of this imbalance may reveal novel strategies. In addition to conventional receptor/ion channel pharmacotherapies, epigenetic factors that control neuroadaptation to chronic ethanol treatment can be targeted as an avenue for development of therapeutic approaches to restore the balance. Furthermore, when exploring therapies to address reward/aversion imbalance in the action of alcohol in the VTA, sex differences have to be taken into account to ensure effective treatment for both men and women. These principles apply to a VTA-centric approach to therapies, but should hold true when thinking about the overall approach in the development of neuroactive drugs to treat alcohol use disorders. Although the functions of the VTA itself are complex, it is a useful model system to evaluate the reward/aversion imbalance that occurs with ethanol exposure and could be used to provide new leads in the efforts to develop novel drugs to treat alcoholism.
Assuntos
Alcoolismo/tratamento farmacológico , Etanol/administração & dosagem , Neurônios/efeitos dos fármacos , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Alcoolismo/metabolismo , Animais , Fármacos do Sistema Nervoso Central/farmacologia , Fármacos do Sistema Nervoso Central/uso terapêutico , Dopamina/metabolismo , Humanos , Canais Iônicos/metabolismo , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Neurônios/metabolismo , Área Tegmentar Ventral/metabolismoRESUMO
Gender differences in psychiatric disorders such as addiction may be modulated by the steroid hormone estrogen. For instance, 17ß-estradiol (E2), the predominant form of circulating estrogen in pre-menopausal females, increases ethanol consumption, suggesting that E2 may affect the rewarding properties of ethanol and thus the development of alcohol use disorder in females. The ventral tegmental area (VTA) is critically involved in the rewarding and reinforcing effects of ethanol. In order to determine the role of E2 in VTA physiology, gonadally intact female mice were sacrificed during diestrus II (high E2) or estrus (low E2) for electrophysiology recordings. We measured the excitation by ethanol and inhibition by dopamine (DA) of VTA DA neurons and found that both excitation by ethanol and inhibition by dopamine were greater in diestrus II compared with estrus. Treatment of VTA slices from mice in diestrus II with an estrogen receptor antagonist (ICI 182,780) reduced ethanol-stimulated neuronal firing, but had no effect on ethanol-stimulated firing of neurons in slices from mice in estrus. Surprisingly, ICI 182,780 did not affect the inhibition by DA, indicating different mechanisms of action of estrogen receptors in altering ethanol and DA responses. We also examined the responses of VTA DA neurons to ethanol and DA in ovariectomized mice treated with E2 and found that E2 treatment enhanced the responses to ethanol and DA in a manner similar to what we observed in mice in diestrus II. Our data indicate that E2 modulates VTA neuron physiology, which may contribute to both the enhanced reinforcing and rewarding effects of alcohol and the development of other psychiatric disorders in females that involve alterations in DA neurotransmission.
Assuntos
Dopamina/metabolismo , Dopamina/farmacologia , Estradiol/farmacologia , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Diestro , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Ovariectomia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismoRESUMO
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase associated with alcohol dependence in humans and behavioral responses to ethanol in mice. To characterize the ability of ALK to control ethanol consumption, we treated mice with the ALK inhibitors TAE684 or alectinib before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with ALK inhibitors drank less ethanol than controls. In addition, TAE684 treatment abolished ethanol conditioned place preference, indicating that ALK regulates the rewarding properties of ethanol. Because the ventral tegmental area (VTA) is a key brain region involved in the rewarding effects of ethanol, we determined if Alk expression in the VTA is important for binge-like ethanol consumption. Mice expressing a short hairpin ribonucleic acid targeting Alk in the VTA drank less ethanol compared with controls. ALK is expressed on dopamine (DA) neurons in the VTA, suggesting that ALK might regulate their firing properties. Extracellular recordings of putative DA neurons in VTA slices demonstrated that ALK inhibition did not affect the ability of ethanol to stimulate, or DA to inhibit, the firing of DA neurons. However, inhibiting ALK attenuated the time-dependent reversal of inhibition produced by moderate concentrations of DA, suggesting that ALK affects DA D2 autoreceptor (D2R) desensitization. Altered desensitization of the D2R changes the firing of DA neurons and is predicted to affect DA levels and alcohol drinking. These data support the possibility that ALK might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.
