RESUMO
Nucleus basalis magnocellularis (NBM) cholinergic projections to the basolateral amygdala (BLA) regulate the acquisition and consolidation of fear-like and anxiety-like behaviors. However, it is unclear whether the alterations in the NBM-BLA circuit promote negative affect during ethanol withdrawal (WD). Therefore, we performed ex vivo whole-cell patch-clamp electrophysiology in both the NBM and the BLA of male Sprague Dawley rats following 10 d of chronic intermittent ethanol (CIE) exposure and 24 h of WD. We found that CIE exposure and withdrawal enhanced the neuronal excitability of NBM putative "cholinergic" neurons. We subsequently used optogenetics to directly manipulate NBM terminal activity within the BLA and measure cholinergic modulation of glutamatergic afferents and BLA pyramidal neurons. Our findings indicate that CIE and withdrawal upregulate NBM cholinergic facilitation of glutamate release via activation of presynaptic nicotinic acetylcholine receptors (AChRs). Ethanol withdrawal-induced increases in NBM terminal activity also enhance BLA pyramidal neuron firing. Collectively, our results provide a novel characterization of the NBM-BLA circuit and suggest that CIE-dependent modifications to NBM afferents enhance BLA pyramidal neuron activity during ethanol withdrawal.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Síndrome de Abstinência a Substâncias , Animais , Ratos , Masculino , Etanol/farmacologia , Ratos Sprague-Dawley , Tonsila do Cerebelo/fisiologia , Núcleo Basal de MeynertRESUMO
Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.
RESUMO
The basolateral amygdala (BLA) is intimately involved in the development of neuropsychiatric disorders such as anxiety and alcohol use disorder (AUD). These disorders have clear sex biases, with women more likely to develop an anxiety disorder and men more likely to develop AUD. Preclinical models have largely confirmed these sex-specific vulnerabilities and emphasize the effects of sex hormones on behaviors influenced by the BLA. This review will discuss sex differences in BLA-related behaviors and highlight potential mechanisms mediated by altered BLA structure and function, including the composition of GABAergic interneuron subpopulations, glutamatergic pyramidal neuron morphology, glutamate/GABA neurotransmission, and neuromodulators. Further, sex hormones differentially organize dimorphic circuits during sensitive developmental periods (organizational effects) and initiate more transient effects throughout adulthood (activational effects). Current literature indicates that estradiol and allopregnanolone, a neuroactive progestogen, generally reduce BLA-related behaviors through a variety of mechanisms, including activation of estrogen receptors or facilitation of GABAA-mediated inhibition, respectively. This enhanced GABAergic inhibition may protect BLA pyramidal neurons from the excitability associated with anxiety and alcohol withdrawal. Understanding sex differences and the effects of sex hormones on BLA structure and function may help explain sex-specific vulnerabilities in BLA-related behaviors and ultimately improve treatments for anxiety and AUD.
Assuntos
Alcoolismo , Complexo Nuclear Basolateral da Amígdala , Síndrome de Abstinência a Substâncias , Adulto , Complexo Nuclear Basolateral da Amígdala/fisiologia , Feminino , Hormônios Gonadais/farmacologia , Humanos , Masculino , Transmissão SinápticaRESUMO
This review highlights literature relating the anatomy, physiology, and behavioral contributions by projections between rodent prefrontal cortical areas and the basolateral amygdala. These projections are robustly modulated by both environmental experience and exposure to drugs of abuse including ethanol. Recent literature relating optogenetic and chemogenetic dissection of these circuits within behavior both compliments and occasionally challenges roles defined by more traditional pharmacological or lesion-based approaches. In particular, cortico-amygdala circuits help control both aversive and reward-seeking. Exposure to pathology-producing environments or abused drugs dysregulates the relative 'balance' of these outcomes. Modern circuit-based approaches have also shown that overlapping populations of neurons within a given brain region frequently govern both aversion and reward-seeking. In addition, these circuits often dramatically influence 'local' cortical or basolateral amygdala excitatory or inhibitory circuits. Our understanding of these neurobiological processes, particularly in relation to ethanol research, has just begun and represents a significant opportunity. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Rede Nervosa/efeitos dos fármacos , Animais , HumanosRESUMO
