RESUMO
Contextual drug-associated memories precipitate craving and relapse in cocaine users. Such associative memories can be weakened through interference with memory reconsolidation, a process by which memories are maintained following memory retrieval-induced destabilization. We hypothesized that cocaine-memory reconsolidation requires cannabinoid type 1 receptor (CB1R) signaling based on the fundamental role of the endocannabinoid system in synaptic plasticity and emotional memory processing. Using an instrumental model of cocaine relapse, we evaluated whether systemic CB1R antagonism (AM251; 3 mg/kg, i.p.) during memory reconsolidation altered (1) subsequent drug context-induced cocaine-seeking behavior as well as (2) cellular adaptations and (3) excitatory synaptic physiology in the basolateral amygdala (BLA) in male Sprague Dawley rats. Systemic CB1R antagonism, during, but not after, cocaine-memory reconsolidation reduced drug context-induced cocaine-seeking behavior 3 d, but not three weeks, later. CB1R antagonism also inhibited memory retrieval-associated increases in BLA zinc finger 268 (zif268) and activity regulated cytoskeletal-associated protein (Arc) immediate-early gene (IEG) expression and changes in BLA AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subunit phosphorylation that likely contribute to increased receptor membrane trafficking and synaptic plasticity during memory reconsolidation. Furthermore, CB1R antagonism increased memory reconsolidation-associated spontaneous EPSC (sEPSC) frequency in BLA principal neurons during memory reconsolidation. Together, these findings suggest that CB1R signaling modulates cellular and synaptic mechanisms in the BLA that may facilitate cocaine-memory strength by enhancing reconsolidation or synaptic reentry reinforcement, or by inhibiting extinction-memory consolidation. These findings identify the CB1R as a potential therapeutic target for relapse prevention.SIGNIFICANCE STATEMENT Drug relapse can be triggered by the retrieval of context-drug memories on re-exposure to a drug-associated environment. Context-drug associative memories become destabilized on retrieval and must be reconsolidated into long-term memory stores to persist. Hence, targeted interference with memory reconsolidation can weaken maladaptive context-drug memories and reduce the propensity for drug relapse. Our findings indicate that cannabinoid type 1 receptor (CB1R) signaling is critical for context-cocaine memory reconsolidation and subsequent drug context-induced reinstatement of cocaine-seeking behavior. Furthermore, cocaine-memory reconsolidation is associated with CB1R-dependent immediate-early gene (IEG) expression and changes in excitatory synaptic proteins and physiology in the basolateral amygdala (BLA). Together, our findings provide initial support for CB1R as a potential therapeutic target for relapse prevention.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Cocaína/farmacologia , Comportamento de Procura de Droga/efeitos dos fármacos , Consolidação da Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Receptor CB1 de Canabinoide/efeitos dos fármacos , Animais , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Transtornos Relacionados ao Uso de Cocaína/psicologia , Endocanabinoides/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , AutoadministraçãoRESUMO
The endocannabinoid system plays important roles in brain development, but mechanistic studies have focused on neuronal differentiation, migration, and synaptogenesis, with less attention to transcellular interactions that coordinate neurodevelopmental processes across developing neural networks. We determined that, in the developing rodent cerebellar cortex (of both sexes), there is a transient window when the dominant brain cannabinoid receptor, CB1R, is expressed on afferent terminals instead of output neuron Purkinje cell synapses that dominate the adult cerebellum. Activation of these afferent CB1Rs suppresses synaptic transmission onto developing granule cells, and consequently also suppresses excitation of downstream neurons in the developing cortical network, including nonsynaptic, migrating neurons. Application of a CB1R antagonist during afferent stimulation trains and depolarizing voltage steps caused a significant, sustained potentiation of synaptic amplitude. Our data demonstrate that transiently expressed afferent CB1Rs regulate afferent synaptic strength during synaptogenesis, which enables coordinated dampening of transcortical developmental signals.SIGNIFICANCE STATEMENT The endogenous cannabinoid system plays diverse roles in brain development, which, combined with the rapidly changing legal and medical status of cannabis-related compounds, makes understanding how exogenous cannabinoids affect brain development an important biomedical objective. The cerebellum is a key brain region in a variety of neurodevelopmental disorders, and the adult cerebellum has one of the highest expression levels of CB1R, but little is known about CB1R in the developing cerebellum. Here we report a developmentally distinct expression and function of CB1R in the cerebellum, in which endogenous or exogenous activation of CB1Rs modifies afferent synaptic strength and coordinated downstream network signaling. These findings have implications for recreational and medical use of exogenous cannabinoids by pregnant and breastfeeding women.
