RESUMEN

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1FA+TL/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

Asunto(s)

Glicina , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , Dominios Proteicos , Glicina/farmacología , Sitios de Unión

RESUMEN

Exposure to trauma is a risk factor for the development of a number of mood disorders, and may enhance vulnerability to future adverse life events. Recent data demonstrate that ventral tegmental area (VTA) neurons expressing the vesicular glutamate transporter 2 (VGluT2) signal and causally contribute to behaviors that involve aversive or threatening stimuli. However, it is unknown whether VTA VGluT2 neurons regulate transsituational outcomes of stress and whether these neurons are sensitive to stressor controllability. This work adapted an operant mouse paradigm to examine the impact of stressor controllability on VTA VGluT2 neuron function as well as the role of VTA VGluT2 neurons in mediating transsituational stressor outcomes. Uncontrollable (inescapable) stress, but not physically identical controllable (escapable) stress, produced social avoidance and exaggerated fear in male mice. Uncontrollable stress in females led to exploratory avoidance of a novel brightly lit environment. Both controllable and uncontrollable stressors increased VTA VGluT2 neuronal activity, and chemogenetic silencing of VTA VGluT2 neurons prevented the behavioral sequelae of uncontrollable stress in male and female mice. Further, we show that stress activates multiple genetically-distinct subtypes of VTA VGluT2 neurons, especially those that are VGluT2+VGaT+, as well as lateral habenula neurons receiving synaptic input from VTA VGluT2 neurons. Our results provide causal evidence that mice can be used for identifying stressor controllability circuitry and that VTA VGluT2 neurons contribute to transsituational stressor outcomes, such as social avoidance, exaggerated fear, or anxiety-like behavior that are observed within trauma-related disorders.

RESUMEN

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its efferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their efferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct efferent projections may serve as a way for the POMC population to organize its many functions.

Asunto(s)

Núcleo Arqueado del Hipotálamo/metabolismo , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/citología , Señalización del Calcio , Vías Eferentes/metabolismo , Femenino , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proopiomelanocortina/genética

RESUMEN

To maintain metabolic homeostasis, motivated behaviors are driven by neuronal circuits that process information encoding the animal's energy state. Such circuits likely include ventromedial hypothalamus (VMH) glutamatergic neurons that project throughout the brain to drive food intake and energy expenditure. Targets of VMH glutamatergic neurons include proopiomelanocortin (POMC) neurons in the arcuate nucleus that, when activated, inhibit food intake. Although an energy-state-sensitive, glutamate circuit between the VMH and POMC neurons has been previously indicated, the significance and details of this circuit have not been fully elucidated. Thus, the goal of the present work was to add to the understanding of this circuit. Using a knockout strategy, the data show that the VMH glutamate→POMC neuron circuit is important for the inhibition of food intake. Conditional deletion of the vesicular glutamate transporter (VGLUT2) in the VMH results in increased bodyweight and increased food intake following a fast in both male and female mice. Additionally, the targeted blunting of glutamate release from the VMH resulted in an ∼32% reduction in excitatory inputs to POMC cells, suggesting that this circuit may respond to changes in energy state to affect POMC activity. Indeed, we found that glutamate release is increased at VMH-to-POMC synapses during feeding and POMC AMPA receptors switch from a calcium-permeable state to a calcium-impermeable state during fasting. Collectively, these data indicate that there is an energy-balance-sensitive VMH-to-POMC circuit conveying excitatory neuromodulation onto POMC cells at both pre- and postsynaptic levels, which may contribute to maintaining appropriate food intake and body mass.NEW & NOTEWORTHY Despite decades of research, the neurocircuitry underlying metabolic homeostasis remains incompletely understood. Specifically, the roles of amino acid transmitters, particularly glutamate, have received less attention than hormonal signals. Here, we characterize an energy-state-sensitive glutamate circuit from the ventromedial hypothalamus to anorexigenic proopiomelanocortin (POMC) neurons that responds to changes in energy state at both sides of the synapse, providing novel information about how variations in metabolic state affect excitatory drive onto POMC cells.