Chronic intermittent ethanol (CIE) exposure dysregulates glutamatergic and GABAergic neurotransmission, facilitating basolateral amygdala (BLA) pyramidal neuron hyperexcitability and the expression of anxiety during withdrawal. It is unknown whether ethanol-induced alterations in nucleus basalis magnocellularis (NBM) cholinergic projections to the BLA mediate anxiety-related behaviors through direct modulation of GABA and glutamate afferents. Following 10â¯days of CIE exposure and 24â¯h of withdrawal, we recorded GABAergic and glutamatergic synaptic responses in BLA pyramidal neurons with electrophysiology, assessed total protein expression of cholinergic markers, and quantified acetylcholine and choline concentrations using a colorimetric assay. We measured α7 nicotinic acetylcholine receptor (nAChR) dependent modulation of presynaptic function at distinct inputs in AIR- and CIE-exposed BLA coronal slices as a functional read-out of cholinergic neurotransmission. CIE/withdrawal upregulates the endogenous activity of α7 nAChRs, facilitating release at both GABAergic' local' interneuron and glutamatergic synaptic responses to stria terminalis (ST) stimulation, with no effect at GABAergic lateral paracapsular cells (LPCs). CIE caused a three-fold increase in BLA acetylcholine concentration, with no changes in α7 nAChR or cholinergic marker expression. These data illustrate that α7 nAChR-dependent changes in presynaptic function serve as a proxy for CIE-dependent alterations in synaptic acetylcholine levels. Thus, cholinergic projections appear to mediate CIE-induced alterations at GABA/glutamate inputs.
Assuntos
Tonsila do Cerebelo , Complexo Nuclear Basolateral da Amígdala , Etanol , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiopatologia , Animais , Colinérgicos/farmacologia , Etanol/farmacologia , Potenciais Pós-Sinápticos Excitadores , Masculino , Ratos , Ratos Sprague-Dawley , Sinapses , Transmissão SinápticaRESUMO
A key feature of alcohol use disorder (AUD) is negative affect during withdrawal, which often contributes to relapse and is thought to be caused by altered brain function, especially in circuits that are important mediators of emotional behaviors. Both the agranular insular cortex (AIC) and the basolateral amygdala (BLA) regulate emotions and are sensitive to ethanol-induced changes in synaptic plasticity. The AIC and BLA are reciprocally connected; and the effects of chronic ethanol exposure on this circuit have yet to be explored. Here, we use a combination of optogenetics and electrophysiology to examine the pre- and postsynaptic changes that occur to AIC-BLA synapses following withdrawal from 7- or 10-days of chronic intermittent ethanol (CIE) exposure. While CIE/withdrawal did not alter presynaptic glutamate release probability from AIC inputs, withdrawal from 10, but not 7, days of CIE increased AMPA receptor-mediated postsynaptic function at these synapses. Additionally, NMDA receptor-mediated currents evoked by electrical stimulation of the external capsule, which contains AIC afferents, were also increased during withdrawal. Notably, a single subanesthetic dose of ketamine administered at the onset of withdrawal prevented the withdrawal-induced increases in both AMPAR and NMDAR postsynaptic function. Ketamine also prevented the withdrawal-induced increases in anxiety-like behavior measured using the elevated zero maze. Together, these findings suggest that chronic ethanol exposure increases postsynaptic function within the AIC-BLA circuit and that ketamine can prevent ethanol withdrawal-induced alterations in synaptic plasticity and negative affect.
