Aberrant synaptic activation of N-methyl-D-aspartate receptors underlies ethanol withdrawal hyperexcitability.

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-d-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr(2+)-supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.

Sindbis viral-mediated expression of eGFP-dopamine D1 receptors in situ with real-time two-photon microscopic detection.

Dopamine D1 receptors (D1DRs) mediate a major component of dopaminergic neurotransmission, and alterations in their synaptic and subcellular distribution may underlie a variety of neurological diseases. In order to monitor D1DR localization in real time, we subcloned a sindbis virus containing an enhanced-GFP coding region inserted at the C-terminal region of a dopamine D1 receptor (eGFP-D1DR). Two-photon excitation of expressed eGFP-D1DRs was monitored in a variety of viable neural preparations. Infection of primary cultured rat ventral striatal neurons, verified for neuronal phenotype using patch clamp electrophysiology, was induced by the simple addition of the virus to media. Parasagittal slice cultures, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), were infected by manual injection below the glia surface. NAc-containing parasagittal slices prepared from mice in which the virus was administered via stereotaxic injection in vivo also displayed robust eGFP-D1DR expression. Expression of functional D1DRs following infection in baby hamster kidney (BHK) cells was monitored by DA-stimulated cAMP production that was also blocked by a selective D1 antagonist. Taken together, these findings provide the first demonstration of the functional expression and real-time imaging of eGFP-D1DRs, and indicate that sindbis virus is an effective method for D1 receptor expression in a variety of native neuronal preparations.

Dopamine activity in the nucleus accumbens during consummatory phases of oral ethanol self-administration.

UNLABELLED: BACKGROUND This present study was designed to clarify the role of dopamine in the nucleus accumbens during operant ethanol self-administration by separating bar pressing (ethanol seeking) from ethanol consumption. Furthermore, we sought to define the relationship between ethanol in the brain and the accumbal dopamine response after oral self-administration of ethanol. METHODS: Two separate groups of male Long-Evans rats were trained to bar press with 10% ethanol or water. Rats were trained to elicit an escalating number of bar presses across daily sessions before gaining access to the drinking solution for 20 min. Microdialysis was performed before (during a waiting period), during, and after bar pressing and drinking. A handling control group was included, but did not receive training. RESULTS: A significant increase in dopamine occurred during placement of the rats into the operant chamber in trained rats and handling controls. The lever-pressing period did not produce an increase in dialysate dopamine. Accumbal dopamine was increased in the first 5 min of ethanol, but not water, consumption. Ethanol appeared in the dialysate sample following ethanol availability, and peak concentrations were reached at 10 min. Most of the ethanol and water consumption occurred within 5 min of fluid access. The probes were distributed in the core (32%), shell (32%), and core plus shell (36%) regions of the nucleus accumbens. CONCLUSIONS: The enhancement of dopamine during transfer into the operant chamber does not depend on anticipation or operant training with ethanol or water reinforcement. Furthermore, the difference between the time course of accumbal dopamine and ethanol in dialysates suggests that the dopamine response is not solely due to pharmacological effects of ethanol. The dopamine response may be associated with the stimulus properties of ethanol presentation, which would be strongest during consumption.