Assuntos
Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Dopaminérgicos/metabolismo , Área Tegmentar Ventral/metabolismo , Quinase do Linfoma Anaplásico , Animais , Comportamento Animal/efeitos dos fármacos , Consumo Excessivo de Bebidas Alcoólicas/genética , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Drugs of abuse increase the activity of dopaminergic neurons of the ventral tegmental area (VTA), and output from the VTA is critical for both natural and drug-induced reward and reinforcement. Ethanol and the abused inhalant toluene both enhance VTA neuronal firing, but the mechanisms of this effect is not fully known. In this study, we used extracellular recordings to compare the actions of toluene and ethanol on DA VTA neurons. Both ethanol and toluene increased the firing rate of DA neurons, although toluene was ~100 times more potent than ethanol. The mixed ion channel blocker quinine (100 µM) blocked the increases in firing produced by ethanol and toluene, indicating some similarity in mechanisms of excitation. A mixture of antagonists of GABA and cholinergic receptors did not prevent toluene-induced or ethanol-induced excitation, and toluene-induced excitation was not altered by co-administration of ethanol, suggesting independent mechanisms of excitation for ethanol and toluene. Concurrent blockade of NMDA, AMPA, and metabotropic glutamate receptors enhanced the excitatory effect of toluene while having no significant effect on ethanol excitation. Nicotine increased firing of DA VTA neurons, and this was blocked by the nicotinic antagonist mecamylamine (1 µM). Mecamylamine did not alter ethanol or toluene excitation of firing but the muscarinic antagonist atropine (5 µM) or a combination of GABA antagonists (bicuculline and CGP35348, 10 µM each) reduced toluene-induced excitation without affecting ethanol excitation. The Ih current blocker ZD7288 abolished the excitatory effect of toluene but unlike the block of ethanol excitation, the effect of ZD7288 was not reversed by the GIRK channel blocker barium, but was reversed by GABA antagonists. These results demonstrate that the excitatory effects of ethanol and toluene have some similarity, such as block by quinine and ZD7288, but also indicate that there are important differences between these two drugs in their modulation by glutamatergic, cholinergic, and GABAergic receptors. These findings provide important information regarding the actions of abused inhalants on central reward pathways, and suggest that regulation of the activation of central dopamine pathways by ethanol and toluene partially overlap.
RESUMO
Neurons of the ventral tegmental area (VTA) are the source of dopaminergic (DAergic) input to important brain regions related to addiction. Prolonged exposure of these VTA neurons to moderate concentrations of dopamine (DA) causes a time-dependent decrease in DA-induced inhibition, a complex desensitization called DA inhibition reversal (DIR). DIR is mediated by conventional protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of some Gq-linked receptors. Corticotropin releasing factor (CRF) acts via Gq, and can modulate glutamater neurotransmission in the VTA. In the present study, we used brain slice electrophysiology to characterize the interaction of DA, glutamate antagonists, and CRF agonists in the induction and maintenance of DIR in the VTA. Glutamate receptor antagonists blocked induction but not maintenance of DIR. Putative blockers of neurotransmitter release and store-operated calcium channels blocked and reversed DIR. CRF and the CRF agonist urocortin reversed inhibition produced by the D2 agonist quinpirole, consistent with our earlier work indicating that Gq activation reverses quinpirole-mediated inhibition. In whole cell recordings, the combination of urocortin and quinpirole, but not either agent alone, increased spontaneous excitatory postsynaptic currents (sEPSCs) in VTA neurons. Likewise, the combination of a D1-like receptor agonist and quinpirole, but not either agent alone, increased sEPSCs in VTA neurons. In summary, desensitization of D2 receptors induced by dopamine or CRF on DAergic VTA neurons is associated with increased glutamatergic signaling in the VTA.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Receptores de Dopamina D2/metabolismo , Área Tegmentar Ventral/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Fármacos do Sistema Nervoso Central/farmacologia , Hormônio Liberador da Corticotropina/agonistas , Agonistas de Dopamina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Quimpirol/farmacologia , Ratos Endogâmicos F344 , Ratos Wistar , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/agonistas , Receptores de Glutamato/metabolismo , Urocortinas/farmacologia , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
Putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons have an important role in alcohol addiction. Acute ethanol increases the activity of pDAergic neurons, and withdrawal from repeated ethanol administration produces a decreased sensitivity of pDAergic VTA neurons to GABA. Recent studies show that behavioral changes induced by chronic alcohol are reversed by inhibitors of histone deacetylases (HDACs). Whether HDAC-induced histone modifications regulate changes in GABA sensitivity of VTA pDAergic neurons during withdrawal is unknown. Here, we investigated modulation of withdrawal-induced changes in GABA sensitivity of pDAergic VTA neurons by HDAC inhibitors (HDACi), and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-Aα1 receptor (GABA (A-α1) R) subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally (ip) with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDAergic VTA neurons in brain slices from ethanol-withdrawn mice, sensitivity to GABA (50-500 µM) was reduced. In brain slices from ethanol-withdrawn mice incubated with the HDACi SAHA (vorinostat) or trichostatin A (TSA) for 2 h, the hyposensitivity of pDAergic VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDAergic VTA neurons from saline-treated mice. In addition, ethanol withdrawal was associated with an increase in levels of HDAC2 and a decrease in histone (H3-K9) acetylation and levels of GABA (A-α1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDACi normalizes GABA hyposensitivity of pDAergic neurons developed during withdrawal after chronic ethanol treatment, which suggests the possibility that inhibition of HDACs can reverse ethanol-induced neuroadaptational changes in reward circuitry.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Síndrome de Abstinência a Substâncias/fisiopatologia , Área Tegmentar Ventral/fisiologia , Ácido gama-Aminobutírico/farmacologia , Acetilação , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanol/farmacologia , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Receptores de GABA-A/metabolismo , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Área Tegmentar Ventral/efeitos dos fármacos , VorinostatRESUMO
Selective suppression of protein function in the brain can be achieved using specific silencing RNAs administered in vivo. A viral delivery system is often employed to transfect neurons with small hairpin RNA (shRNA) directed against specific proteins, and intervals of several days are allowed between microinjection of the shRNA-containing virus into the brain and experiments to assess suppression of gene function. Here we report studies using extracellular recording of dopaminergic neurons of the ventral tegmental area (DA VTA neurons) recorded in brain slices in which lentivirus containing shRNA directed against Gq was included in the recording pipette, and suppression of Gq-related function was observed within the time frame of the recording. The action of neurotensin (NT) is associated with activation of Gq, and the firing rate of DA VTA neurons is increased by NT. With shRNA directed against Gq in the pipette, there was a significant reduction of NT excitation within 2 h. Likewise, time-dependent dopamine desensitization, which we have hypothesized to be Gq-dependent, was not observed when shRNA directed against Gq was present in the pipette and dopamine was tested 2 h after initiation of recording. As the time interval (2 h) is relatively short, we tested whether blockade of protein synthesis with cycloheximide delivered via the recording pipette would alter Gq-linked responses similarly. Both NT-induced excitation and dopamine desensitization were inhibited in the presence of cycloheximide. Inclusion of shRNA in the recording pipette may be an efficient and selective way to dampen responses linked to Gq, and, more generally, the use of lentiviral-packaged shRNA in the recording pipette is a means to produce selective inhibition of the function of specific proteins in experiments.
RESUMO
Neurons of the ventral tegmental area (VTA) play a key role in the rewarding and reinforcing effects of drugs of abuse, including alcohol. Ethanol directly increases the firing rate of dopaminergic (DAergic) VTA neurons, but modulation of the firing rate of DAergic VTA neurons can be controlled by a number of factors, including some that are under the control of protein kinase C (PKC). Application of phorbol esters activates PKC and the present study assessed the effect of a phorbol ester, phorbol 12-myristate 13-acetate (PMA), on ethanol-induced excitation of DA VTA neurons. Ethanol-induced excitation of DAergic VTA neurons was reduced significantly in the presence of PMA. This action of PMA was antagonized by chelerythrine chloride, a non-selective antagonist of PKC, but not by moderate concentrations of antagonists of conventional PKC isoforms (Gö6976 and Gö6983). A PKC δ/θ inhibitor antagonized PMA-induced reduction of ethanol excitation. Since PKCδ antagonist Gö6983 did not antagonize the effect of PMA on ethanol excitation, the PMA reduction of ethanol excitation is most likely to be mediated by PKCθ. Antagonists of intracellular calcium pathways were ineffective in antagonizing PMA action on ethanol excitation, consistent with the lack of calcium dependence of PKCθ. In summary, ethanol-induced excitation of VTA neurons is attenuated in the presence of PMA, and this attenuation appears to be mediated by PKCθ. This novel mechanism for interfering with ethanol activation of reward-related neurons could provide a new target for pharmacotherapy to ameliorate alcoholism.
