Identification and confirmation of quantitative trait loci regulating alcohol consumption in congenic strains of mice.

BACKGROUND: C57BL/6 inbred mice prefer alcohol whereas DBA/2 mice avoid it. We describe the construction of congenic strains of mice in which DBA/2 alleles for alcohol avoidance were placed on a C57BL/6 background using phenotypic selection. METHODS: Mice were primed to drink 10% v/v ethanol in water for 2 days before a two-bottle choice paradigm. N2 males who demonstrated an alcohol-avoidance phenotype were backcrossed to B6 females to construct 15 independent lines. RESULTS: Eight of these lines were lost due to failure to breed or absence of males with an alcohol-avoidance phenotype. The remaining sublines were split to form a total of 21 sublines. In the N7 and N9 generations, a genome scan located provisional quantitative trait loci (QTLs) on chromosomes 1, 2, 3, 6, and 9. Progeny testing confirmed QTLs on chromosomes 1 and 2. CONCLUSIONS: The QTL on chromosome 2 overlaps the 95% confidence interval of Alcp1 whereas that on chromosome 1 is new and has been called Alcp5. Marker-assisted selection was used in the N9 and subsequent generations to maintain the congenic lines and produce congenic strains.

Stable expression of human glycine alpha1 and alpha2 homomeric receptors in mouse L(tk-) cells.

We report the development of two mouse fibroblast-like stably-transfected cell lines (alpha1-62-4 and alpha2-B36-1) that express human alpha1 or alpha2 glycine receptor subunits, respectively. Transfected cDNAs were cloned into the pMSGneo expression vector, for which transcription is controlled by the dexamethasone-inducible MMTV promoter. Patch-clamp electrophysiological recordings revealed that the alpha1 or alpha2 glycine receptor subunits expressed in these cells form functional glycine receptors that are inhibited by strychnine and picrotoxin. Glycine activated currents in these cells with EC50s of 101+/-7 or 112+/-23 microM for cells stably expressing alpha1 or alpha2 receptors, respectively. As indicated by assays of glycine-stimulated 36Cl-- uptake, these cells express glycine receptors only after treatment with dexamethasone. In order to measure expression of the glycine alpha1 or alpha2 receptor protein, we produced a new anti-alpha1/alpha2 glycine receptor antibody (anti-alpha GR). Western blot analysis with this antibody showed a band of approximately 48 kDa only in homogenates from cells which had been transfected with the glycine alpha1 or alpha2 receptor cDNAs. Thus, through use of this stable expression system, we successfully produced cell lines expressing strychnine-sensitive glycine receptors that display similar functional characteristics to homomeric glycine receptors expressed in other systems. These stably transfected cells should provide a useful in vitro system for the study of the physiology and pharmacology of strychnine-sensitive glycine receptors.

Colchicine is a competitive antagonist at human recombinant gamma-aminobutyric acidA receptors.

Colchicine is an alkaloid that is used clinically in the treatment of arthritic gout. This potent microtubule disrupting agent has also been used extensively as an experimental tool in studies characterizing the role of the cytoskeleton in a variety of cellular processes. Colchicine has also been used as a selective neurotoxin and in animal models of Alzheimer's disease and epilepsy. Although the mechanism(s) mediating the neurotoxic actions of colchicine have not been established, most studies have attributed these effects to its microtubule depolymerizing actions. Here we report another central nervous system action of colchicine, competitive antagonism of gamma-aminobutyric acid (GABA)A receptor function. By use of a rapid drug perfusion system, colchicine (10-1000 microM) significantly inhibited GABA currents recorded from L(tk-) cells stably transfected with human alpha 1 beta 2 gamma 2L GABAA receptor subunits. The inhibition was rapid and reversible, with 100 microM colchicine shifting the GABA EC50 from 2.5 to 5.1 microM with no effect on currents evoked by saturating concentrations of GABA. Colchicine also significantly inhibited binding of the competitive GABAA receptor antagonist [3H]SR-95531. Other microtubule disrupting agents (10 microM vinblastine, 10 micrograms/ml nocodazole, 1 microM taxol) had no acute effects on GABA currents, nor did the inactive analog gamma-lumicolchicine (100 microM). Moreover, pretreating cells with colchicine, vinblastine, nocodazole or taxol for 1 to 4 hr did not occlude the acute inhibitory action of colchicine. We conclude that, in addition to its well characterized effects on microtubule assembly, colchicine can also inhibit GABAA receptor function through a direct interaction with the receptor/ion channel complex.