Asunto(s)

Metabolismo Energético/fisiología , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Sinapsis/metabolismo , Núcleo Hipotalámico Ventromedial/metabolismo , Animales , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Metabolismo Energético/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Núcleo Hipotalámico Ventromedial/efectos de los fármacos , Proteína 2 de Transporte Vesicular de Glutamato/deficiencia , Proteína 2 de Transporte Vesicular de Glutamato/genética

RESUMEN

Proopiomelanocortin (POMC) neurons contribute to the regulation of many physiological processes; the majority of which have been attributed to the release of peptides produced from the POMC prohormone such as α-MSH, which plays key roles in food intake and metabolism. However, it is now clear that POMC neurons also release amino acid transmitters that likely contribute to the overall function of POMC cells. Recent work indicates that constitutive deletion of these transmitters can affect metabolic phenotypes, but also that the expression of GABAergic or glutamatergic markers changes throughout development. The goal of the present study was to determine whether the release of glutamate or GABA from POMC neurons in the adult mouse contributes notably to energy balance regulation. Disturbed release of glutamate or GABA specifically from POMC neurons in adult mice was achieved using a tamoxifen-inducible Cre construct (Pomc-CreERT2) expressed in mice also carrying floxed versions of Slc17a6 (vGlut2) or Gad1 and Gad2, encoding the vesicular glutamate transporter type 2 and GAD67 and GAD65 proteins, respectively. All mice in the experiments received tamoxifen injections, but control mice lacked the tamoxifen-inducible Cre sequence. Body weight was unchanged in Gad1- and Gad2- or vGlut2-deleted female and male mice. Additionally, no significant differences in glucose tolerance or refeeding after an overnight fast were observed. These data collectively suggest that the release of GABA or glutamate from POMC neurons in adult mice does not significantly contribute to the metabolic parameters tested here. In light of prior work, the data also suggest that amino acid transmitter release from POMC cells may contribute to separate functions in the adult versus the developing mouse.

Asunto(s)

Dieta Alta en Grasa , Metabolismo Energético/efectos de los fármacos , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Peso Corporal , Metabolismo Energético/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Intolerancia a la Glucosa , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Masculino , Ratones , Ratones Noqueados

RESUMEN

Neuronal circuits regulating hunger and satiety synthesize information encoding the energy state of the animal and translate those signals into motivated behaviors to meet homeostatic needs. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus are activated by energy surfeits and inhibited by energy deficits. When activated, these cells inhibit food intake and facilitate weight loss. Conversely, decreased activity in POMC cells is associated with increased food intake and obesity. Circulating nutrients and hormones modulate the activity of POMC neurons over protracted periods of time. However, recent work indicates that calcium activity in POMC cells changes in response to food cues on times scales consistent with the rapid actions of amino acid transmitters. Indeed, the frequency of spontaneous IPSCs (sIPSCs) onto POMC neurons increases during caloric deficits. However, the afferent brain regions responsible for this inhibitory modulation are currently unknown. Here, through the use of brain region-specific deletion of GABA release in both male and female mice we show that neurons in the dorsomedial hypothalamus (DMH) are responsible for the majority of sIPSCs in POMC neurons as well as the fasting-induced increase in sIPSC frequency. Further, the readily releasable pool of GABA vesicles and the release probability of GABA is increased at DMH-to-POMC synapses following an overnight fast. Collectively these data provide evidence that DMH-to-POMC GABA circuitry conveys inhibitory neuromodulation onto POMC cells that is sensitive to the animal's energy state.SIGNIFICANCE STATEMENT Activation of proopiomelanocortin (POMC) cells signals satiety, whereas GABAergic cells in the dorsomedial hypothalamus (DMH) can increase food consumption. However, communication between these cells, particularly in response to changes in metabolic state, is unknown. Here, through targeted inhibition of DMH GABA release, we show that DMH neurons contribute a significant portion of spontaneously released GABA onto POMC cells and are responsible for increased GABAergic inhibition of POMC cells during fasting, likely mediated through increased release probability of GABA at DMH terminals. These data provide important information about inhibitory modulation of metabolic circuitry and provide a mechanism through which POMC neurons could be inhibited, or disinhibited, rapidly in response to food availability.

Asunto(s)

Núcleo Hipotalámico Dorsomedial/metabolismo , Neuronas GABAérgicas/metabolismo , Potenciales Postsinápticos Inhibidores/fisiología , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos

RESUMEN

BACKGROUND: Recent work with long-term ethanol (EtOH) self-administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre- and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long-term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long-term EtOH self-administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward-seeking behaviors. METHODS: Basolateral amygdala samples from control and EtOH-drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE-associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration. RESULTS: Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE-associated proteins. Across the range of EtOH-drinking phenotypes, SNAP25 and Munc13-1 proteins levels were significantly different between males and females, and Munc13-2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy-drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males). CONCLUSIONS: Protein expression analysis of basolateral amygdala total protein from controls and animals following long-term EtOH self-administration suggests a number of alterations in core SNARE or SNARE-associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.