RESUMO
Alcohol abuse and alcoholism represent substantial problems that affect a large portion of individuals throughout the world. Extensive research continues to be conducted in an effort to identify the biological basis of the reinforcing properties of alcohol in order to develop effective pharmacotherapeutic and behavioral interventions. One theory that has developed within the alcohol field over the past four decades postulates that the reinforcing properties of alcohol are due to the action of the metabolites/products of alcohol within the central nervous system (CNS). The most extreme version of this theory suggests that the biologically active metabolites/products of alcohol, created from the breakdown from alcohol, are the ultimate source of the reinforcing properties of alcohol. The contrary theory proposes that the reinforcing properties of alcohol are mediated completely through the interaction of the ethanol molecule with several neurochemical systems within the CNS. While there are scientific findings that offer support for both of these stances, the reinforcing properties of alcohol are most likely generated through a complex series of peripheral and central effects of both alcohol and its metabolites. Nonetheless, the development of a greater understanding for how the metabolites/products of alcohol contribute to the reinforcing properties of alcohol is an important factor in the development of efficacious pharmacotherapies for alcohol abuse and alcoholism. This chapter is intended to provide a historical perspective of the role of acetaldehyde (the first metabolite of alcohol) in alcohol reinforcement as well as review the basic research literature on the effects of acetaldehyde (and acetaldehyde metabolites/products) within the CNS and how these function with regard to alcohol reward.
Assuntos
Acetaldeído/administração & dosagem , Consumo de Bebidas Alcoólicas/epidemiologia , Alcoolismo/epidemiologia , Etanol/administração & dosagem , Isoquinolinas/administração & dosagem , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , HumanosRESUMO
Dopaminergic neurons of the ventral tegmental area are important components of brain pathways related to addiction. Prolonged exposure of these neurons to moderate concentrations of dopamine (DA) decreases their sensitivity to inhibition by DA, a process called DA-inhibition reversal (DIR). DIR is mediated by phospholipase C and conventional subtype of protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of 5-HT(2) or neurotensin receptors. In the present study, we further characterized this phenomenon by use of extracellular recordings in brain slices to examine whether DIR is linked to G protein-coupled receptor kinase-2 (GRK2) or dynamin by assessing DIR in the presence of antagonists of these enzymes. DIR was blocked by ß-ARK1 inhibitor, which inhibits GRK2, and by dynasore, which blocks dynamin. Reversal of inhibition by D2 agonist quinpirole was produced by serotonin (50 µM) and by neurotensin (5-10 nM). Serotonin-induced or neurotensin-induced reversal was blocked by ß-ARK1 inhibitor, dynasore, or cPKC antagonist 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4c]carbazole-12-propanenitrile (Gö6976). This further characterization of DIR indicates that cPKC, GRK2, and dynamin play important roles in the desensitization of D2 receptors. As drugs of abuse produce persistent increases in DA concentration in the ventral tegmental area, reduction of D2 receptor sensitivity as a result of drug abuse may be a critical factor in the processes of addiction.
Assuntos
Agonistas de Dopamina/farmacologia , Dinaminas/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Proteína Quinase C/fisiologia , Receptores de Dopamina D2/agonistas , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Técnicas In Vitro , Masculino , Peptídeos/farmacologia , Fosforilação , Quimpirol/antagonistas & inibidores , Quimpirol/farmacologia , Ratos , Ratos Endogâmicos F344 , Receptores de Neurotensina/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Área Tegmentar Ventral/citologiaRESUMO
BACKGROUND: Dopaminergic (DAergic) neurons of the ventral tegmental area (VTA) are important for the rewarding and reinforcing properties of alcohol and other drugs of abuse. Regulation of the firing of DAergic VTA neurons is controlled by a number of factors, including autoregulation of firing by D2 dopamine (DA) receptors. The inhibitory effects of DA on these neurons exhibit concentration- and time-dependent desensitization, which we have termed dopamine inhibition reversal (DIR), as it requires concurrent stimulation of D1/D5 and D2 receptors. METHODS: Extracellular recording of DAergic VTA neurons in brain slices was used to test the effects of ethanol (EtOH) (10 to 80 mM) on DIR. RESULTS: DIR was reduced by concentrations of EtOH as low as 10 mM and was blocked by higher EtOH concentrations. In addition, as we have shown that reversal of inhibition by the selective D2 agonist quinpirole can be observed in the presence of an activator of protein kinase C (PKC), we tested whether EtOH could antagonize the reversal of quinpirole inhibition in the presence of phorbol 12-myristate 13-acetate (PMA). EtOH (80 mM) blocked the reversal of quinpirole seen in the presence of PMA, suggesting that the antagonism of DIR by EtOH is owing to an action at a stage in the mechanism at or distal to PKC. Once achieved, DIR is not antagonized by EtOH. CONCLUSIONS: The blockade by relatively low concentrations of EtOH of DIR may play an important role in the spectrum of action of EtOH on DAergic neurons of the VTA and may be important in the acute and chronic actions of EtOH on the excitability of these brain reward/reinforcement neurons.