Assuntos
Alcoolismo/fisiopatologia , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Vias Neurais/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/metabolismo , Administração por Inalação , Animais , Estimulação Elétrica , Fenômenos Eletrofisiológicos , Etanol/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ketamina/farmacologia , Masculino , Optogenética , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
The medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) have strong reciprocal connectivity. Projections from the BLA to the mPFC can drive innate, anxiety-related behaviors, but it is unclear whether reciprocal projections from the mPFC to BLA have similar roles. Here, we use optogenetics and chemogenetics to characterize the neurophysiological and behavioral alterations produced by chronic ethanol exposure and withdrawal on dorsal mPFC (dmPFC) and ventral mPFC (vmPFC) medial prefrontal cortical terminals in the BLA. We exposed adult male Sprague Dawley rats to chronic intermittent ethanol (CIE) using vapor chambers, measured anxiety-like behavior on the elevated zero maze, and used electrophysiology to record glutamatergic and GABAergic responses in BLA principal neurons. We found that withdrawal from a 7 d CIE exposure produced opposing effects at dmPFC (increased glutamate release) and vmPFC (decreased glutamate release) terminals in the BLA. Chemogenetic inhibition of dmPFC terminals in the BLA attenuated the increased anxiety-like behavior we observed during withdrawal. These data demonstrate that chronic ethanol exposure and withdrawal strengthen the synaptic connections between the dmPFC and BLA but weakens the vmPFC-BLA pathway. Moreover, facilitation of the dmPFC-BLA pathway during withdrawal contributes to anxiety-like behavior. Given the opposing roles of dmPFC-BLA and vmPFC-BLA pathways in fear conditioning, our results suggest that chronic ethanol exposure simultaneously facilitates circuits involved in the acquisition of and diminishes circuits involved with the extinction of withdrawal-related aversive behaviors.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Tonsila do Cerebelo , Animais , Etanol , Ácido Glutâmico , Masculino , Neurônios , Córtex Pré-Frontal , Ratos , Ratos Sprague-DawleyRESUMO
Adolescent alcohol use in human populations dramatically increases the likelihood of adult alcohol use disorder. This adolescent vulnerability is recapitulated in preclinical models which provide important opportunities to understand basic neurobiological mechanisms. We provide here an overview of GABAergic and glutamatergic neurotransmission and our current understanding of the sensitivity of these systems to adolescent ethanol exposure. As a whole, the preclinical literature suggests that adolescent vulnerability may be directly related to region-specific neurobiological processes that continue to develop during adolescence. These processes include the activity of intrinsic circuits within diverse brain regions (primarily represented by GABAergic neurotransmission) and activity-dependent regulation of synaptic strength at glutamatergic synapses. Furthermore, GABAergic and glutamatergic neurotransmission within regions/circuits that regulate cognitive function, emotion, and their integration appears to be the most vulnerable to adolescent ethanol exposure. Finally, using documented behavioral differences between adolescents and adults with respect to acute ethanol, we highlight additional circuits and regions for future study.
Assuntos
Alcoolismo/fisiopatologia , Encéfalo/efeitos dos fármacos , Transmissão Sináptica , Adolescente , Encéfalo/fisiopatologia , Neurônios GABAérgicos/fisiologia , Ácido Glutâmico , Humanos , SinapsesRESUMO
Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. ß-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ß-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ß-arrestin expression and ß-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ß-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ß-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ß-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ß-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ß-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced ß-arrestin2 levels and delayed agonist-induced ß-arrestin2 recruitment to the membrane. We conclude that ß-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ß-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.
Assuntos
Endocitose , Etanol/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , Ubiquitinação/efeitos dos fármacos , beta-Arrestina 2/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular , Masculino , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , beta-Arrestina 2/antagonistas & inibidores , beta-Arrestina 2/genéticaRESUMO
Chronic peri-adolescent stress in humans increases risk to develop a substance use disorder during adulthood. Rats reared in social isolation during peri-adolescence (aSI; 1 rat/cage) period show greater ethanol and cocaine intake compared to group housed (aGH; 4 rats/cage) rats. In addition, aSI rats have a heightened dopamine response in the nucleus accumbens (NAc) to rewarding and aversive stimuli. Furthermore, single pulse electrical stimulation in slices containing NAc core elicits greater dopamine release in aSI rats. Here, we further investigated dopamine release kinetics and machinery following aSI. Dopamine release, across a wide range of stimulation intensities and frequencies, was significantly greater in aSI rats. Interestingly, subthreshold intensity stimulations also resulted in measurable dopamine release in accumbal slices from aSI but not aGH rats. Extracellular [Ca2+] manipulations revealed augmented calcium sensitivity of dopamine release in aSI rats. The readily releasable pools of dopamine, examined by bath application of Ro-04-1284/000, a vesicular monoamine transporter 2 (VMAT2) inhibitor, were depleted faster in aGH rats. Western blot analysis of release machinery proteins (VMAT2, Synaptogyrin-3, Syntaxin-1, and Munc13-3) showed no difference between the two groups. Tyrosine hydroxylase (TH) protein expression levels, however, were elevated in aSI rats. The greater dopamine release could potentially be explained by higher levels of TH, the rate-limiting step for dopamine synthesis. This augmented responsivity of the dopamine system and heightened dopamine availability post-aSI may lead to an increased risk of addiction vulnerability.
