The neural mechanisms underlying alcohol dependence are not well-understood. GABAergic neurons in the ventral tegmental area (VTA) are a relevant target for ethanol. They are inhibited by ethanol at physiologically-relevant levels in vivo and display marked hyperexcitability during withdrawal. In the present study, we examined the effects of the GABA(A) receptor agonist muscimol on VTA neurons ex vivo following withdrawal from acute and chronic ethanol exposure. We used standard cell-attached mode electrophysiology in the slice preparation to evaluate the effects of muscimol on VTA GABA neuron firing rate following exposure to acute and chronic ethanol in male CD-1 GAD-67 GFP mice. In the acute condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after a single in vivo dose of saline or ethanol. In the chronic condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after either 2 weeks of twice-daily IP ethanol or saline or following exposure to chronic intermittent ethanol (CIE) vapor or air for 3 weeks. VTA GABA neuron firing rate was more sensitive to muscimol than DA neuron firing rate. VTA GABA neurons, but not DA neurons, were resistant to the inhibitory effects of muscimol recorded 24 h after a single ethanol injection or chronic ethanol exposure. Administration of the NMDA receptor antagonist MK-801 before ethanol injection restored the sensitivity of VTA GABA neurons to muscimol inhibition. Seven days after ethanol exposure, VTA GABA neuron firing rate was again susceptible to muscimol's inhibitory effects in the acute condition, but the resistance persisted in the chronic condition. These findings suggest that VTA GABA neurons exclusively undergo a shift in GABA(A) receptor function following acute and chronic exposure. There appears to be transient GABA(A) receptor-mediated plasticity after a single exposure to ethanol that is mediated by NMDA glutamate receptors. In addition, the resistance to muscimol inhibition in VTA GABA neurons persists in the dependent condition, which may contribute to the the hyperexcitability of VTA GABA neurons and inhibition of VTA DA neurons during withdrawal as well as the motivation to seek alcohol.

Objective/Background: Restless Legs Syndrome (RLS) is a dopamine-dependent disorder characterized by a strong urge to move. The objective of this study was to evalulate blood levels of dopamine and other catecholamines and blood D2-subtype dopamine receptors (D2Rs) in RLS. Patients/Methods: Dopamine levels in blood samples from age-matched unmedicated RLS subjects, medicated RLS subjects and Controls were evaluated with high performance liquid chromatography and dopamine D2R white blood cell (WBC) expression levels were determined with fluorescence-activated cell sorting and immunocytochemistry. Results: Blood plasma dopamine levels, but not norepinepherine or epinephrine levels, were significantly increased in medicated RLS subjects vs unmedicated RLS subjects and Controls. The percentage of lymphocytes and monocytes expressing D2Rs differed between Control, RLS medicated and RLS unmedicated subjects. Total D2R expression in lymphocytes, but not monocytes, differed between Control, RLS medicated and RLS unmedicated subjects. D2Rs in lymphocytes, but not monocytes, were sensitive to dopamine in Controls only. Conclusion: Downregulation of WBCs D2Rs occurs in RLS. This downregulation is not reversed by medication, although commonly used RLS medications increase plasma dopamine levels. The insensitivity of monocytes to dopamine levels, but their downregulation in RLS, may reflect their utility as a biomarker for RLS and perhaps brain dopamine homeostasis.

Growing evidence suggests that mechanical stimulation modulates substrates in the supraspinal central nervous system (CNS) outside the canonical somatosensory circuits. We evaluate mechanical stimulation applied to the cervical spine at the C7-T1 level (termed "MStim") on neurons and neurotransmitter release in the mesolimbic dopamine (DA) system, an area implicated in reward and motivation, utilizing electrophysiological, pharmacological, neurochemical and immunohistochemical techniques in Wistar rats. Low frequency (45-80 Hz), but not higher frequency (115 Hz), MStim inhibited the firing rate of ventral tegmental area (VTA) GABA neurons (52.8% baseline; 450 s) while increasing the firing rate of VTA DA neurons (248% baseline; 500 s). Inactivation of the nucleus accumbens (NAc), or systemic or in situ antagonism of delta opioid receptors (DORs), blocked MStim inhibition of VTA GABA neuron firing rate. MStim enhanced both basal (178.4% peak increase at 60 min) and evoked DA release in NAc (135.0% peak increase at 40 min), which was blocked by antagonism of DORs or acetylcholine release in the NAc. MStim enhanced c-FOS expression in the NAc, but inhibited total expression in the VTA, and induced translocation of DORs to neuronal membranes in the NAc. These findings demonstrate that MStim modulates neuron firing and DA release in the mesolimbic DA system through endogenous opioids and acetylcholine in the NAc. These findings demonstrate the need to explore more broadly the extra-somatosensory effects of peripheral mechanoreceptor activation and the specific role for mechanoreceptor-based therapies in the treatment of substance abuse.

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure underlying the synaptic and behavioral consequences of ethanol dependence. While accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, there is a limited understanding of the role astrocytes play in ethanol dependence. The present study used a combination of viral labeling, super resolution confocal microscopy, 3D image analysis, and slice electrophysiology to determine the effects of chronic intermittent ethanol (CIE) exposure on astrocyte plasticity in the CeA. During withdrawal from CIE exposure, we observed increased GABA transmission, an upregulation in astrocytic GAT3 levels, and an increased proximity of astrocyte processes near CeA synapses. Furthermore, GAT3 levels and synaptic proximity were positively associated with voluntary ethanol drinking in dependent rats. Slice electrophysiology confirmed that the upregulation in astrocytic GAT3 levels was functional, as CIE exposure unmasked a GAT3-sensitive tonic GABA current in the CeA. A causal role for astrocytic GAT3 in ethanol dependence was assessed using viral-mediated GAT3 overexpression and knockdown approaches. However, GAT3 knockdown or overexpression had no effect on somatic withdrawal symptoms, dependence-escalated ethanol intake, aversion-resistant drinking, or post-dependent ethanol drinking in male or female rats. Moreover, intra-CeA pharmacological inhibition of GAT3 did not alter dependent ethanol drinking. Together, these findings indicate that ethanol dependence induces GABAergic dysregulation and astrocyte plasticity in the CeA. However, these changes in astrocytic GAT3 do not appear to be necessary for the drinking related phenotypes associated with dependence.