Assuntos
Potenciais Pós-Sinápticos Excitadores , Neurogênese , Neurônios Aferentes/metabolismo , Células de Purkinje/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Potenciais de Ação , Animais , Antagonistas de Receptores de Canabinoides/farmacologia , Movimento Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/fisiologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Preterm infants are at risk for a broad spectrum of neurobehavioral disabilities associated with diffuse disturbances in cortical growth and development. During brain development, subplate neurons (SPNs) are a largely transient population that serves a critical role to establish functional cortical circuits. By dynamically integrating into developing cortical circuits, they assist in consolidation of intracortical and extracortical circuits. Although SPNs reside in close proximity to cerebral white matter, which is particularly vulnerable to oxidative stress, the susceptibility of SPNs remains controversial. We determined SPN responses to two common insults to the preterm brain: hypoxia-ischemia and hypoxia. We used a preterm fetal sheep model using both sexes that reproduces the spectrum of human cerebral injury and abnormal cortical growth. Unlike oligodendrocyte progenitors, SPNs displayed pronounced resistance to early or delayed cell death from hypoxia or hypoxia-ischemia. We thus explored an alternative hypothesis that these insults alter the maturational trajectory of SPNs. We used DiOlistic labeling to visualize the dendrites of SPNs selectively labeled for complexin-3. SPNs displayed reduced basal dendritic arbor complexity that was accompanied by chronic disturbances in SPN excitability and synaptic activity. SPN dysmaturation was significantly associated with the level of fetal hypoxemia and metabolic stress. Hence, despite the resistance of SPNs to insults that trigger white matter injury, transient hypoxemia disrupted SPN arborization and functional maturation during a critical window in cortical development. Strategies directed at limiting the duration or severity of hypoxemia during brain development may mitigate disturbances in cerebral growth and maturation related to SPN dysmaturation.SIGNIFICANCE STATEMENT The human preterm brain commonly sustains blood flow and oxygenation disturbances that impair cerebral cortex growth and cause life-long cognitive and learning disabilities. We investigated the fate of subplate neurons (SPNs), which are a master regulator of brain development that plays critical roles in establishing cortical connections to other brain regions. We used a preterm fetal sheep model that reproduces key features of brain injury in human preterm survivors. We analyzed the responses of fetal SPNs to transient disturbances in fetal oxygenation. We discovered that SPNs are surprisingly resistant to cell death from low oxygen states but acquire chronic structural and functional changes that suggest new strategies to prevent learning problems in children and adults that survive preterm birth.
Assuntos
Hipóxia/patologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Animais , Dendritos/fisiologia , Feminino , Hipóxia/complicações , Masculino , Degeneração Neural/etiologia , Degeneração Neural/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Ovinos , Fatores de TempoRESUMO
In the brain, fast inhibitory neurotransmission is mediated primarily by the ionotropic subtype of the gamma-aminobutyric acid (GABA) receptor subtype A (GABAAR). It is well established that the brain's GABAAR system mediates many aspects of neurobehavioral responses to alcohol (ethanol; EtOH). Accordingly, in both preclinical studies and some clinical scenarios, pharmacologically targeting the GABAAR system can alter neurobehavioral responses to acute and chronic EtOH consumption. However, many of the well-established interactions of EtOH and the GABAAR system have been identified at concentrations of EtOH ([EtOH]) that would only occur during abusive consumption of EtOH (≥40 mM), and there are still inadequate treatment options for prevention of or recovery from alcohol use disorder (AUD, including abuse and dependence). Accordingly, there is a general acknowledgement that more research is needed to identify and characterize: (1) neurobehavioral targets of lower [EtOH] and (2) associated brain structures that would involve such targets in a manner that may influence the development and maintenance of AUDs.Nearly 15 years ago it was discovered that the GABAAR system of the cerebellum is highly sensitive to EtOH, responding to concentrations as low as 10 mM (as would occur in the blood of a typical adult human after consuming 1-2 standard units of EtOH). This high sensitivity to EtOH, which likely mediates the well-known motor impairing effects of EtOH, combined with recent advances in our understanding of the role of the cerebellum in non-motor, cognitive/emotive/reward processes has renewed interest in this system in the specific context of AUD. In this chapter we will describe recent advances in our understanding of cerebellar processing, actions of EtOH on the cerebellar GABAAR system, and the potential relationship of such actions to the development of AUD. We will finish with speculation about how cerebellar specific GABAAR ligands might be effective pharmacological agents for treating aspects of AUD.