Assuntos
Dopamina/metabolismo , Núcleo Accumbens/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isolamento Social , Estresse Psicológico/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Fatores Etários , Animais , Doença Crônica , Inibidores da Captação de Dopamina/farmacologia , Masculino , Núcleo Accumbens/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Long-Evans , Isolamento Social/psicologia , Estresse Psicológico/psicologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidoresRESUMO
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.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Consumo de Bebidas Alcoólicas/tendências , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Complexo Nuclear Basolateral da Amígdala/metabolismo , Etanol/administração & dosagem , Proteínas SNARE/biossíntese , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Complexo Nuclear Basolateral da Amígdala/química , Etanol/efeitos adversos , Feminino , Macaca mulatta , Masculino , Proteínas SNARE/análise , Autoadministração , Fatores de TempoRESUMO
The basolateral amygdala (BLA) controls numerous behaviors, like anxiety and reward seeking, via the activity of glutamatergic principal neurons. These BLA neurons receive excitatory inputs primarily via two major anatomical pathways - the external capsule (EC), which contains afferents from lateral cortical structures, and the stria terminalis (ST), containing synapses from more midline brain structures. Chronic intermittent ethanol (CIE) exposure/withdrawal produces distinct alterations in these pathways. Specifically, 10â¯days of CIE (via vapor inhalation) increases presynaptic function at ST synapses and postsynaptic function at EC synapses. Given that 10-day CIE/withdrawal also increases anxiety-like behavior, we sought to examine the development of these alterations at these inputs using an exposure time-course in both male and female rats. Specifically, using 3, 7, and 10â¯days CIE exposure, we found that all three durations increase anxiety-like behavior in the elevated plus maze. At BLA synapses, increased presynaptic function at ST inputs required shorter exposure durations relative to post-synaptic alterations at EC inputs in both sexes. But, synaptic alterations in females required longer ethanol exposures compared to males. These data suggest that presynaptic alteration at ST-BLA afferents is an early neuroadaptation during repeated ethanol exposures. And, the similar patterns of presynaptic-then-postsynaptic facilitation across the sexes suggest the former may be required for the latter. These cooperative interactions may contribute to the increased anxiety-like behavior that is observed following CIE-induced withdrawal and may provide novel therapeutic targets to reverse withdrawal-induced anxiety.
Assuntos
Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Caracteres Sexuais , Administração por Inalação , Animais , Ansiedade/induzido quimicamente , Ansiedade/fisiopatologia , Complexo Nuclear Basolateral da Amígdala/fisiopatologia , Ciclo Estral/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Cápsula Externa/efeitos dos fármacos , Cápsula Externa/fisiopatologia , Feminino , Ácido Glutâmico/metabolismo , Cápsula Interna/efeitos dos fármacos , Cápsula Interna/fisiopatologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Ratos Sprague-Dawley , Síndrome de Abstinência a Substâncias/fisiopatologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Chronic exposure to alcohol produces adaptations within the basolateral amygdala (BLA) that are associated with the development of anxiety-like behaviors during withdrawal. In part, these adaptations are mediated by plasticity in glutamatergic synapses occurring through an AMPA receptor mediated form of post-synaptic facilitation in addition to a unique form of presynaptic facilitation. In comparison to the post-synaptic compartment, relatively less is understood about the mechanisms involved in the acute and chronic effects of ethanol in the presynaptic terminal. Previous research has demonstrated that glutamatergic terminals in the mouse BLA are sensitive to ethanol mediated inhibition of synaptic vesicle recycling in a strain-dependent fashion. Importantly, the strain-dependent differences in presynaptic ethanol sensitivity are in accordance with known strain-dependent differences in ethanol/anxiety interactions. In the present study, we have used a short-hairpin RNA to knockdown the expression of the presynaptic Munc13-2 protein in C57BL/6J mice, whose BLA glutamate terminals are normally ethanol-insensitive. We injected this shRNA, or a scrambled control virus, into the medial prefrontal cortex (mPFC) which sends dense projections to the BLA. Accordingly, this knockdown strategy reduces the expression of the Munc13-2 isoform in mPFC terminals within the BLA and alters presynaptic terminal function in C57BL/6J mice in a manner that phenocopies DBA/2J glutamate terminals which are normally ethanol-sensitive. Here, we provide evidence that manipulation of this single protein, Munc13-2, renders C57BL/6J terminals sensitive to ethanol mediated inhibition of synaptic vesicle recycling and post-tetanic potentiation. Furthermore, we found that this ethanol inhibition was dose dependent. Considering the important role of Munc13 proteins in synaptic plasticity, this study potentially identifies a molecular mechanism regulating the acute presynaptic effects of ethanol to the long lasting adaptations in the BLA that occur during chronic ethanol exposure.