Assuntos
Dopamina/fisiologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Etanol/farmacologia , Inibição Neural/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Antagonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/fisiologia , Relação Dose-Resposta a Droga , Inibição Neural/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos F344 , Área Tegmentar Ventral/fisiologiaRESUMO
Putative dopaminergic (pDAergic) ventral tegmental area neurons play an important role in brain pathways related to addiction. Extended exposure of pDAergic neurons to moderate concentrations of dopamine (DA) results in a time-dependent decrease in sensitivity of pDAergic neurons to DA inhibition, a process called dopamine inhibition reversal (DIR). We have shown that DIR is mediated by phospholipase C and conventional protein kinase C through concurrent stimulation of D2 and D1-like receptors. In the present study, we further characterized this phenomenon by using extracellular recordings in brain slices to examine whether DIR is linked to phosphatidylinositol (PI) or adenylate cyclase (AC) second-messenger pathways. A D1-like dopaminergic agonist associated with PI turnover (SKF83959), but not one linked to AC (SKF83822), promoted reversal of inhibition produced by quinpirole, a dopamine D2-selective agonist. Other neurotransmitter receptors linked to PI turnover include serotonin 5-HT(2), α(1)-adrenergic, neurotensin, and group I metabotropic glutamate (mGlu) receptors. Both serotonin and neurotensin produced significant reversal of quinpirole inhibition, but agonists of α(1)-adrenergic and group I mGlu receptors failed to significantly reverse quinpirole inhibition. These results indicate that some agonists that stimulate PI turnover can facilitate desensitization of D2 receptors but that there may be other factors in addition to PI that control that interaction.
Assuntos
Dopaminérgicos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositóis/metabolismo , Quimpirol/farmacologia , Área Tegmentar Ventral/citologia , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Ketanserina/farmacologia , Neurotensina/farmacologia , Fenilefrina/farmacologia , Ratos , Ratos Endogâmicos F344 , Serotonina/farmacologiaRESUMO
Adaptation of putative dopaminergic (pDA) neurons in the ventral tegmental area (VTA) to drugs of abuse may alter information processing related to reward and reinforcement and is an important factor in the development of addiction. We have demonstrated that prolonged increases in the concentration of dopamine (DA) result in a time-dependent decrease in sensitivity of pDA neurons to DA, which we termed DA inhibition reversal (DIR). In this study, we used extracellular recordings to examine factors mediating DIR. A 40 min administration of DA (2.5-10 µM), but not the DA D2 receptor agonist quinpirole (50-200 nM), resulted in inhibition of neuronal firing followed by DIR. In the presence of 100 nM cocaine, inhibition followed by DIR was seen with much lower DA concentrations. Reversal of quinpirole inhibition could be induced by an activator of protein kinase C, but not of protein kinase A. Inhibitors of protein kinase C or phospholipase C blocked the development of DIR. Disruption of intracellular calcium release also prevented DIR. Reduction of extracellular calcium or inhibition of store-operated calcium entry blocked DIR, but the L-type calcium channel blocker nifedipine did not. DIR was age-dependent and not seen in pDA VTA neurons from rat pups younger than 15 days postnatally. Our data indicate that DIR is mediated by protein kinase C, and implicate a conventional protein kinase C. This characterization of DIR gives insight into the regulation of autoinhibition of pDA VTA neurons, and the resulting long-term alteration in information processing related to reward and reinforcement.