Assuntos
Transtornos Relacionados ao Uso de Álcool/tratamento farmacológico , Cerebelo/fisiologia , Antagonistas de Receptores de GABA-A/farmacologia , Etanol , Humanos , Receptores de GABA-A , Ácido gama-AminobutíricoRESUMO
Variation in cerebellar sensitivity to alcohol/ethanol (EtOH) is a heritable trait associated with alcohol use disorder in humans and high EtOH consumption in rodents, but the underlying mechanisms are poorly understood. A recently identified cellular substrate of cerebellar sensitivity to EtOH, the GABAergic system of cerebellar granule cells (GCs), shows divergent responses to EtOH paralleling EtOH consumption and motor impairment phenotype. Although GCs are the dominant afferent integrator in the cerebellum, such integration is shared by unipolar brush cells (UBCs) in vestibulocerebellar lobes. UBCs receive both GABAergic and glycinergic inhibition, both of which may mediate diverse neurological effects of EtOH. Therefore, the impact of recreational concentrations of EtOH (~10-50 mM) on GABAA receptor (GABAAR)- and glycine receptor (GlyR)-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) of UBCs in cerebellar slices was characterized. Sprague-Dawley rat (SDR) UBCs exhibited sIPSCs mediated by GABAARs, GlyRs, or both, and EtOH dose-dependently (10, 26, 52 mM) increased their frequency and amplitude. EtOH increased the frequency of glycinergic and GABAergic sIPSCs and selectively enhanced the amplitude of glycinergic sIPSCs. This GlyR-specific enhancement of sIPSC amplitude resulted from EtOH actions at presynaptic Golgi cells and via protein kinase C-dependent direct actions on postsynaptic GlyRs. The magnitude of EtOH-induced increases in UBC sIPSC activity varied across SDRs and two lines of mice, in parallel with their respective alcohol consumption/motor impairment phenotypes. These data indicate that Golgi cell-to-UBC inhibitory synapses are targets of EtOH, which acts at pre- and postsynaptic sites, via Golgi cell excitation and direct GlyR enhancement.NEW & NOTEWORTHY Genetic variability in cerebellar alcohol/ethanol sensitivity (ethanol-induced ataxia) predicts ethanol consumption phenotype in rodents and humans, but the cellular and molecular mechanisms underlying genetic differences are largely unknown. Here it is demonstrated that recreational concentrations of alcohol (10-30 mM) enhance glycinergic and GABAergic inhibition of unipolar brush cells through increases in glycine/GABA release and postsynaptic enhancement of glycine receptor-mediated responses. Ethanol effects varied across rodent genotypes parallel to ethanol consumption and motor sensitivity phenotype.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Depressores do Sistema Nervoso Central/farmacologia , Cerebelo/efeitos dos fármacos , Etanol/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores , Sinapses/efeitos dos fármacos , Animais , Depressores do Sistema Nervoso Central/administração & dosagem , Cerebelo/citologia , Cerebelo/fisiologia , Relação Dose-Resposta a Droga , Etanol/administração & dosagem , Feminino , Neurônios GABAérgicos/citologia , Glicina/metabolismo , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
BACKGROUND: Evidence indicates that the cerebellum plays a role in genetic predilection to excessive alcohol (ethanol [EtOH]) consumption in rodents and humans, but the molecular mechanisms mediating such predilection are not understood. We recently determined that EtOH has opposite actions (enhancement or suppression) on tonic GABAA receptor (GABAA R) currents in cerebellar granule cells (GCs) in low- and high-EtOH-consuming rodents, respectively, and proposed that variation in GC tonic GABAA R current responses to EtOH contributes to genetic variation in EtOH consumption phenotype. METHODS: Voltage-clamp recordings of GCs in acutely prepared slices of cerebellum were used to evaluate the effect of EtOH on GC tonic GABAA R currents in another high-EtOH-consuming rodent, prairie voles (PVs). RESULTS: EtOH (52 mM) suppressed the magnitude of the tonic GABAA R current in 57% of cells, had no effect in 38% of cells, and enhanced the tonic GABAA R current in 5% of cells. This result is similar to GCs from high-EtOH-consuming C57BL/6J (B6) mice, but it differs from the enhancement of tonic GABAA R currents by EtOH in low-EtOH-consuming DBA/2J (D2) mice and Sprague Dawley (SD) rats. EtOH suppression of tonic GABAA R currents was not affected by the sodium channel blocker, tetrodotoxin (500 nM), and was independent of the frequency of phasic GABAA R-mediated currents, suggesting that suppression is mediated by postsynaptic actions on GABAA Rs, rather than a reduction of GABA release. Finally, immunohistochemical analysis of neuronal nitric oxide synthase (nNOS; which can mediate EtOH enhancement of GABA release) demonstrated that nNOS expression in the GC layer of PV cerebellum was similar to the levels seen in B6 mice, both being significantly reduced relative to D2 mice and SD rats. CONCLUSIONS: Combined, these data highlight the GC GABAA R response to EtOH in another species, the high-EtOH-consuming PV, which correlates with EtOH consumption phenotype and further implicates the GC GABAA R system as a contributing mechanism to high EtOH consumption.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Cerebelo/citologia , Cerebelo/metabolismo , Etanol/administração & dosagem , Genótipo , Receptores de GABA-A/metabolismo , Animais , Arvicolinae , Cerebelo/efeitos dos fármacos , Feminino , Masculino , Técnicas de Cultura de Órgãos , Especificidade da EspécieRESUMO