RESUMO
C57BL/6J (B6) and DBA/2J (D2) mice are well known to differentially express a number of behavioral phenotypes, including anxiety-like behavior, fear conditioning, and drug self-administration. However, the cellular mechanisms contributing to these differences remain unclear. Given the basolateral amygdala (BLA) contributes to these behaviors, we characterized strain-dependent differences in presynaptic and postsynaptic function in BLA neurons by integrating electrophysiological, biochemical, and genetic approaches to identify specific molecular mechanisms. We found that D2 glutamatergic synapses expressed enhanced release probability and lower sensitivity to both the inhibitory effects of low extracellular calcium and facilitation by phorbol esters. Furthermore, repetitive stimulation of BLA afferents at low (2 Hz) or high (40 Hz) frequencies revealed that B6 terminals, relative to D2 terminals, were more sensitive to synaptic fatigue principally because of reduced vesicle recycling rates. Additionally, B6 synapses exhibited more robust augmentation of spontaneous release after repetitive stimulation relative to the D2 strain. In silico analysis of the inheritance of synaptic physiology from an array of BXD recombinant inbred strains (Jansen et al., 2011) identified a segment on chromosome 4 containing the gene encoding Munc13-2, which has calcium-/phorbol ester-binding domains and controls presynaptic function. We subsequently found that B6 mice express substantially higher levels of Munc13-2 compared with the D2 strain whereas expression of several release-related proteins, including Munc13-1, was equivalent. We then knocked down the expression of Munc13-2 in B6 mice using a short hairpin RNA and found this recapitulated the presynaptic phenotype of D2 BLA synapses. SIGNIFICANCE STATEMENT: DBA/2J and C57BL/6J mice have been used to understand the genetic mechanisms controlling behaviors related to a number of psychiatric illnesses. However, the fundamental neurobiological mechanisms producing these behavioral characteristics remain unresolved. Here we identify a critical family of presynaptic proteins differentially expressed by these strains that control strain-dependent synaptic physiology. This family of proteins regulates excitation/secretion coupling, vesicle recycling, and short-term plasticity throughout the CNS. Thus, differential inheritance of proteins like Munc13-2 has broad implications for genetic control over a wide variety of pathological behaviors. Importantly, these proteins also contain a large number of modulatory sites, making them attractive potential targets for the development of novel neuropharmaceutical treatments.
Assuntos
Complexo Nuclear Basolateral da Amígdala/metabolismo , Ácido Glutâmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/classificação , Sinapses/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Especificidade da EspécieRESUMO
Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced ß-arrestin membrane recruitment; however, it prevents ß-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time ß-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of ß-arrestin post-endocytic fate revealed that quinpirole treatment caused ß-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of ß-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired ß-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for ß-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.
