Acute alcohol tolerance is intrinsic to the BKCa protein, but is modulated by the lipid environment

Ethanol tolerance, in which exposure leads to reduced sensitivity, is an important component of alcohol abuse and addiction. The molecular mechanisms underlying this process remain poorly understood. The BKCa channel plays a central role in the behavioral response to ethanol in Caenorhabditis elegans (Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele, T. R., Bonci, A., Bargmann, C. I., and McIntire, S. L. (2003) Cell 115, 655-666) and Drosophila (Cowmeadow, R. B., Krishnan, H. R., and Atkinson, N. S. (2005) Alcohol. Clin. Exp. Res. 29, 1777-1786) . In neurons, ethanol tolerance in BKCa channels has two components: a reduced number of membrane channels and decreased potentiation of the remaining channels (Pietrzykowski, A. Z., Martin, G. E., Puig, S. I., Knott, T. K., Lemos, J. R., and Treistman, S. N. (2004) J. Neurosci. 24, 8322-8332) . Here, heterologous expression coupled with planar bilayer techniques examines two additional aspects of tolerance in human BKCa channels. 1) Is acute tolerance observed in a single channel protein complex within a lipid environment reduced to only two lipids? 2) Does lipid bilayer composition affect the appearance of acute tolerance? We found that tolerance was observable in BKCa channels in membrane patches pulled from HEK cells and when they are placed into reconstituted 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine/1-palmitoyl-2-o leoyl-sn-glycero-3-phosphatidylserine membranes. Furthermore, altering bilayer thickness by incorporating the channel into lipid mixtures of 1,2-dioleoyl-3-phosphatidylethanolamine with phosphatidylcholines of increasing chain length, or with sphingomyelin, strongly affected the sensitivity of the channel, as well as the time course of the acute response. Ethanol sensitivity changed from a strong potentiation in thin bilayers to inhibition in thick sphingomyelin/1,2-dioleoyl-3-phosphatidylethanolamine bilayers. Thus, tolerance can be an intrinsic property of the channel protein-lipid complex, and bilayer thickness plays an important role in shaping the pattern of response to ethanol. As a consequence of these findings the protein-lipid complex should be treated as a unit when studying ethanol action

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

Impulsivity and comorbid traits: a multi-step approach for finding putative responsible microRNAs in the amygdala

Malfunction of synaptic plasticity in different brain regions, including the amygdala plays a role in impulse control deficits that are characteristics of several psychiatric disorders, such as ADHD, schizophrenia, depression and addiction. Previously, we discovered a locus for impulsivity (Impu1) containing the neuregulin 3 (Nrg3) gene, of which the level of expression determines levels of inhibitory control. MicroRNAs (miRNAs) are potent regulators of gene expression, and have recently emerged as important factors contributing to the development of psychiatric disorders. However, their role in impulsivity, as well as control of Nrg3 expression or malfunction of the amygdala, is not well established. Here, we used the GeneNetwork database of BXD mice to search for correlated traits with impulsivity using an overrepresentation analysis to filter for biologically meaningful traits. We determined that inhibitory control was significantly correlated with expression of miR-190b, -28a, -340, -219a, and -491 in the amygdala, and that the overrepresented correlated traits showed a specific pattern of coregulation with these miRNAs. A bioinformatics analysis iDentified that miR-190b, by targeting an Nrg3-related network, could affect synaptic plasticity in the amygdala, targeting bot impulsive and compulsive traits. Moreover, miR-28a, -340, -219a, and possibly -491 could act on synaptic function by determining the balance between neuronal outgrowth and differentiation. We propose that these miRNAs are attractive candidates of regulation of amygdala synaptic plasticity, possibly during development but also in maintaining the impulsive phenotype. These results can help us to better understand mechanisms of synaptic dysregulation in psychiatric disorders

