Summary 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 post-transcriptional 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 regulation of additional miR-9 targets relevant to alcohol abuse. Our results describe a novel mechanism of multiplex regulation of stability of alternatively spliced mRNA by miRNA in drug adaptation and neuronal plasticity.

Formalin injected subcutaneously into the paw is a frequently used pain assay; it evokes an initial period of licking and flinching followed by a period of quiescence and last by a second period of intense and protracted licking and flinching. The prominent second phase is believed to reflect the development of a central (spinal cord) facilitation. This conclusion is based on the assumption that formalin evokes an initial burst of activity in fine afferent fibers, followed by prolonged low levels of activity in C fibers. Detailed reports substantiating this essential assumption have not been published. Thus, we recorded in situ from single sural nerve fibers, identified by their conduction velocity and modality, in the barbiturate anesthetized rat. Following formalin (2.5%, 50 microliters) injection into their receptive fields, phase-1 activity was prominent in A beta and A delta fibers as well as in high-threshold C nociceptive afferent fibers. Phase-2 activity was observed in A delta fibers with receptive fields in hairy skin and in all mechanically sensitive C fibers. Mean phase-2 activity in these fibers was 1/2-2/3 of the magnitude achieved in phase 1. Mechanically insensitive fibers and A delta and C fibers with receptive field centers outside of the injection zone began firing 15 min or more post-injection and would contribute to phase-2, but not phase-1, behavioral activity. Intravenous infusion of low doses of lidocaine yielding plasma levels of 3.6-7.9 micrograms/ml administered during phase 2 blocked formalin-evoked activity in primary afferent fibers in a dose-related fashion without blocking either electrically or mechanically evoked activity. Effective plasma doses were comparable to those found to relieve neuropathic pain. These data indicate that phase 2 in the formalin test is more closely related to ongoing afferent input than had previously been thought.

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 ␤4 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 ␤1 and ␤4 subunit mRNAs are found in the NAcc, although the signal for ␤1 is significantly weaker. Immunohistochemistry revealed that ␤1 subunits were found in both soma and dendrites, whereas ␤4 appeared restricted to the soma. These findings suggest that the ␤4 subunit may confer EtOH sensitivity to somatic BK channels, whereas the absence of ␤4 in the dendrite results in insensitivity to the drug. Consistent with this idea, acute EtOH potentiated ␣␤4 BK currents in transfected human embryonic kidney cells, whereas it failed to alter ␣␤1 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.

Tolerance is an important element of drug addiction and provides a model for understanding neuronal plasticity. The hypothalamicneurohypophysial 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 ␣-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.

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.

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed ␣␤1 versus ␣␤4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary ␤1 subunits to the somatic and dendritic compartments and ␤4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate ␤1 or ␤4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.Ion channel compartmentalization between specific brain regions and various neuronal populations has been known for many years. Technological advances recently have permitted researchers to probe the distribution of channel subtypes on a subcellular level. Here, we have utilized a unique system, the hypothalamic-neurohypophysial system (HNS), which allows us to examine dendrites, cell bodies, and individual nerve terminals within the same population of magnocellular neurons. The HNS is an ideal model system to study compartmentalization of channel properties because the three neuronal domains (dendrite, cell body, and nerve terminal) can be easily distinguished from one another. The large (20 -30 m) magnocellular neurons of the supraoptic nucleus (SON) send axonal projections to the posterior pituitary (neurohypophysis), where they terminate in thousands of nerve endings that release oxytocin (OXT) or vasopressin (AVP) into systemic circulation. Magnocellular neuron dendrites, on the other hand, project toward the ventral surface of the brain, forming a dense interlaced network that releases OXT or AVP centrally. HNS axons morphologically have few, if any, collaterals, allowin...

Touch evoked agitation (allodynia) can be induced by spinal delivery of strychnine and this effect is antagonized by intrathecal NMDA and non-NMDA receptor antagonists, but not by mu-opiate receptor agonists. In this study, we sought to characterize the effect of focal glycine-receptor inhibition on spontaneous and evoked activity in dorsal horn neurons of the chloralose-anesthetized cat. Strychnine (1 mM) applied near the neurons through a dialysis fiber caused an enhanced response to hair deflection, enlargement of the low threshold receptive fields and in some cells, an increase in afterdischarge. These changes were observed only in cells that were activated by both hair deflection and high intensity mechanical stimulation. Subsequent co-administration of an NMDA receptor antagonist (AP-7, 2.0 mM) preferentially blocked strychnine-associated effects without changing the original receptive field characteristics. Co-administration of a non-NMDA excitatory amino acid receptor antagonist (CNQX, 1 mM) with the strychnine served to block low (brush) and high intensity (pinch) afferent input. In contrast, addition of a mu-opiate receptor agonist (alfentanil 2.4 mM) to the strychnine perfusate selectively reduced responsiveness to high intensity stimulation, while having no effect on the exaggerated response to hair deflection. Given the functional and pharmacological similarity of the effects of spinal strychnine to post-nerve injury states in man, disinhibition due to a loss of glycinergic input may be associated with large myelinated fiber-mediated nociceptive states. Consistent with these data is the contention that under normal circumstances, afferent hair follicle input onto convergent neurons is regulated by a tonic glycinergic circuit. Removal of this regulatory influence leads to a magnification of low threshold tactile throughput in dorsal horn. This model may help to provide pharmacological insights into more efficacious treatments for such pain states that are relatively refractory to opioid therapies.

These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.