OBJECTIVE: Recently, we reported that the neocortex displays impaired growth after transient cerebral hypoxia-ischemia (HI) at preterm gestation that is unrelated to neuronal death but is associated with decreased dendritic arbor complexity of cortical projection neurons. We hypothesized that these morphological changes constituted part of a more widespread neuronal dysmaturation response to HI in the caudate nucleus (CN), which contributes to motor and cognitive disability in preterm survivors. METHODS: Ex vivo magnetic resonance imaging (MRI), immunohistochemistry, and Golgi staining defined CN growth, cell death, proliferation, and dendritic maturation in preterm fetal sheep 4 weeks after HI. Patch-clamp recording was used to analyze glutamatergic synaptic currents in CN neurons. RESULTS: MRI-defined growth of the CN was reduced after ischemia compared to controls. However, no significant acute or delayed neuronal death was seen in the CN or white matter. Nor was there significant loss of calbindin-positive medium spiny projection neurons (MSNs) or CN interneurons expressing somatostatin, calretinin, parvalbumin, or tyrosine hydroxylase. Morphologically, ischemic MSNs showed a markedly immature dendritic arbor, with fewer dendritic branches, nodes, endings, and spines. The magnitude and kinetics of synaptic currents, and the relative contribution of glutamate receptor subtypes in the CN were significantly altered. INTERPRETATION: The marked MSN dendritic and functional abnormalities after preterm cerebral HI, despite the marked resistance of immature CN neurons to cell death, are consistent with widespread susceptibility of projection neurons to HI-induced dysmaturation. These global disturbances in dendritic maturation and glutamatergic synaptic transmission suggest a new mechanism for long-term motor and behavioral disabilities in preterm survivors via widespread disruption of neuronal connectivity.
Assuntos
Isquemia Encefálica/patologia , Núcleo Caudado/patologia , Hipóxia Fetal/patologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/patologia , Nascimento Prematuro/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Animais , Isquemia Encefálica/sangue , Caspase 3/metabolismo , Dendritos/patologia , Dendritos/ultraestrutura , Modelos Animais de Doenças , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , GABAérgicos/farmacologia , Cabras , Antígeno Ki-67/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Gravidez , Fatores de TempoRESUMO
The brain adapts to chronic ethanol intoxication by altering synaptic and ion-channel function to increase excitability, a homeostatic counterbalance to inhibition by alcohol. Delirium tremens occurs when those adaptations are unmasked during withdrawal, but little is known about whether the primate brain returns to normal with repeated bouts of ethanol abuse and abstinence. Here, we show a form of bidirectional plasticity of pacemaking currents induced by chronic heavy drinking within the inferior olive of cynomolgus monkeys. Intracellular recordings of inferior olive neurons demonstrated that ethanol inhibited the tail current triggered by release from hyperpolarization (I(tail)). Both the slow deactivation of hyperpolarization-activated cyclic nucleotide-gated channels conducting the hyperpolarization-activated inward current and the activation of Ca(v)3.1 channels conducting the T-type calcium current (I(T)) contributed to I(tail), but ethanol inhibited only the I(T) component of I(tail). Recordings of inferior olive neurons obtained from chronically intoxicated monkeys revealed a significant up-regulation in I(tail) that was induced by 1 y of daily ethanol self-administration. The up-regulation was caused by a specific increase in I(T) which (i) greatly increased neurons' susceptibility for rebound excitation following hyperpolarization and (ii) may have accounted for intention tremors observed during ethanol withdrawal. In another set of monkeys, sustained abstinence produced the opposite effects: (i) a reduction in rebound excitability and (ii) a down-regulation of I(tail) caused by the down-regulation of both the hyperpolarization-activated inward current and I(T). Bidirectional plasticity of two hyperpolarization-sensitive currents following chronic ethanol abuse and abstinence may underlie persistent brain dysfunction in primates and be a target for therapy.