Asunto(s)

Consumo de Bebidas Alcohólicas/metabolismo , Consumo de Bebidas Alcohólicas/tendencias , Complejo Nuclear Basolateral/efectos de los fármacos , Complejo Nuclear Basolateral/metabolismo , Etanol/administración & dosificación , Proteínas SNARE/biosíntesis , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Complejo Nuclear Basolateral/química , Etanol/efectos adversos , Femenino , Macaca mulatta , Masculino , Proteínas SNARE/análisis , Autoadministración , Factores de Tiempo

RESUMEN

Products of the proopiomelanocortin (POMC) prohormone regulate aspects of analgesia, reward, and energy balance; thus, the neurons that produce POMC in the hypothalamus have received considerable attention. However, there are also cells in the nucleus of the solitary tract (NTS) that transcribe Pomc, although low levels of Pomc mRNA and relative lack of POMC peptide products in the adult mouse NTS have hindered the study of these cells. Therefore, studies of NTS POMC cells have largely relied on transgenic mouse lines. Here, we set out to determine the amino acid (AA) transmitter phenotype of NTS POMC neurons by using Pomc-Gfp transgenic mice to identify POMC cells. We found that cells expressing the green fluorescent protein (GFP) represent a mix of GABAergic and glutamatergic cells as indicated by Gad2 and vesicular Glut2 ( vGlut2) mRNA expression, respectively. We then examined the AA phenotype of POMC cells labeled by a Pomc-Cre transgene and found that these are also a mix of GABAergic and glutamatergic cells. However, the NTS cells labeled by the Gfp- and Cre-containing transgenes represented distinct populations of cells in three different Pomc-Cre mouse lines. Consistent with previous work, we were unable to reliably detect Pomc mRNA in the NTS despite clear expression in the hypothalamus. Thus, it was not possible to determine which transgenic tool most accurately identifies NTS cells that may express Pomc or release POMC peptides, although the results indicate the transgenic tools for study of these NTS neurons can label disparate populations of cells with varied AA phenotypes.

Asunto(s)

Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Núcleo Solitario/metabolismo , Animales , Femenino , Genotipo , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Proopiomelanocortina/genética , Núcleo Solitario/citología

RESUMEN

Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA release from AgRP neurons either by cell type-specific deletion of the vesicular GABA transporter or by expression of botulinum toxin in AgRP neurons to prevent vesicle-associated membrane protein 2-dependent vesicle fusion. Additionally, there was no difference in the ability of µ-opioid receptor (MOR) agonists to inhibit sIPSCs in POMC neurons when MORs were deleted from AgRP neurons, and activation of the inhibitory designer receptor hM4Di on AgRP neurons did not affect sIPSCs recorded from POMC neurons. These approaches collectively indicate that AgRP neurons do not significantly contribute to the strong spontaneous GABA input to POMC neurons. Despite these observations, optogenetic stimulation of AgRP neurons reliably produced evoked IPSCs in POMC neurons, leading to the inhibition of POMC neuron firing. Thus, AgRP neurons can potently affect POMC neuron function without contributing a significant source of spontaneous GABA input to POMC neurons. Together, these results indicate that the relevance of GABAergic inputs from AgRP to POMC neurons is state dependent and highlight the need to consider different types of transmitter release in circuit mapping and physiologic regulation.SIGNIFICANCE STATEMENT Agouti-related peptide (AgRP) neurons play an important role in driving food intake, while proopiomelanocortin (POMC) neurons inhibit feeding. Despite the importance of these two well characterized neuron types in maintaining metabolic homeostasis, communication between these cells remains poorly understood. To provide clarity to this circuit, we made electrophysiological recordings from mouse brain slices and found that AgRP neurons do not contribute spontaneously released GABA onto POMC neurons, although when activated with channelrhodopsin AgRP neurons inhibit POMC neurons through GABA-mediated transmission. These findings indicate that the relevance of AgRP to POMC neuron GABA connectivity depends on the state of AgRP neuron activity and suggest that different types of transmitter release should be considered when circuit mapping.

