Desformylflustrabromine (dFBr), a positive allosteric modulator of α4ß2 nicotinic acetylcholine receptors decreases voluntary ethanol consumption and preference in male and female Sprague-Dawley rats.

Alcohol use disorder is a medical condition that impacts millions of individuals worldwide. Although there are a few pharmacotherapeutic options for alcohol-dependent individuals; there is a need for the development of novel and more effective therapeutic approaches. Alcohol and nicotine are commonly co-abused, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4ß2 nAChRs has been shown to reduce nicotine intake, compulsive-like behavior and neuropathic pain in animal models. dFBr has also been previously shown to cross the blood-brain-barrier. We have recently shown that dFBr can attenuate the response to an acute, hypnotic dose of ethanol, via ß2 nAchR. Here, we have investigated the effect of dFBr in modulating ethanol consumption using the intermittent access two-bottle choice (IA2BC) model of voluntary ethanol consumption in male and female Sprague Dawley rats. We show that dFBr selectively reduced ethanol but not sucrose consumption in the IA2BC model. Furthermore, dFBr decreased preference for ethanol in both male and female rats. No rebound increase in ethanol intake was observed after the washout period after dFBr treatment. The ability of dFBr to decrease ethanol consumption, along with its previously demonstrated ability to decrease nicotine self-administration in rodents, suggest that dFBr is an attractive therapeutic candidate to target both nicotine and alcohol abuse.

The Use of Direct-to-Consumer (DTC) Pharmaceutical Advertisements in Televised and Print Formats as a Teaching Tool in a Pharmacy Curriculum.

The overall goal of this study was to employ direct-to-consumer advertisements (DTCAs) as a teaching tool in a Doctor of Pharmacy (PharmD) curriculum. The objectives of this pilot study were to investigate the following questions: 1. Do DTCAs generate student curiosity about the advertised drug and associated disease? 2. Can DTCAs help students understand and reinforce various pharmacological aspects of the drug? 3. How do students perceive DTCAs? A DTCA-based teaching tool was employed in a pharmacology course taken by P2 (second professional year) PharmD and final year (U4) Bachelor of Science (BS) in Pharmacology-Toxicology students. A voluntary online survey was administered to students to determine the effectiveness of this tool. Survey data were analyzed quantitatively and qualitatively. 70-85% of responding students indicated that this teaching tool was an effective visual aid for learning pharmacology and correlating the drug to disease state, mechanism of action, and adverse effects. Moreover, themes identified from the qualitative analysis suggest that this teaching tool may be useful to enhance patient counseling skills in students. The initial implementation of this DTCA-based teaching tool proved to be successful, and a similar approach can be easily implemented in other pharmacotherapy and laboratory courses. Further studies are needed to determine if this approach can improve patient counseling skills.

Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4ß2 nicotinic receptor modulates the hypnotic response to ethanol.

BACKGROUND: Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α4ß2 nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α4ß2 nAChR in vitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol. METHODS: Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The ß2 nAChR selective antagonist dihydro-ß-erythroidine (DHßE) was used to study the involvement of the ß2 subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α4 nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α4ß2 nAChR. RESULTS: Pretreatment with 6 mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHßE suggest that dFBr reduced ethanol-induced LORR duration via the ß2 nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α4 nAChR subunit protein levels. This ethanol-induced α4 nAChR upregulation was significantly reduced in rats pretreated with 6 mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α4ß2 nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents. CONCLUSIONS: Our results suggest that thalamic nAChRs containing the α4 subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α4ß2 nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on ß2 nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.

Ethanol-induced plasticity of GABAA receptors in the basolateral amygdala.