Assuntos
Alcoolismo/fisiopatologia , Etanol/farmacologia , Macaca fascicularis/anatomia & histologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Núcleo Olivar/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Feminino , Macaca fascicularis/fisiologia , Masculino , Núcleo Olivar/anatomia & histologia , Núcleo Olivar/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenótipo , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
Genetic differences in cerebellar sensitivity to alcohol (EtOH) influence EtOH consumption phenotype in animal models and contribute to risk for developing an alcohol use disorder in humans. We previously determined that EtOH enhances cerebellar granule cell (GC) tonic GABAAR currents in low EtOH consuming rodent genotypes, but suppresses it in high EtOH consuming rodent genotypes. Moreover, pharmacologically counteracting EtOH suppression of GC tonic GABAAR currents reduces EtOH consumption in high alcohol consuming C57BL/6J (B6J) mice, suggesting a causative role. In the low EtOH consuming rodent models tested to date, EtOH enhancement of GC tonic GABAAR currents is mediated by inhibition of neuronal nitric oxide synthase (nNOS) which drives increased vesicular GABA release onto GCs and a consequent enhancement of tonic GABAAR currents. Consequently, genetic variation in nNOS expression across rodent genotypes is a key determinant of whether EtOH enhances or suppresses tonic GABAAR currents, and thus EtOH consumption. We used behavioral, electrophysiological, and immunocytochemical techniques to further explore the relationship between EtOH consumption and GC GABAAR current responses in C57BL/6N (B6N) mice. B6N mice consume significantly less EtOH and achieve significantly lower blood EtOH concentrations than B6J mice, an outcome not mediated by differences in taste. In voltage-clamped GCs, EtOH enhanced the GC tonic current in B6N mice but suppressed it in B6J mice. Immunohistochemical and electrophysiological studies revealed significantly higher nNOS expression and function in the GC layer of B6N mice compared to B6Js. Collectively, our data demonstrate that despite being genetically similar, B6N mice consume significantly less EtOH than B6J mice, a behavioral difference paralleled by increased cerebellar nNOS expression and opposite EtOH action on GC tonic GABAAR currents in each genotype.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Alcoolismo/fisiopatologia , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebelar , Fenômenos Eletrofisiológicos , Etanol/farmacologia , Óxido Nítrico Sintase Tipo I , Receptores de GABA-A , Animais , Comportamento Animal/fisiologia , Depressores do Sistema Nervoso Central/administração & dosagem , Córtex Cerebelar/efeitos dos fármacos , Córtex Cerebelar/metabolismo , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Etanol/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , Óxido Nítrico Sintase Tipo I/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Especificidade da EspécieRESUMO
Brain ischemia results from cardiac arrest, stroke or head trauma. These conditions can cause severe brain damage and are a leading cause of death and long-term disability. Neurons are far more susceptible to ischemic damage than neighboring astrocytes, but astrocytes have diverse and important functions in many aspects of ischemic brain damage. Here we review three main roles of astrocytes in ischemic brain damage. First, we consider astrocyte glycogen stores, which can defend the brain against hypoglycemic brain damage but may aggravate brain damage during ischemia due to enhanced lactic acidosis. Second, we review recent breakthroughs in understanding astrocytic mechanisms of transmitter release, particularly for those transmitters with known roles in ischemic brain damage: glutamate, D-serine, ATP and adenosine. Third, we discuss the role of gap-junctionally connected networks of astrocytes in mediating the spread of damaging molecules to healthy 'bystanders' during infarct expansion in stroke.
Assuntos
Astrócitos/metabolismo , Isquemia Encefálica , Transdução de Sinais/fisiologia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Humanos , Modelos BiológicosRESUMO
Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.