Assuntos
Proteínas RGS/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clatrina/metabolismo , AMP Cíclico/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Camundongos , Fosforilação/fisiologia , Proteínas RGS/genética , beta-Arrestinas/metabolismoRESUMO
The endogenous cannabinoid (eCB) system has been suggested to play a key role in ethanol preference and intake, the acute effects of ethanol, and in the development of withdrawal symptoms following ethanol dependence. Ethanol-dependent alterations in glutamatergic signaling within the lateral/basolateral nucleus of the amygdala (BLA) are critical for the development and expression of withdrawal-induced anxiety. Notably, the eCB system significantly regulates both glutamatergic and GABAergic synaptic activity within the BLA. Chronic ethanol exposure significantly alters eCB system expression within regions critical to the expression of emotionality and anxiety-related behavior, including the BLA. Here, we investigated specific interactions between the BLA eCB system and its functional regulation of synaptic activity during acute and chronic ethanol exposure. In tissue from ethanol naïve-rats, a prolonged acute ethanol exposure caused a dose dependent inhibition of glutamatergic synaptic activity via a presynaptic mechanism that was occluded by CB1 antagonist/inverse agonists SR141716a and AM251. Importantly, this acute ethanol inhibition was attenuated following 10 day chronic intermittent ethanol vapor exposure (CIE). CIE exposure also significantly down-regulated CB1-mediated presynaptic inhibition at glutamatergic afferent terminals but spared CB1-inhibition of GABAergic synapses arising from local inhibitory-interneurons. CIE also significantly elevated BLA N-arachidonoylethanolamine (AEA or anandamide) levels and decreased CB1 receptor protein levels. Collectively, these data suggest a dynamic regulation of the BLA eCB system by acute and chronic ethanol.
Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiologia , Etanol/administração & dosagem , Ácido Glutâmico/metabolismo , Receptor CB1 de Canabinoide/fisiologia , Sinapses/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Sinapses/efeitos dos fármacosRESUMO
BACKGROUND: Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined. METHODS: Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens. RESULTS: Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group. CONCLUSIONS: These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety/compulsive-like behaviors may be driven by greater kappa opioid receptor sensitivity and a hypodopaminergic state of the nucleus accumbens.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Transtornos do Sistema Nervoso Induzidos por Álcool/metabolismo , Comportamento Animal , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Etanol , Núcleo Accumbens/metabolismo , Receptores Opioides kappa/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Transmissão Sináptica , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/fisiopatologia , Consumo de Bebidas Alcoólicas/psicologia , Transtornos do Sistema Nervoso Induzidos por Álcool/fisiopatologia , Transtornos do Sistema Nervoso Induzidos por Álcool/psicologia , Analgésicos Opioides/farmacologia , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Ansiedade/psicologia , Comportamento Animal/efeitos dos fármacos , Comportamento Compulsivo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Antagonistas de Entorpecentes/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Receptores Opioides kappa/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/fisiopatologia , Síndrome de Abstinência a Substâncias/psicologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
The current experiment examined the effects of 10 days of chronic intermittent ethanol (CIE) exposure on anxiety-like behavior and home cage ethanol intake using a 20% intermittent access (M, W, F) paradigm in male and female Long-Evans rats. Withdrawal from alcohol dependence contributes to relapse in humans and increases in anxiety-like behavior and voluntary ethanol consumption in preclinical models. Our laboratory has shown that 10 days of CIE exposure produces both behavioral and neurophysiological alterations associated with withdrawal in male rats; however, we have yet to examine the effects of this exposure regime on ethanol intake in females. During baseline, females consumed more ethanol than males but, unlike males, did not show escalations in intake. Rats were then exposed to CIE and were again given intermittent access to 20% ethanol. CIE males increased their intake compared to baseline, whereas air-exposed males did not. Ethanol intake in females was unaffected by CIE exposure. Notably, both sexes expressed significantly elevated withdrawal-associated anxiety-like behavior in the plus maze. Finally, rats were injected with the cannabinoid CB1 receptor antagonist, SR141716A (0, 1, 3, 10mg/kg, i.p.) which reduced ethanol intake in both sexes. However, females appear to be more sensitive to lower doses of this CB1 receptor antagonist. Our results show that females consume more ethanol than males; however, they did not escalate their intake using the intermittent access paradigm. Unlike males, CIE exposure had no effect on drinking in females. It is possible that females may be less sensitive than males to ethanol-induced increases in drinking after a short CIE exposure. Lastly, our results demonstrate that males and females may have different pharmacological sensitivities to CB1 receptor blockade on ethanol intake, at least under the current conditions.
