RESUMEN
Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABAB receptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABAB R2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants. SIGNIFICANCE STATEMENT: Exposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABAB receptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.
Asunto(s)
Dopaminérgicos/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Metanfetamina/farmacología , Receptores de GABA-B/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/citología , Animales , Animales Recién Nacidos , Baclofeno/farmacología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Agonistas de Receptores GABA-B/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de GABA-B/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/genéticaRESUMEN
G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.
Asunto(s)
Analgésicos Opioides/farmacología , Neuronas Dopaminérgicas/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Neuronas GABAérgicas/fisiología , Actividad Motora/fisiología , Animales , Neuronas Dopaminérgicas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Neuronas GABAérgicas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Subunidades de Proteína/fisiología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/fisiologíaRESUMEN
G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Etanol/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Activación del Canal Iónico/fisiología , Motivación/genética , Análisis de Varianza , Animales , Consumo Excesivo de Bebidas Alcohólicas/genética , Cartilla de ADN/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/deficiencia , Hibridación in Situ , Activación del Canal Iónico/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microdiálisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , RecompensaRESUMEN
The K(+)/Cl(-) cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl(-) levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl(-) extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.
Asunto(s)
Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Simportadores/metabolismo , Animales , Cloruros/metabolismo , Endocitosis , Técnicas de Sustitución del Gen , Glutamatos/farmacología , Ratones , Ratones Mutantes Neurológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/metabolismo , Simportadores/genética , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.
Asunto(s)
Cocaína/toxicidad , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/biosíntesis , Nexinas de Clasificación/fisiología , Animales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Técnicas de Cultivo de Órganos , Unión Proteica/genética , Transducción de Señal/genética , Nexinas de Clasificación/deficiencia , Área Tegmental Ventral/citología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
G-protein-coupled inwardly rectifying potassium (GIRK) channels contribute to the resting membrane potential of many neurons, including dopamine (DA) neurons in the ventral tegmental area (VTA). VTA DA neurons are bistable, firing in two modes: one characterized by bursts of action potentials, the other by tonic firing at a lower frequency. Here we provide evidence that these firing modes drive bidirectional plasticity of GIRK channel-mediated currents. In acute midbrain slices of mice, we observed that in vitro burst activation of VTA DA neurons potentiated GIRK currents whereas tonic firing depressed these currents. This plasticity was not specific to the metabotropic receptor activating the GIRK channels, as direct activation of GIRK channels by nonhydrolyzable GTP also potentiated the currents. The plasticity of GIRK currents required NMDA receptor and CaMKII activation, and involved protein trafficking through specific PDZ domains of GIRK2c and GIRK3 subunit isoforms. Prolonged tonic firing may thus enhance the probability to switch into burst-firing mode, which then potentiates GIRK currents and favors the return to baseline. In conclusion, activity-dependent GIRK channel plasticity may represent a slow destabilization process favoring the switch between the two firing modes of VTA DA neurons.
Asunto(s)
Potenciales de Acción , Neuronas Dopaminérgicas/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Plasticidad Neuronal , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Femenino , Guanosina Trifosfato/metabolismo , Potenciales Postsinápticos Inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismo , Área Tegmental Ventral/citología , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/fisiologíaRESUMEN
Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.
Asunto(s)
Estimulantes del Sistema Nervioso Central/farmacología , Regulación hacia Abajo/efectos de los fármacos , Metanfetamina/farmacología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Área Tegmental Ventral/citología , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Baclofeno/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Channelrhodopsins , Dopamina/farmacología , Dopaminérgicos/farmacología , Interacciones Farmacológicas , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/ultraestructura , Agonistas de Receptores GABA-B/farmacología , Glutamato Descarboxilasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Inmunoelectrónica/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Compuestos Organofosforados/farmacología , Fosforilación , Receptores de GABA-A/ultraestructura , Factores de Transcripción/genética , Área Tegmental Ventral/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
RATIONALE: We previously colocalized a quantitative trait locus (QTL) for sensitivity to the locomotor stimulant effects of methamphetamine (MA) with a QTL for expression of casein kinase 1 epsilon (Csnk1-epsilon) in the nucleus accumbens (NAc). Subsequently, we identified a single nucleotide polymorphism in CSNK1E (rs135745) that was associated with increased sensitivity to the subjective effects of d-amphetamine in healthy human subjects. Based on these results, we hypothesized that differential expression of Csnk1-epsilon causes differential sensitivity to MA-induced locomotor activity in mice. OBJECTIVE: In the present study, we used PF-670462 (PF), which is a selective inhibitor of Csnk1-epsilon, to directly evaluate the role of Csnk1-epsilon in the locomotor stimulant response to MA in male C57BL/6J mice. METHODS: We administered vehicle, PF, MA, or MA + PF, either via intraperitoneal injections or bilateral intra-NAc microinjections. We also examined Darpp-32 phosphorylation in mice receiving intraperitoneal injections. RESULTS: Intraperitoneal PF (20-40 mg/kg) attenuated the locomotor stimulant response to MA (2 mg/kg) without affecting baseline activity. The high dose of PF also significantly inhibited MA-induced phosphorylation of Darpp-32, providing a potential mechanism by which Csnk1-epsilon contributes to MA-induced locomotor activity. Furthermore, microinjection of PF (5 microg/side) into the NAc completely blocked the locomotor stimulant response to MA (2.5 microg/side) without affecting baseline activity. CONCLUSIONS: These results provide direct evidence that Csnk1-epsilon is crucial for the locomotor stimulant response to a moderate dose of MA and suggest that genetic polymorphisms affecting Csnk1-epsilon expression or function could influence sensitivity to amphetamines in both mice and humans.
Asunto(s)
Caseína Cinasa 1 épsilon/genética , Estimulantes del Sistema Nervioso Central/farmacología , Metanfetamina/farmacología , Actividad Motora/efectos de los fármacos , Animales , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Fosforilación , Polimorfismo de Nucleótido Simple , Pirimidinas/farmacología , Sitios de Carácter CuantitativoRESUMEN
We used short-term selection to produce outbred mouse lines with differences in contextual fear conditioning. Within two generations of selection all low selected mice were homozygous for the recessive tyrc allele and showed the corresponding albino coat color. Freezing differed in the high and low selected lines across a range of parameters. We identified several QTLs for the selection response, including a highly significant QTL at the tyr locus (p < 9.6(-10)). To determine whether the tyrc allele was directly responsible for the response to selection, we examined B6 mice that have a mutant tyr allele (tyr(c-2j-)) and an AJ congenic strain that has the wild-type B6 allele for tyr. These studies showed that the tyr allele had a small influence on fear learning. We used Affymetrix microarrays to identify many differentially expressed genes in the amygdala and hippocampus of the selected lines. We conclude that tyr is one of many alleles that influence fear conditioning.