Assuntos
Citoesqueleto de Actina/metabolismo , Isquemia Encefálica/fisiopatologia , Região CA1 Hipocampal/fisiologia , Cálcio/fisiologia , Glutamatos/fisiologia , Vesículas Sinápticas/fisiologia , Citoesqueleto de Actina/química , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Quelantes/farmacologia , Depsipeptídeos/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes , Fura-2/análogos & derivados , Técnicas In Vitro , Técnicas de Patch-Clamp , Faloidina/farmacologia , Polímeros , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/citologia , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Sensitivity to anticonvulsant effects of the γ-aminobutyric acidA receptor-active neurosteroid allopregnanolone (ALLO) during ethanol withdrawal varies across genotypes, with high sensitivity in genotypes with mild withdrawal and low sensitivity in genotypes with high withdrawal. The present studies determined whether the resistance to ALLO during withdrawal in mouse genotypes with high handling-induced convulsions (HICs) during withdrawal could be overcome with use of ganaxolone (GAN), the metabolically stable derivative of ALLO. In separate studies, male and female Withdrawal Seizure-Prone (WSP-1) and DBA/2J (D2) mice were exposed to air (controls) or 72-h ethanol vapor and then were scored for HICs during withdrawal (hourly for the first 12â¯h, then at hours 24 and 25). After the HIC scoring at hours 5 and 9, mice were injected with 10â¯mg/kg GAN or vehicle. Area under the HIC curve (AUC) for hours 5-12 was analyzed. In control WSP-1 mice, GAN significantly reduced AUC by 52% (males) and 63% (females), with effects that were absent or substantially reduced during withdrawal. In contrast, GAN significantly reduced AUC in both control and ethanol-withdrawing male and female D2 mice. AUC was decreased by 81% (males) and 70% (females) in controls and by 35% (males) and 21% (females) during withdrawal. The significant anticonvulsant effect of GAN during withdrawal in D2 but not WSP-1 mice suggests that different mechanisms may contribute to ALLO insensitivity during withdrawal in these two genotypes. Importantly, the results in D2 mice suggest that GAN may be a useful treatment for ethanol withdrawal-induced seizures.
Assuntos
Convulsões por Abstinência de Álcool/tratamento farmacológico , Convulsões por Abstinência de Álcool/genética , Anticonvulsivantes/farmacologia , Pregnanolona/análogos & derivados , Animais , Feminino , Predisposição Genética para Doença , Genótipo , Masculino , Camundongos Endogâmicos DBA , Pregnanolona/farmacologia , Fatores Sexuais , Especificidade da EspécieRESUMO
We show that information flow through the adult cerebellar cortex, from the mossy fiber input to the Purkinje cell output, is controlled by furosemide-sensitive, diazepam- and neurosteroid-insensitive GABA(A) receptors on granule cells, which are activated both tonically and by GABA spillover from synaptic release sites. Tonic activation of these receptors contributes a 3-fold larger mean inhibitory conductance than GABA released synaptically by high-frequency stimulation. Tonic and spillover inhibition reduce the fraction of granule cells activated by mossy fiber input, generating an increase of coding sparseness, which is predicted to improve the information storage capacity of the cerebellum.
Assuntos
Córtex Cerebelar/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Bumetanida/farmacologia , Córtex Cerebelar/citologia , Córtex Cerebelar/efeitos dos fármacos , Diazepam/farmacologia , Estimulação Elétrica , Furosemida/farmacologia , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Fibras Nervosas/ultraestrutura , Inibição Neural/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacos , Esteroides/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Cotransportadores de K e Cl-RESUMO
OBJECTIVE: Roux-en-Y gastric bypass (RYGB) surgery reduces appetite and stimulates new onset alcohol misuse; however, the genesis of these behavioral changes is unclear. This study is hypothesized that new onset alcohol intake is a behavioral adaptation that occurs secondary to reduced appetite and correlates with altered central ghrelin signaling. METHODS: Hedonic high-fat diet (HFD) intake was evaluated prior to the assessment of alcohol intake behaviors in RYGB and control rats. Measurements were also taken of circulating ghrelin and ghrelin receptor (GHSR) regulation of neuronal firing in ventral tegmental area (VTA) dopamine (DA) neurons. RESULTS: RYGB rats displayed reduced HFD intake relative to controls. Sham and RYGB rats consumed more alcohol and preferred lower concentrations of alcohol, whereas only RYGB rats escalated alcohol intake during acute withdrawal. Remarkably, GHSR activity, independent of peripheral ghrelin release, set the tonic firing of VTA DA neurons, a response selectively diminished in RYGB rats. CONCLUSIONS: This study indicates that gut manipulations lead to increased alcohol intake, whereas RYGB promotes behaviors that may maintain alcohol misuse. Reductions in hedonic feeding and diminished GHSR control of VTA firing further distinguish gut manipulation from complete bypass and present a potential mechanism linking reduced appetite with alcohol misuse after RYGB surgery.