Dynamic nongenomic actions of thyroid hormone in the developing rat brain

Two well-characterized nongenomic actions of thyroid hormone in cultured brain tissues are: 1) regulation of type 2 iodothyronine 5\u27deiodinase (D2) activity and 2) regulation of actin polymerization. In particular, the latter is likely to have profound effects on neuronal migration in the developing brain. In this study, we determined whether these nongenomic actions also occurred in vivo during brain development. Neonatal hypothyroidism was induced by propylthiouracil given to pregnant dams beginning on d17 of gestation and continued throughout the neonatal period. On postnatal d 14, rats were injected with either cold or [(125)I]-labeled iodothyronines and killed sequentially after injection. In contrast to reports in the adult rat, all three iodothyronines readily and equally entered developing brain tissues. As expected, cerebrocortical D2 activity was markedly elevated in the hypothyroid brain and both reverse T(3) (rT(3)) and T(4) rapidly decreased D2 to euthyroid levels within 3 h. Furthermore, cerebellar G-actin content in the hypothyroid rat was approximately 5-fold higher than in the euthyroid rat. Again, both rT(3) and T(4) rapidly decreased the G-actin content by approximately 50%, with a reciprocal increase in F-actin content to euthyroid levels without altering total actin. Neither T(3) nor vehicle had any effect on D2 activity in the cortex or G- or F-actin content in the cerebellum. The thyroid hormone-dependent regulation of actin polymerization in the rat brain provides a mechanism by which this morphogenic hormone can influence neuronal migration independent of the need for altered gene transcription. Furthermore, these data suggest a prominent role for rT(3) during brain development

Immediate-early alcohol-responsive miRNA expression in <i>Drosophila</i>

<p>At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in the <i>Drosophila</i> model organism. In our analysis, we combine the power of next-generation sequencing with <i>Drosophila</i> genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 known <i>Drosophila</i> miRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.</p

Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and 3,3\u27,5\u27-triiodothyronine

The timing of granule cell migration in the developing cerebellum is regulated by thyroid hormone. Granule cell migration depends on the recognition of extracellular neuronal guidance molecule(s), such as laminin, and this, in turn, requires cell surface adhesion molecules (integrins) that are anchored on the cell membrane by the actin cytoskeleton. While many of the actions of thyroid hormone, specifically 3,5,3\u27-triiodothyronine (T3), are mediated by regulated gene expression, both thyroxine (T4) and 3,3\u27,5\u27-triiodothyronine (rT3) also exert direct, positive control of the quantity of polymerized actin in cultured astrocytes without affecting gene expression. T4-dependent actin polymerization has been shown to (i) participate in the immobilization of laminin to the cell surface, (ii) help deposit laminin in the molecular layer of the developing cerebellum, and (iii) anchor integrin(s) that recognize laminin present in the extracellular matrix. In this study, we show that both T4 and rT3, but not T3, directly regulate the F-actin content of elongating neurites of cerebellar neurons. T4 and rT3 also promoted extensive granule cell migration from cerebellar explants, as well as, dense cell clustering and extensive neuronal process formation when granule cells were grown on a laminin-coated surface. Both granule cell migration and neuronal process outgrowth were markedly attenuated by the addition of integrin-blocking antibodies or binding peptides, by the absence of thyroid hormone or the presence of T3. These data suggest that the T4-dependent actin polymerization in developing neurons is necessary for these migrating cells to recognize the laminin guidance molecule, thereby providing a novel molecular mechanism for the profound influence of thyroid hormone on brain development that is independent of regulated gene expression

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 �-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. Key words: addiction; alcohol; ethanol; explant; tolerance; BK channel; terminal

Interleukin-4 activates large-conductance, calcium-activated potassium (BKCa) channels in human airway smooth muscle cells

Large-conductance, calcium-activated potassium (BK(Ca)) channels are regulated by voltage and near-membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper-2 (Th2) cytokine, interleukin (IL)-4, is an important mediator of airway inflammation, we investigated whether IL-4 rapidly regulated BK(Ca) activity in normal airway smooth muscle cells. On-cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin-4 (50 ng ml(-1)), IL-13 (50 ng ml(-1)) or histamine (10 microm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the alpha and beta1 subunits of BK(Ca) were also performed. Both approaches demonstrated that HBSMC membranes contained large-conductance channels (\u3e200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BK(Ca) channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL-4 stimulated rapid, large increases in BK(Ca) channel activity (77.2 +/- 63.3-fold increase, P \u3c 0.05, n = 18). This large potentiation depended on the presence of external calcium. In contrast, IL-13 (50 ng ml(-1)) had little effect on BK(Ca) channel activity, but inhibited the effect of IL-4. Thus, HBSMC contain functional BK(Ca) channels whose activity is rapidly potentiated by the cytokine, IL-4, but not by IL-13. These findings are consistent with a model in which IL-4 rapidly increases near-membrane calcium concentrations to regulate BK(Ca) activity