Asunto(s)

Potenciales de Acción/fisiología , Proteína Relacionada con Agouti/metabolismo , Potenciales Evocados/fisiología , Proopiomelanocortina/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurotransmisores/metabolismo , Sinapsis/fisiología

RESUMEN

Adolescence represents a particularly vulnerable period during which exposure to stressors can precipitate the onset of psychiatric disorders and addiction. The basolateral amygdala (BLA) plays an integral role in the pathophysiology of anxiety and addiction. Acute and chronic stress promote increases in BLA pyramidal cell firing, and decreasing BLA excitability alleviates anxiety measures in humans and rodents. Notably, the impact of early-life stress on the mechanisms that govern BLA excitability is unknown. To address this gap in our knowledge, we used a rodent model of chronic early-life stress that engenders robust and enduring increases in anxiety-like behaviors and ethanol intake and examined the impact of this model on the intrinsic excitability of BLA pyramidal cells. Adolescent social isolation was associated with a significant increase in the intrinsic excitability of BLA pyramidal cells and a blunting of the medium component of the afterhyperpolarization potential, a voltage signature of calcium-activated potassium (Kca) channel activity. Western blot analysis revealed reduced expression of small-conductance Kca (SK) channel protein in the BLA of socially isolated (SI) rats. Bath application of a positive SK channel modulator (1-EBIO) normalized firing in ex vivo recordings from SI rats, and in vivo intra-BLA 1-EBIO infusion reduced anxiety-like behaviors. These findings reveal that chronic adolescent stress impairs SK channel function, which contributes to an increase in BLA pyramidal cell excitability and highlights BLA SK channels as promising targets for the treatment of anxiety disorders and comorbid addiction. SIGNIFICANCE STATEMENT: Although anxiety disorders and alcohol addiction frequently co-occur, the mechanisms that contribute to this comorbidity are poorly understood. Here, we used a rodent early-life stress model that leads to robust and longlasting increases in behaviors associated with elevated risk of anxiety disorders and addiction to identify novel neurobiological substrates that may underlie these behaviors. Our studies focused on the primary output neurons of the basolateral amygdala, a brain region that plays a key role in anxiety and addiction. We discovered that early-life stress decreases the activity of a specific class of potassium channels and increases the intrinsic excitability of BLA neurons and present evidence that enhancing the function of these channels normalizes BLA excitability and attenuates anxiety-like behaviors.

Asunto(s)

Potenciales de Acción/fisiología , Complejo Nuclear Basolateral/patología , Células Piramidales/fisiología , Estrés Psicológico/patología , Potenciales de Acción/efectos de los fármacos , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Complejo Nuclear Basolateral/efectos de los fármacos , Bencimidazoles/farmacología , Agonistas de los Canales de Calcio/farmacología , Modelos Animales de Enfermedad , Quinasas del Centro Germinal , Técnicas In Vitro , Masculino , Microinyecciones , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/metabolismo , Células Piramidales/efectos de los fármacos , Ratas , Ratas Long-Evans , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Aislamiento Social/psicología , Estrés Psicológico/etiología

RESUMEN

BACKGROUND: The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons. METHODS: Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A(2A) receptor modulation on intrinsic excitability. RESULTS: Activation of adenosine A(2A) receptors with the selective A(2A) receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A(2A) receptor-mediated effects were blocked by preapplication of a selective A(2A) receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A(2A) receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A(2A) receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons. CONCLUSIONS: Collectively, these data suggest that adenosine, via activation of A(2A) receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A1 receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells.

Asunto(s)

Adenosina/metabolismo , Complejo Nuclear Basolateral/metabolismo , Células Piramidales/metabolismo , Receptor de Adenosina A2A/metabolismo , Membranas Sinápticas/metabolismo , Transmisión Sináptica , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Complejo Nuclear Basolateral/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Células Piramidales/efectos de los fármacos , Ratas Long-Evans , Receptor de Adenosina A2A/efectos de los fármacos , Membranas Sinápticas/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Factores de Tiempo

RESUMEN

Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine signaling or cocaine self-administration, particularly under high effort conditions. To address this, we examined the effects of hypocretin receptor 1, and/or hypocretin receptor 2 blockade on dopamine signaling and cocaine reinforcement. We used in vivo fast scan cyclic voltammetry to test the effects of hypocretin antagonists on dopamine signaling in the nucleus accumbens core and a progressive ratio schedule to examine the effects of these antagonists on cocaine self-administration. Results demonstrate that blockade of either hypocretin receptor 1 or both hypocretin receptor 1 and 2 significantly reduces the effects of cocaine on dopamine signaling and decreases the motivation to take cocaine. In contrast, blockade of hypocretin receptor 2 alone had no significant effects on dopamine signaling or self-administration. These findings suggest a differential involvement of the two hypocretin receptors, with hypocretin receptor 1 appearing to be more involved than hypocretin receptor 2 in the regulation of dopamine signaling and cocaine self-administration. When considered with the existing literature, these data support the hypothesis that hypocretins exert a permissive influence on dopamine signaling and motivated behavior via preferential actions on hypocretin receptor 1.