Acute and chronic ethanol (EtOH) administration is known to affect function, surface expression, and subunit composition of γ-aminobutyric acid (A) receptors (GABAARs) in different parts of the brain, which is believed to play a major role in alcohol dependence and withdrawal symptoms. The basolateral amygdala (BLA) participates in anxiety-like behaviors including those induced by alcohol withdrawal. In the present study we assessed the changes in cell surface levels of select GABAAR subunits in the BLA of a rat model of alcohol dependence induced by chronic intermittent EtOH (CIE) treatment and long-term (>40 days) withdrawal and investigated the time-course of such changes after a single dose of EtOH (5 g/kg, gavage). We found an early decrease in surface expression of α4 and δ subunits at 1 h following single dose EtOH treatment. At 48 h post-EtOH and after CIE treatment there was an increase in α4 and γ2, while α1, α2, and δ surface expression were decreased. To relate functional changes in GABAARs to changes in their subunit composition we analyzed miniature inhibitory postsynaptic currents (mIPSCs) and the picrotoxin-sensitive tonic current (Itonic) 48 h after EtOH intoxication. The Itonic magnitude and most of the mIPSC kinetic parameters (except faster mIPSC decay) were unchanged at 48 h post-EtOH. At the same time, Itonic potentiation by acute EtOH was greatly reduced, whereas mIPSCs became significantly more sensitive to potentiation by acute EtOH. These results suggest that EtOH intoxication-induced GABAAR plasticity in the BLA might contribute to the diminished sedative/hypnotic and maintained anxiolytic effectiveness of EtOH.

Plasticity of GABA(A) receptor-mediated neurotransmission in the nucleus accumbens of alcohol-dependent rats.

Chronic alcohol exposure-induced changes in reinforcement mechanisms and motivational state are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Here we describe the long-lasting alterations of γ-aminobutyric acid type A receptors (GABA(A)Rs) of medium spiny neurons (MSNs) in the NAcc after chronic intermittent ethanol (CIE) treatment, a rat model of alcohol dependence. CIE treatment and withdrawal (>40 days) produced decreases in the ethanol and Ro15-4513 potentiation of extrasynaptic GABA(A)Rs, which mediate the picrotoxin-sensitive tonic current (I(tonic)), while potentiation of synaptic receptors, which give rise to miniature inhibitory postsynaptic currents (mIPSCs), was increased. Diazepam sensitivity of both I(tonic) and mIPSCs was decreased by CIE treatment. The average magnitude of I(tonic) was unchanged, but mIPSC amplitude and frequency decreased and mIPSC rise time increased after CIE treatment. Rise-time histograms revealed decreased frequency of fast-rising mIPSCs after CIE treatment, consistent with possible decreases in somatic GABAergic synapses in MSNs from CIE rats. However, unbiased stereological analysis of NeuN-stained NAcc neurons did not detect any decreases in NAcc volume, neuronal numbers, or neuronal cell body volume. Western blot analysis of surface subunit levels revealed selective decreases in α1 and δ and increases in α4, α5, and γ2 GABA(A)R subunits after CIE treatment and withdrawal. Similar, but reversible, alterations occurred after a single ethanol dose (5 g/kg). These data reveal CIE-induced long-lasting neuroadaptations in the NAcc GABAergic neurotransmission.

Subunit Compensation and Plasticity of Synaptic GABA(A) Receptors Induced by Ethanol in α4 Subunit Knockout Mice.

There is considerable evidence that ethanol (EtOH) potentiates γ-aminobutyric acid type A receptor (GABA(A)R) action, but only GABA(A)Rs containing δ subunits appear sensitive to low millimolar EtOH. The α4 and δ subunits co-assemble into GABA(A)Rs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABA(A)R function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4ßδ-GABA(A)Rs. Based on this hypothesis, any highly EtOH-sensitive GABA(A)Rs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABA(A)Rs in mice with a global deletion of the α4 subunit (KO). The dentate granule cells of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT) controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABA(A)Rs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p.) revealed increased intracellular fraction of the α1, α4, and δ, but not α2, α5, or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 µM, requiring α1/α2, inactive at α4/5 GABA(A)Rs) than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice were significantly less sensitive to zolpidem, suggesting decreases in functional α1/2ßγ GABA(A)Rs. These data further support our hypothesis that EtOH intoxication induces GABA(A)R plasticity via internalization of highly EtOH-sensitive GABA(A)Rs.

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons.

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18 days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4 h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCß isoform with PKCß pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.