Assuntos
Consumo de Bebidas Alcoólicas , Apetite , Derivação Gástrica , Grelina/sangue , Área Tegmentar Ventral/metabolismo , Animais , Dieta Hiperlipídica , Neurônios Dopaminérgicos/metabolismo , Masculino , Ratos , Ratos Long-Evans , Receptores de Grelina/sangueRESUMO
Cocaine addiction is a disease characterized by chronic relapse despite long periods of abstinence. The lateral orbitofrontal cortex (lOFC) and basolateral amygdala (BLA) promote cocaine-seeking behavior in response to drug-associated conditioned stimuli (CS) and share dense reciprocal connections. Hence, we hypothesized that monosynaptic projections between these brain regions mediate CS-induced cocaine-seeking behavior. Male Sprague-Dawley rats received bilateral infusions of a Cre-dependent adeno-associated viral (AAV) vector expressing enhanced halorhodopsin 3.0 fused with a reporter protein (NpHR-mCherry) or a control AAV (mCherry) plus optic fiber implants into the lOFC (Experiment 1) or BLA (Experiment 2). The same rats also received bilateral infusions of a retrogradely transported AAV vector expressing Cre recombinase (Retro-Cre-GFP) into the BLA (Experiment 1) or lOFC (Experiment 2). Thus, NpHR-mCherry or mCherry expression was targeted to lOFC neurons that project to the BLA or to BLA neurons that project to the lOFC in different groups. Rats were trained to lever press for cocaine infusions paired with 5-s CS presentations. Responding was then extinguished. At test, response-contingent CS presentation was discretely coupled with optogenetic inhibition (5-s laser activation) or no optogenetic inhibition while lever responding was assessed without cocaine/food reinforcement. Optogenetic inhibition of lOFC to BLA, but not BLA to lOFC, projections in the NpHR-mCherry groups disrupted CS-induced reinstatement of cocaine-seeking behavior relative to (i) no optogenetic inhibition or (ii) manipulations in mCherry control or (iii) NpHR-mCherry food control groups. These findings suggest that the lOFC sends requisite input to the BLA, via monosynaptic connections, to promote CS-induced cocaine-seeking behavior.
Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiopatologia , Comportamento Animal/fisiologia , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Cocaína/farmacologia , Sinais (Psicologia) , Inibidores da Captação de Dopamina/farmacologia , Optogenética , Córtex Pré-Frontal/fisiopatologia , Animais , Modelos Animais de Doenças , Comportamento de Procura de Droga , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Endogenous γ-aminobutyric acidA receptor (GABAAR)-active neurosteroids (e.g., allopregnanolone) regulate central nervous system excitability and many physiological functions, so fluctuations are implicated in several neuropsychiatric disorders. Pertinently, evidence supports an inverse relationship between endogenous GABAAR-active neurosteroid levels and behavioral changes in excitability during ethanol withdrawal (WD). OBJECTIVES: The present studies determined mouse genotype differences in ten neurosteroid levels in plasma, cortex, and hippocampus over the time course of ethanol WD in the WD Seizure-Prone (WSP) and WD Seizure-Resistant (WSR) selected lines and in the DBA/2J (DBA) inbred strain. METHODS: Gas chromatography-mass spectrometry was utilized to simultaneously quantify neurosteroid levels from control-treated male WSP-1, WSR-1, and DBA mice and during 8 and 48 h of WD. RESULTS: Combined with our prior work, there was a consistent decrease in plasma allopregnanolone levels at 8 h WD in all three genotypes, an effect that persisted at 48 h WD only in DBA mice. WSR-1 and WSP-1 mice exhibited unexpected divergent changes in cortical neurosteroids at 8 h WD, with the majority of neurosteroids (including allopregnanolone) being significantly decreased in WSR-1 mice, but unaffected or significantly increased in WSP-1 mice. In DBA mice, hippocampal allopregnanolone and tetrahydrodeoxycorticosterone were significantly decreased at 8 h WD. The pattern of significant correlations between allopregnanolone and other GABAAR-active neurosteroid levels differed between controls and withdrawing mice. CONCLUSIONS: Ethanol WD dysregulated neurosteroid synthesis. Results in WSP-1 mice suggest that diminished GABAAR function is more important for their high WD phenotype than fluctuations in neurosteroid levels.