Asunto(s)

Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Dopamina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuropéptidos/metabolismo , Receptores de Orexina/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Varianza , Animales , Benzoxazoles/farmacología , Relación Dosis-Respuesta a Droga , Técnicas Electroquímicas , Masculino , Naftiridinas , Antagonistas de los Receptores de Orexina , Orexinas , Ratas , Ratas Sprague-Dawley , Autoadministración , Urea/análogos & derivados , Urea/farmacología

RESUMEN

The basolateral amygdala (BLA) plays an integral role in the etiology of anxiety disorders and alcoholism. Although much is known about the intrinsic circuitry that governs BLA excitability, our understanding of the neuromodulators that control BLA excitation is incomplete. In many brain regions, adenosine (ADO) regulates neuronal excitability, primarily via A1 receptor inhibition of glutamate release, and basal adenosinergic tone is high enough to tonically inhibit neuronal excitation. Although ADO signaling modulates many anxiety- and alcohol-related behaviors, little is known about ADO regulation of BLA neurotransmission. To that end, we used patch clamp methods in rodent brain slices to characterize adenosinergic modulation of excitatory neurotransmission onto BLA pyramidal cells. ADO significantly inhibited EPSCs evoked by stimulation of either medial or external glutamatergic inputs into the BLA. This effect was mimicked by an A1, but not by an A(2a), agonist. Paired-pulse ratio and miniature EPSC experiments revealed that A1 receptors reside at a presynaptic locus on BLA glutamatergic synapses. Moreover, bath application of an A1 receptor antagonist significantly enhanced EPSCs, providing evidence of tonic adenosinergic tone at BLA glutamatergic synapses. In addition, tonic ADO was regulated by adenosine kinase, but not adenosine deaminase. Finally, activation of A1 receptors had no direct effects on the intrinsic excitability of BLA pyramidal cells. Collectively, these data suggest that tonic A1 receptor signaling may play an important role in regulating BLA excitability and suggest a possible neurobiological substrate through which ADO may contribute to the pathophysiology of anxiety disorders and alcohol addiction.

Asunto(s)

Agonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Adenosina/farmacología , Amígdala del Cerebelo/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Receptor de Adenosina A1/metabolismo , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Amígdala del Cerebelo/metabolismo , Animales , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

BACKGROUND: In vivo magnetic resonance spectroscopy (MRS) has previously been used to directly monitor brain ethanol (EtOH). It has been proposed that the EtOH methyl ¹H resonance intensity is larger in EtOH-tolerant individuals than in sensitive individuals. To characterize the relationship between long-term EtOH exposure and the brain EtOH MRS intensity, we present data from a longitudinal experiment conducted using nonhuman primate subjects. METHODS: In vivo MRS was used to measure the gray matter (GM) and white matter (WM) EtOH methyl ¹H MRS intensity in 18 adult male rhesus macaques at 4 time points throughout the course of a chronic drinking experiment. Time points were prior to EtOH drinking, following a 3-month EtOH induction procedure, and following 6, and 12 subsequent months of 22 h/d of "open access" to EtOH (4% w/v) and water. RESULTS: The EtOH methyl ¹H MRS intensity, which we observed to be independent of age over the range examined, increased with chronic EtOH exposure in GM and WM. In GM, MRS intensity increased from naïve level following the EtOH induction period (90 g/kg cumulative EtOH intake). In WM, MRS intensity was not significantly different from the EtOH-naïve state until after 6 months of 22-hour free access (110 to 850 g/kg cumulative intake range). The WM MRS intensity in the EtOH-naïve state was positively correlated with future drinking, and the increase in WM MRS intensity was negatively correlated with the amount of EtOH consumed throughout the experiment. CONCLUSIONS: Chronic exposure to EtOH is associated with brain changes that result in differential increases in EtOH MRS intensity in GM and WM. The EtOH-naïve WM MRS intensity pattern is consistent with its previously proposed relationship to innate tolerance to the intoxicating effects of EtOH. EtOH-dependent MRS intensity changes in GM required less EtOH exposure than was necessary to produce changes in WM. Within WM, an unexpected, potentially age dependent, enhanced sensitivity to EtOH in light drinkers relative to heavy drinkers was observed.