Ethanol reduces GABAA alpha1 subunit receptor surface expression by a protein kinase Cgamma-dependent mechanism in cultured cerebral cortical neurons.

Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA(A) receptors composed of alpha1beta2/3gamma2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA(A) alpha1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) beta, gamma, and epsilon isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABA- and zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA(A) receptor alpha1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKCgamma with alpha1 subunits, whereas PKCbeta/alpha1 association and PKCepsilon/alpha1 colocalization were not altered by ethanol exposure. Selective PKCgamma inhibition by transfection of selective PKCgamma small interfering RNAs blocked ethanol-induced internalization of GABA(A) receptor alpha1 subunits, whereas PKCbeta inhibition using pseudo-PKCbeta had no effect. These findings suggest that ethanol exposure selectively alters PKCgamma translocation to GABA(A) receptors and PKCgamma regulates GABA(A) alpha1 receptor trafficking after ethanol exposure.

A comprehensive study on the 5-hydroxytryptamine(3A) receptor binding of agonists serotonin and m-chlorophenylbiguanidine.

Serotonin type 3 receptors (5-HT(3)R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT(3A)R were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT(3A)Rs. Using this data we have constructed a refined homology model of the 5-HT(3A)R that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.

Normal acute behavioral responses to moderate/high dose ethanol in GABAA receptor alpha 4 subunit knockout mice.

BACKGROUND: gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) have been implicated in mediating some of the behavioral effects of ethanol (EtOH), but the contribution of specific GABA(A)-R subunits is not yet fully understood. The GABA(A)-R alpha 4 subunit often partners with beta2/3 and delta subunits to form extrasynaptic GABA(A)-Rs that mediate tonic inhibition. Several in vitro studies have suggested that these extrasynaptic GABA(A)-Rs may be particularly relevant to the intoxicating effects of low doses of EtOH. In alpha 4 subunit knockout mice, tonic inhibition was greatly reduced, as were the potentiating effects of EtOH. We therefore hypothesized that those behavioral responses to EtOH that are mediated by alpha 4-containing GABA(A)-Rs would be diminished in alpha 4 knockout mice. METHODS: We investigated behavioral responses to acute administration of moderate/high dose EtOH or pentylenetetrazol in alpha 4 subunit knockout mice. We compared behavioral responses to EtOH in alpha 4 knockout and wild-type littermates in the elevated plus maze (0.0, 1.0 g/kg EtOH), screen test (1.5, 2.0 g/kg), hypothermia (1.5, 2.0 g/kg), fixed speed rotarod (1.5, 2.0, 2.5 g/kg), open field (0.0, 1.0, 2.0 g/kg), radiant tail flick (2.0 g/kg), loss of righting reflex (3.5 g/kg), and EtOH metabolism and clearance assays. Sensitivity to pentylenetetrazol-induced seizures was also analyzed. RESULTS: No differences were observed between alpha 4 knockout mice and wild-type controls in terms of the baseline behavior in the absence of EtOH treatment or in the behavioral effects of EtOH in the assays tested. In contrast, alpha 4 knockout mice were significantly more sensitive to pentylenetetrazol-induced seizures. CONCLUSIONS: We conclude that GABA(A)-Rs containing the alpha 4 subunit are not absolutely required for the acute behavioral responses to moderate/high dose EtOH that were assessed with the elevated plus maze, screen test, hypothermia, fixed speed rotarod, open field, radiant tail flick, and loss of right reflex assays. We further suggest that these findings are complicated by the demonstrated compensatory alterations in synaptic GABA(A)-R EtOH sensitivity and function in alpha 4 knockout mice.

Functional consequences of GABAA receptor alpha 4 subunit deletion on synaptic and extrasynaptic currents in mouse dentate granule cells.