Microtubule depolymerization inhibits ethanol-induced enhancement of GABAA responses in stably transfected cells.

We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk-) cells stably transfected with GABAA receptor alpha 1 beta 1 gamma 2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. beta-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicin did not alter the potentiating actions of flunitrazeparn or pentobarbital on muscimol-stimulated chloride uptakes. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.

The cytoskeleton and neurotransmitter receptors.

The neuronal cytoskeleton consists of microtubules and microfilaments that can interact with membrane proteins including neurotransmitter receptors and ion channels. Ligand-gated ion channels, such as nicotinic acetylcholine receptors, glycine receptors, glutamate receptors and gamma-aminobutryic acidA (GABAA) receptors, are known to cluster in plasma membranes. Studies suggest that postsynaptic ligand-gated channels form clusters that are anchored in the plasma membrane by interacting with cytoskeletal components and these clusters may serve to optimize delivery of neurotransmitters to the channels. Other findings indicate that the interaction of clustered ligand-gated ion channels with cytoskeletal components may also play a role in channel function. For example, studies suggest that the interaction of microtubules with GABAA receptors regualtes GABA binding affinity. Regulation of neurotransmitter function may be significant in the study of neuropathological processes, such as Alzheimer's disease, neurotrauma, and experimental epilepsy, in which the cytoskeleton is vulnerable to disruption.

Gamma-aminobutyric acidA receptor function is inhibited by microtubule depolymerization.

Microtubules are present at postsynaptic densities in brain and are proposed to be involved in anchoring neurotransmitter receptor clusters at postsynaptic membranes. However, the influence of microtubules on gamma-aminobutyric acidA (GABAA) receptors has not been studied. Microtubule-affecting agents were tested for their actions on GABAA receptor function, by measuring muscimol-stimulated chloride uptake into cerebral cortical microsacs and proteoliposomes and GABA-mediated currents in Xenopus laevis oocytes expressing GABAA receptors. Colchicine, nocodazole, vinblastine, and taxol inhibited muscimol-stimulated chloride uptake. beta- and gamma-lumicolchicine did not inhibit GABAA ergic function. Colchicine decreased the potency of muscimol, a GABA agonist, to stimulate chloride uptake without affecting the specific binding of [3H]flunitrazepam or t-[35S]butylbicyclophosphorothionate to the GABAA receptor, or the allosteric modulation of binding of these ligands by muscimol. The function of purified GABAA receptors reconstituted in proteoliposomes, a preparation not containing microtubule components, was not affected by colchicine. In contrast to the results seen in human monocytes by other investigators, we found that colchicine decreased, rather than increased, protein kinase A activity in cortical microsacs. Thus, protein kinase A modulation of the GABAA receptor is not a likely mechanism for the actions of colchicine. We propose that microtubule-depolymerizing agents inhibit GABAA ergic function by disrupting the interaction of GABAA receptors with microtubules.

beta-Lumicolchicine interacts with the benzodiazepine binding site to potentiate GABAA receptor-mediated currents.

An analogue of colchicine, beta-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. beta-Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol-stimulated 36Cl- uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopus oocytes expressing human alpha 1 beta 2 gamma 2S, but not alpha 1 beta 2, GABAA receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of beta-lumicolchicine on the GABAA receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, beta-lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.