Asunto(s)

Consumo de Bebidas Alcohólicas/metabolismo , Encéfalo/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Hidrógeno , Trastornos Relacionados con Alcohol/etiología , Animales , Encéfalo/metabolismo , Depresores del Sistema Nervioso Central/metabolismo , Enfermedad Crónica , Etanol/metabolismo , Macaca mulatta , Espectroscopía de Resonancia Magnética , Masculino

RESUMEN

Although alcoholism is a worldwide problem resulting in millions of deaths, only a small percentage of alcohol users become addicted. The specific neural substrates responsible for individual differences in vulnerability to alcohol addiction are not known. In this study, we used rodent models to study behavioral and synaptic correlates related to individual differences in the development of ethanol locomotor sensitization, a form of drug-dependent behavioral plasticity associated with addiction vulnerability. Male Swiss Webster mice were treated daily with saline or 1.8 g/kg ethanol for 21 d. Locomotor activity tests were performed once a week for 15 min immediately after saline or ethanol injections. After at least 11 d of withdrawal, cohorts of saline- or ethanol-treated mice were used to characterize the relationships between locomotor sensitization, ethanol drinking, and glutamatergic synaptic transmission in the nucleus accumbens. Ethanol-treated mice that expressed locomotor sensitization to ethanol drank significantly more ethanol than saline-treated subjects and ethanol-treated animals resilient to this form of behavioral plasticity. Moreover, ethanol-sensitized mice also had reduced accumbal NMDA receptor function and expression, as well as deficits in NMDA receptor-dependent long-term depression in the nucleus accumbens core after a protracted withdrawal. These findings suggest that disruption of accumbal core NMDA receptor-dependent plasticity may represent a synaptic correlate associated with ethanol-induced locomotor sensitization and increased propensity to consume ethanol.

Asunto(s)

Intoxicación Alcohólica/patología , Depresores del Sistema Nervioso Central/administración & dosificación , Etanol/administración & dosificación , Locomoción/fisiología , Núcleo Accumbens/patología , Receptores de N-Metil-D-Aspartato/metabolismo , Intoxicación Alcohólica/etiología , Análisis de Varianza , Animales , Bicuculina/farmacología , Biofisica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Antagonistas de Receptores de GABA-A/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Locomoción/efectos de los fármacos , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Técnicas de Placa-Clamp , Autoadministración , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

Alcoholism and alcohol use disorders are characterized by several months to decades of heavy and problematic drinking, interspersed with periods of abstinence and relapse to heavy drinking. This alcohol-drinking phenotype was modeled using macaque monkeys to explore neuronal adaptations in the striatum, a brain region controlling habitual behaviors. Prolonged drinking with repeated abstinence narrowed the variability in daily intake, increased the amount of ethanol consumed in bouts, and led to higher blood ethanol concentrations more than twice the legal intoxication limit. After the final abstinence period of this extensive drinking protocol, we found a selective increase in dendritic spine density and enhanced glutamatergic transmission in the putamen, but not in the caudate nucleus. Intrinsic excitability of medium-sized spiny neurons was also enhanced in the putamen of alcohol-drinking monkeys in comparison with non-drinkers, and GABAeric transmission was selectively suppressed in the putamen of heavy drinkers. These morphological and physiological changes indicate a shift in the balance of inhibitory/excitatory transmission that biases the circuit toward an enduring increase in synaptic activation of putamen output as a consequence of prolonged heavy drinking/relapse. The resultant potential for increased putamen activation may underlie an alcohol-drinking phenotype of regulated drinking and sustained intoxication.

Asunto(s)

Adaptación Fisiológica/fisiología , Consumo de Bebidas Alcohólicas/metabolismo , Putamen/metabolismo , Putamen/patología , Sinapsis/metabolismo , Sinapsis/patología , Adaptación Fisiológica/efectos de los fármacos , Consumo de Bebidas Alcohólicas/patología , Animales , Etanol/administración & dosificación , Macaca fascicularis , Masculino , Putamen/efectos de los fármacos , Recurrencia , Síndrome de Abstinencia a Sustancias/metabolismo , Síndrome de Abstinencia a Sustancias/patología , Sinapsis/efectos de los fármacos , Factores de Tiempo