BACKGROUND: The alpha 4 subunit-containing gamma-aminobutyric acid (A) receptors (GABA(A)Rs) are highly expressed primarily at extrasynaptic sites in the dentate gyrus (DG) and thalamus and are suspected to contribute to tonic inhibition that is sensitive to potentiation by gaboxadol and ethanol (EtOH). Global alpha 4 subunit knockout (KO) mice exhibit greatly reduced tonic currents and insensitivity to ataxic, sedative and analgesic effects of gaboxadol compared to wild type (WT) controls. The alpha 4 KO mice were also significantly more sensitive to pentylenetetrazol-induced seizures. However, no differences were observed between alpha 4 KO and WT mice in other baseline behaviors or in the effects of EtOH on these behaviors. To examine possible functional and pharmacological GABA(A)R alterations, and search for causes for the lack of differences in EtOH behaviors we studied the effects of acute EtOH application on GABA(A)R-currents of DG cells from alpha 4 KO and WT control mice complemented by Western blot measurements. METHODS: We studied the consequences of alpha 4 subunit deletion using Western immunoblotting and whole cell patch recordings from DG cells in brain slices from alpha 4 KO and WT mice. RESULTS: The magnitude of tonic current and its potentiation by EtOH (10 to 100 mM), alphaxalone (3 microM), and Ro15-4513 (0.3 microM) was greatly attenuated in alpha 4 KO mice. The kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in alpha 4 KO mice were significantly slower compared to WT mice. Potentiation of mIPSCs by alphaxalone was greatly reduced in alpha 4 KO mice. Ro15-4513 had no effect on mIPSCs from WT or KO mice. However, mIPSCs of alpha 4 KO mice were significantly more sensitive to EtOH than those from WT mice. The gamma 2 subunit protein levels were selectively increased in hippocampus and thalamus, but not cortex of alpha 4 KO mice. CONCLUSIONS: These data suggest that the global loss of alpha 4 subunits leads to region- and cell location-specific compensatory increases in gamma 2 subunits, which in turn alter the pharmacological sensitivity of synaptic and extrasynaptic GABA(A)R-currents. Our data also suggests that while enhancement of tonic inhibitory currents by gaboxadol, alphaxalone, and EtOH are reduced, and behavioral sensitivity to gaboxadol and alphaxalone may be reduced, compensatory changes in synaptic GABA(A)R subunits may prevent similar reductions in behavioral sensitivity to EtOH.

Mechanisms of reversible GABAA receptor plasticity after ethanol intoxication.

The time-dependent effects of ethanol (EtOH) intoxication on GABA(A) receptor (GABA(A)R) composition and function were studied in rats. A cross-linking assay and Western blot analysis of microdissected CA1 area of hippocampal slices obtained 1 h after EtOH intoxication (5 g/kg, gavage), revealed decreases in the cell-surface fraction of alpha4 and delta, but not alpha1, alpha5, or gamma2 GABA(A)R subunits, without changes in their total content. This was accompanied (in CA1 neuron recordings) by decreased magnitude of the picrotoxin-sensitive tonic current (I(tonic)), but not miniature IPSCs (mIPSCs), and by reduced enhancement of I(tonic) by EtOH, but not by diazepam. By 48 h after EtOH dosing, cell-surface alpha4 (80%) and gamma2 (82%) subunit content increased, and cell-surface alpha1 (-50%) and delta (-79%) and overall content were decreased. This was paralleled by faster decay of mIPSCs, decreased diazepam enhancement of both mIPSCs and I(tonic), and paradoxically increased mIPSC responsiveness to EtOH (10-100 mm). Sensitivity to isoflurane- or diazepam-induced loss of righting reflex was decreased at 12 and 24 h after EtOH intoxication, respectively, suggesting functional GABA(A)R tolerance. The plastic GABA(A)R changes were gradually and fully reversible by 2 weeks after single EtOH dosing, but unexplainably persisted long after withdrawal from chronic intermittent ethanol treatment, which leads to signs of alcohol dependence. Our data suggest that early tolerance to EtOH may result from excessive activation and subsequent internalization of alpha4betadelta extrasynaptic GABA(A)Rs. This leads to transcriptionally regulated increases in alpha4 and gamma2 and decreases in alpha1 subunits, with preferential insertion of the newly formed alpha4betagamma2 GABA(A)Rs at synapses.

Interactions of granisetron with an agonist-free 5-HT3A receptor model.

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.

The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide.

Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.