Molecular tolerance of voltage-gated calcium channels is evident after short exposures to alcohol in vasopressin-releasing nerve terminals.

BACKGROUND: Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. METHODS: We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release. RESULTS: We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K(+) -induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K(+) -induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane. CONCLUSIONS: These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

Posttranscriptional regulation of BK channel splice variant stability by miR-9 underlies neuroadaptation to alcohol.

Tolerance represents a critical component of addiction. The large-conductance calcium- and voltage-activated potassium channel (BK) is a well-established alcohol target, and an important element in behavioral and molecular alcohol tolerance. We tested whether microRNA, a newly discovered class of gene expression regulators, plays a role in the development of tolerance. We show that in adult mammalian brain, alcohol upregulates microRNA miR-9 and mediates posttranscriptional reorganization in BK mRNA splice variants by miR-9-dependent destabilization of BK mRNAs containing 3'UTRs with a miR-9 Recognition Element (MRE). Different splice variants encode BK isoforms with different alcohol sensitivities. Computational modeling indicates that this miR-9-dependent mechanism contributes to alcohol tolerance. Moreover, this mechanism can be extended to include regulation of additional miR-9 targets relevant to alcohol abuse. Our results describe a mechanism of multiplex regulation of stability of alternatively spliced mRNA by microRNA in drug adaptation and neuronal plasticity.

Alcohol tolerance in large-conductance, calcium-activated potassium channels of CNS terminals is intrinsic and includes two components: decreased ethanol potentiation and decreased channel density.

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamic-neurohypophysial system (HNS) is an established preparation in which to study the actions of alcohol. Acute application of alcohol to the rat neurohypophysis potentiates large-conductance calcium-sensitive potassium channels (BK), contributing to inhibition of hormone secretion. A cultured HNS explant from adult rat was used to explore the molecular mechanisms of BK tolerance after prolonged alcohol exposure. Ethanol tolerance was intrinsic to the HNS and consisted of: (1) decreased BK potentiation by ethanol, complete within 12 min of exposure, and (2) decreased current density, which was not complete until 24 hr after exposure, indicating that the two components of tolerance represent distinct processes. Single-channel properties were not affected by chronic exposure, suggesting that decreased current density resulted from downregulation of functional channels in the membrane. Indeed, we observed decreased immunolabeling against the BK alpha-subunit on the surface of tolerant terminals. Analysis using confocal microscopy revealed a reduction of BK channel clustering, likely associated with the internalization of the channel.

Somatic localization of a specific large-conductance calcium-activated potassium channel subtype controls compartmentalized ethanol sensitivity in the nucleus accumbens.

Alcohol is an addictive drug that targets a variety of ion channels and receptors. To address whether the effects of alcohol are compartment specific (soma vs dendrite), we examined the effects of ethanol (EtOH) on large-conductance calcium-activated potassium channels (BK) in cell bodies and dendrites of freshly isolated neurons from the rat nucleus accumbens (NAcc), a region known to be critical for the development of addiction. Compartment-specific drug action was indeed observed. Clinically relevant concentrations of EtOH increased somatic but not dendritic BK channel open probability. Electrophysiological single-channel recordings and pharmacological analysis of the BK channel in excised patches from each region indicated a number of differences, suggestive of a compartment-specific expression of the beta4 subunit of the BK channel, that might explain the differential alcohol sensitivity. These parameters included activation kinetics, calcium dependency, and toxin blockade. Reverse transcription-PCR showed that both BK channel beta1 and beta4 subunit mRNAs are found in the NAcc, although the signal for beta1 is significantly weaker. Immunohistochemistry revealed that beta1 subunits were found in both soma and dendrites, whereas beta4 appeared restricted to the soma. These findings suggest that the beta4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of beta4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated alphabeta4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter alphabeta1 BK channel-mediated currents. Finally, an EtOH concentration (50 mm) that increased BK channel open probability strongly decreased the duration of somatic-generated action potential in NAcc neurons.

Spinal bicuculline produces hypersensitivity of dorsal horn neurons: effects of excitatory amino acid antagonists.

In this study, we sought to characterize the effects of focal GABA(A) receptor antagonism on spontaneous and evoked activity in dorsal horn neurons of the alpha-chloralose anesthetized cat. Bicuculline (0.5, 1.0 mM) applied near the neurons through a transparenchymal dialysis fiber resulted in increased evoked activity in nociceptive dorsal horn neurons. Hair deflection was the stimulus most affected, followed by both low and high threshold tonic mechanical stimulation of the receptive field. In addition, neurons displayed increased background discharge and a subpopulation developed an increased afterdischarge to noxious mechanical stimulation. This is in contrast to our previous work with glycine receptor antagonism where only the evoked response to hair follicle activation was significantly enhanced. Subsequent co-administration of an NMDA receptor antagonist (AP-7, 2.0 mM) was without any apparent effect on either basal or bicuculline-enhanced responses. Co-administration of a non-NMDA excitatory amino acid receptor antagonist (CNQX, 1.0 mM) with the bicuculline non-selectively blocked both low and high threshold mechanical input. The inability of AP-7 to reverse the bicuculline-associated hyperreactivity also contrasts with the AP-7 reversal of the strychnine-associated hyperreactivity. These results point out that, while GABA and glycine are frequently co-localized in cells of the spinal dorsal horn and both appear to mediate tonic inhibitory control systems, they are not at all equivalent and are subject to different modulatory pharmacologies. Removal of each influence may model a different component of neuropathic pain.