Assuntos
Consumo de Bebidas Alcoólicas , Ingestão de Líquidos/efeitos dos fármacos , Etanol/administração & dosagem , Etanol/farmacologia , Caracteres Sexuais , Animais , Ansiedade/induzido quimicamente , Comportamento de Escolha/efeitos dos fármacos , Relação Dose-Resposta a Droga , Etanol/antagonistas & inibidores , Etanol/sangue , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Long-Evans , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Rimonabanto , AutoadministraçãoRESUMO
BACKGROUND: Ethanol drinking pattern has emerged as an important factor in the development, maintenance, and health consequences of alcohol use disorders in humans. The goal of these studies was to further our understanding of this important factor through refinement of an operant rodent model of ethanol consumption capable of drinking pattern microstructural analysis. We evaluated measures of total consumption, appetitive behavior, and drinking microstructure for ethanol and water at baseline and assessed alterations induced by two treatments previously shown to significantly alter gross ethanol appetitive and consummatory behaviors in opposing directions. METHODS: Male Long-Evans rats were trained on an FR1 operant paradigm which allowed for continuous liquid access until an 8 second pause in consumption resulted in termination of liquid access. Total appetitive and consummatory behaviors were assessed in addition to microstructural drinking pattern for both ethanol and water during a five day baseline drinking period, after chronic intermittent ethanol vapor exposure, and following administration of a cannabinoid receptor antagonist SR141716a. RESULTS: As in previous operant studies, ethanol vapor exposure resulted in increases in ethanol-directed responding, total consumption, and rate of intake. Further, striking differential alterations to ethanol and water bout size, duration, and lick pattern occurred consistent with alterations in hedonic evaluation. Vapor additionally specifically reduced the number of ethanol-directed lever presses which did not result in subsequent consumption. SR141716a administration reversed many of these effects. CONCLUSIONS: The addition of microstructural analysis to operant self-administration by rodents provides a powerful and translational tool for the detection of specific alterations in ethanol drinking pattern which may enable insights into neural mechanisms underlying specific components of drug consumption.
Assuntos
Consumo de Bebidas Alcoólicas , Condicionamento Operante/fisiologia , Comportamento de Ingestão de Líquido/fisiologia , Etanol/administração & dosagem , Água/administração & dosagem , Análise de Variância , Animais , Antagonistas de Receptores de Canabinoides/farmacologia , Masculino , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Long-Evans , Esquema de Reforço , Rimonabanto , Autoadministração , Fatores de TempoRESUMO
Adolescent social isolation (SI) results in numerous behavioral alterations associated with increased risk of alcoholism. Notably, many of these changes involve the basolateral amygdala (BLA), including increased alcohol seeking. The BLA sends a strong glutamatergic projection to the nucleus accumbens and activation of this pathway potentiates reward-seeking behavior. Dopamine (DA) and norepinephrine (NE) exert powerful excitatory and inhibitory effects on BLA activity and chronic stress can disrupt the excitation-inhibition balance maintained by these catecholamines. Notably, the impact of SI on BLA DA and NE neurotransmission is unknown. Thus the aim of this study was to characterize SI-mediated catecholamine alterations in the BLA. Male Long Evans rats were housed in groups of four (GH) or in SI for 6 weeks during adolescence. DA and NE transporter levels were then measured using Western blot hybridization and baseline and ethanol-stimulated DA and NE levels were quantified using microdialysis. DA transporter levels were increased and baseline DA levels were decreased in SI compared to GH rats. SI also increased DA responses to an acute ethanol (2 g kg(-1)) challenge. While no group differences were noted in NE transporter or baseline NE levels, acute ethanol (2 g kg(-1)) only significantly increased NE levels in SI animals. Collectively, these SI-dependent changes in BLA catecholamine signaling may lead to an increase in BLA excitability and a strengthening of the glutamatergic projection between the BLA and NAc. Such changes may promote the elevated ethanol drinking behavior observed in rats subjected to chronic adolescent stress.