Assuntos
Alcoolismo/metabolismo , Córtex Cerebral/metabolismo , Etanol/administração & dosagem , Hipocampo/metabolismo , Neurotransmissores/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Administração por Inalação , Alcoolismo/genética , Alcoolismo/psicologia , Animais , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hipocampo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Neurotransmissores/sangue , Pregnanolona/sangue , Pregnanolona/metabolismo , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/psicologiaRESUMO
Despite lacking N-methyl-D-aspartate receptors, cerebellar Purkinje cells are highly vulnerable to ischaemic insults, which lead them to die necrotically in an -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor-dependent manner. To investigate the electrical events leading to this cell death, we whole-cell clamped Purkinje cells in cerebellar slices. Simulated ischaemia evoked an initial hyperpolarization of Purkinje cells by 8.5 mV, followed by a regenerative 'anoxic depolarization' (AD) to -14 mV. The AD was prevented by glutamate receptor blockers. In voltage-clamp mode, we used the cells' glutamate receptors to sense the rise of extracellular glutamate concentration induced by ischaemia, with GABA(A) and GABA(B) receptors blocked and Cs+ as the main pipette cation. Ischaemia induced a small (<500 pA) slowly developing inward current in Purkinje cells, followed by a sudden large inward 'AD current' (approximately 6 nA) which was largely prevented by blocking AMPA receptors. Removing extracellular calcium reduced the large glutamate-mediated current by approximately 70% at early times (after 10 min ischaemia), but had no effect at later times (15 min). Blocking the operation of glutamate transporters, by preloading cells with the slowly transported glutamate analogue PDC (L-trans-pyrrolidine-2,4-dicarboxylate), reduced the current by approximately 88% at early and 83% at later times. In Purkinje cells in slices from mice lacking the glial glutamate transporters GLAST or GLT-1, the ischaemia-evoked AD current was indistinguishable from that in wild-type slices. These data suggest that, in cerebellar ischaemia, the dominant cause of the electrophysiological dysfunction of Purkinje cells is an activation of Purkinje cell AMPA receptors. The glutamate activating these receptors is released both by exocytosis (at early times) and by reversal of a glutamate transporter, apparently in neurons.
Assuntos
Cerebelo/irrigação sanguínea , Isquemia/fisiopatologia , Células de Purkinje/fisiologia , Potenciais de Ação/fisiologia , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Cálcio/metabolismo , Cerebelo/fisiopatologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Isquemia/patologia , Camundongos , Camundongos Transgênicos , Necrose , Técnicas de Patch-Clamp/métodos , Células de Purkinje/patologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Ácido gama-AminobutíricoRESUMO
GABA release during cerebral energy deprivation (produced by anoxia or ischemia) has been suggested either to be neuroprotective, because GABA will hyperpolarize neurons and reduce release of excitotoxic glutamate, or to be neurotoxic, because activation of GABA(A) receptors facilitates Cl- entry into neurons and consequent cell swelling. We have used the GABA(A) receptors of hippocampal area CA1 pyramidal cells to sense the rise of [GABA](o) occurring in simulated ischemia. Ischemia evoked, after several minutes, a large depolarization to approximately -20 mV. Before this "anoxic depolarization," there was an increase in GABA release by exocytosis (spontaneous IPSCs). After the anoxic depolarization, there was a much larger, sustained release of GABA that was not affected by blocking action potentials, vesicular release, or the glial GABA transporter GAT-3 but was inhibited by blocking the neuronal GABA transporter GAT-1. Blocking GABA(A) receptors resulted in a more positive anoxic depolarization but decreased cell swelling at the time of the anoxic depolarization. The influence of GABA(A) receptors diminished in prolonged ischemia because glutamate release evoked by the anoxic depolarization inhibited GABA(A) receptor function by causing calcium entry through NMDA receptors. These data show that ischemia releases GABA initially by exocytosis and then by reversal of GAT-1 transporters and that the resulting Cl- influx through GABA(A) receptor channels causes potentially neurotoxic cell swelling.
Assuntos
Isquemia Encefálica/metabolismo , Exocitose/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Tamanho Celular/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA , Antagonistas de Receptores de GABA-A , Hipocampo/citologia , Técnicas In Vitro , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , beta-Alanina/farmacologia , Ácido gama-Aminobutírico/farmacocinéticaRESUMO
RATIONALE: The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. OBJECTIVES: Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABAAR sensitivity to ALLO in Withdrawal Seizure-Prone (WSP) but not in Withdrawal Seizure-Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABAAR-active steroids is not known. METHODS: After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of ten neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. RESULTS: We quantified levels of nine neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of five neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed five neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. CONCLUSIONS: Due to lower basal levels of some GABAAR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.