Distinct Roles for Two Chromosome 1 Loci in Ethanol Withdrawal, Consumption, and Conditioned Place Preference

We previously identified a region on chromosome 1 that harbor quantitative trait loci (QTLs) with large effects on alcohol withdrawal risk using both chronic and acute models in mice. Here, using newly created and existing QTL interval-specific congenic (ISC) models, we report the first evidence that this region harbors two distinct alcohol withdrawal QTLs (Alcw11and Alcw12), which underlie 13% and 3–6%, respectively, of the genetic variance in alcohol withdrawal severity measured using the handling-induced convulsion. Our results also precisely localize Alcw11 and Alcw12 to discreet chromosome regions (syntenic with human 1q23.1–23.3) that encompass a limited number of genes with validated genotype-dependent transcript expression and/or non-synonymous sequence variation that may underlie QTL phenotypic effects. ISC analyses also implicate Alcw11and Alcw12 in withdrawal-induced anxiety-like behavior, representing the first evidence for their broader roles in alcohol withdrawal beyond convulsions; but detect no evidence for Alcw12 involvement in ethanol conditioned place preference (CPP) or consumption. Our data point to high-quality candidates for Alcw12, including genes involved in mitochondrial respiration, spatial buffering, and neural plasticity, and to Kcnj9 as a high-quality candidate for Alcw11. Our studies are the first to show, using two null mutant models on different genetic backgrounds, that Kcnj9−/− mice demonstrate significantly less severe alcohol withdrawal than wildtype littermates using acute and repeated exposure paradigms. We also demonstrate that Kcnj9−/− voluntarily consume significantly more alcohol (20%, two-bottle choice) than wildtype littermates. Taken together with evidence implicating Kcnj9 in ethanol CPP, our results support a broad role for this locus in ethanol reward and withdrawal phenotypes. In summary, our results demonstrate two distinct chromosome 1 QTLs that significantly affect risk for ethanol withdrawal, and point to their distinct unique roles in alcohol reward phenotypes

Genetic Control of a Central Pattern Generator: Rhythmic Oromotor Movement in Mice Is Controlled by a Major Locus near Atp1a2

Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h2≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM = 116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F1 hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na+/K+-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo

Contribution of dopamine receptors to periaqueductal gray-mediated antinociception

RATIONALE: Morphine relieves pain, in part, by acting on neurons within the periaqueductal gray (PAG). Given that the PAG contains a subpopulation of dopamine neurons, dopamine may contribute to the antinociceptive effects mediated by the PAG. METHODS: This hypothesis was tested by measuring the behavioral and electrophysiological effects of administering dopamine agonists and antagonists into the ventrolateral PAG (vPAG). An initial histological experiment verified the existence of dopamine neurons within the vPAG using dopamine transporter and tyrosine hydroxylase antibodies visualized with confocal microscopy. RESULTS: Microinjection of cumulative doses of morphine into the vPAG caused antinociception that was dose-dependently inhibited by the dopamine receptor antagonist α-flupenthixol. α-Flupenthixol had no effect on nociception when administered alone. Injection of the dopamine receptor agonist (-) apomorphine into the vPAG caused a robust antinociception that was inhibited by the D2 antagonist eticlopride but not the D1 antagonist SCH-23390. The effects of dopamine on GABA(A)-mediated evoked inhibitory post-synaptic potentials (eIPSCs) were measured in PAG slices. Administration of met-enkephalin inhibited peak evoked inhibitory post-synaptic potentials (eIPSCs) by 20-50%. Dopamine inhibited eIPSC by approximately 20-25%. Administration of α-flupenthixol (20 μM) attenuated eIPSC inhibition by dopamine, but had no effect on met-enkephalin-induced inhibition. CONCLUSIONS: These data indicate that PAG dopamine has a direct antinociceptive effect in addition to modulating the antinociceptive effect of morphine. The lack of an effect of α-flupenthixol on opioid-inhibition of eIPSCs indicates that this modulation occurs in parallel or subsequent to inhibition of GABA release

Biochemical identification of the dopamine D2 receptor domains interacting with the adenosine A2A receptor

Functional interactions between adenosine A2A and dopamine D2 receptors have been demonstrated both at the D2 agonist-binding and second messenger levels. The present studies use a [3H]dopamine-binding assay as a sensitive measure of A2A receptor-mediated modulation of D2 receptors. Co-incubation with an A2A receptor agonist increased the Kd value of high-affinity [3H]dopamine-binding sites of the D2 receptor without changing their Bmax values in a cotransfected cell line. This interaction was shown to be subtype specific, as the A2A receptor agonist did not modulate the affinity of the D1 receptor for [3H]dopamine. The domains of the D2 receptor important for the A2A/D2 receptor interaction were studied with chimeric dopamine D2/D1 receptors. The results showed that the A2A receptor agonist still strongly reduced the affinity of a D2/D1 chimera with the sixth transmembrane (TM) domain and third extracellular loop from the D1 receptor. However, the A2A receptor agonist was not able to modulate a D2/D1 chimeric receptor containing the fifth and sixth TM domains and the third intracellular and extracellular loops from the D1 receptor, indicating that the fifth TM domain and/or the third intracellular loop may be involved in the interaction between A2A and D2 receptors

Direct dopamine D(2)-receptor-mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca(2+)-mobilizing agent

1. In CHO cells transfected with the rat dopamine D(2) receptor (long isoform), administration of dopamine per se elicited a concentration-dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC(50)=25 nM). The partial D(2) receptor agonist, (−)-(3-hydroxyphenyl)-N-n-propylpiperidine [(−)-3-PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC(50)=91 nM). 2. The AA-releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca(2+) release, 8-(N N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), by excluding calcium from the medium, by the phospholipase A(2) (PLA(2)) inhibitor, quinacrine, and by long-term pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, it was antagonized by the D(2) antagonists, raclopride and (−)-sulpiride–but not by (+)-sulpiride–and absent in sham-transfected CHO cells devoid of D(2) receptors. 3. The results obtained contrast to the previous notion that dopamine and other D(2) receptor agonists require the concomitant administration of calcium-mobilizing agents such as ATP, ionophore A-23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines