Confirmation of correlations and common quantitative trait loci between neurotensin receptor density and hypnotic sensitivity to ethanol.

BACKGROUND: In previous studies, genetic correlations were observed between hypnotic sensitivity to ethanol and high-affinity neurotensin receptor (NTS1) binding. Provisional quantitative trait loci (QTLs) were identified for these traits, and some of these QTLs were found on common chromosomal regions. In continued efforts to examine the relationship between NTS1 binding capacity and hypnotic sensitivity to ethanol, studies were designed to confirm correlations between NTS1 densities in the brain, duration of ethanol-induced loss of righting reflex (LORR), and blood ethanol concentrations at regain of righting reflex (BECRR). Another purpose of the study was to confirm QTLs for these traits. METHODS: ILS X ISS F2 mice and HAS X LAS F2 rats as well as the progenitors were tested for LORR, BECRR, and NTS1 densities. Phenotypic correlations were calculated between LORR and BECRR and between these measures and NTS1 densities in striatum from both mice and rats. The F2 mice were genotyped by using polymorphic markers for five previously reported QTLs for LORR to confirm QTLs for BECRR and NTS1 densities in striatum, ventral midbrain, and frontal cortex. RESULTS: Phenotypic correlations were found between LORR and BECRR (r = -0.66 to -0.74, p < 10(-9)) and between these measures and NTS1 densities in striatum (r = 0.28-0.38, p < 10(-2)) from both mice and rats. QTLs for LORR and BECRR (lod score = 2-6) were found in common regions of chromosomes 1, 2, and 15. By using the combined results from a previous LSXSS RI study and the current results, a suggestive QTL (lod score = 3.1) for striatal NTS1 receptor densities was found on chromosome 15 at approximately 60 cM, in the same region as the chromosome 15 LORR/BECRR QTL. CONCLUSIONS: The results are in agreement with previously reported correlations and QTLs for NTS1 receptor densities and measures of hypnotic sensitivity to ethanol in mice and extend those correlations to another species, the rat. These findings support a role for NTS1 in genetically mediated differences in hypnotic sensitivity to ethanol.

The genetics of acute functional tolerance and initial sensitivity to ethanol for an ataxia test in the LSxSS RI strains.

BACKGROUND: It has been proposed that development of tolerance to the behavioral effects of ethanol depends on the degree of impairment produced by the drug; that is, more sensitive individuals should develop greater tolerance. Tests of this hypothesis with respect to acute functional tolerance have produced contradictory results. We tested the hypothesis by examining the genetic relationship between initial sensitivity and acute functional tolerance in the LSXSS recombinant inbred mice. METHODS: We tested mice for initial sensitivity to the ataxic effects of 1.75 g/kg of ethanol in a stationary dowel balance test by determining blood and brain ethanol concentrations at fall. Acute tolerance to the ataxic effects of ethanol was determined by measuring blood ethanol concentration (BEC) at regain of dowel balance ability after the first injection (BEC1RB) and after a second ethanol injection of 2.0 g/kg (BEC2RB). Acute tolerance was quantified by the difference in ethanol concentration at the two regains of balance (BEC2RB - BEC1RB) or by the difference between the second regain and one of the initial sensitivity measures (BEC2RB - initial sensitivity). RESULTS: Four different measures of initial sensitivity were taken: two that used BEC values and two that used forebrain or hindbrain ethanol concentrations. We calculated acute tolerance values by using each of these initial sensitivity measures plus BEC2RB. No evidence of a genetic relationship between initial sensitivity and acute tolerance was found, which suggests that these are two independent phenomena with respect to stationary dowel balance. CONCLUSIONS: Three conclusions can be drawn from this work: (1) Orbital sinus BEC at early time points (<5 min postinjection) may or may not accurately reflect brain EC in mice, dependent on genotype; (2) there is no genetic relationship between initial sensitivity and acute tolerance to stationary dowel ataxia in the LSXSS RIs; and (3) sex-specific factors affect low-dose ethanol responses on the stationary dowel.

Selectively bred lines of mice show response and drug specificity for genetic regulation of acute functional tolerance to ethanol and pentobarbital.

Genetic regulation of acute tolerance to ethanol may be associated with ethanol consumption and other ethanol-related behaviors in rodents. We have used lines of mice, selectively bred for high and low acute functional tolerance (HAFT and LAFT, respectively) to ethanol-induced loss of balance to test this hypothesis. Replicate HAFT and LAFT lines differ in AFT to ethanol-induced loss of balance by 4.4- and 5-fold, respectively. Frequency distributions and mean AFT scores for those lines, F(1), and backcrosses show a dominance for the HAFT phenotype. Time courses for acquisition and decay showed that AFT to ethanol-induced loss of balance developed rapidly, could be maintained up to 6 h with repeated doses, and decayed 6 h after peak tolerance and discontinuance of ethanol administration. The lines did not differ in initial sensitivity as measured by brain ethanol concentration at loss of balance, indicating that initial sensitivity and AFT to loss of balance were not coselected traits. Surprisingly, HAFT versus LAFT lines did not differ in development of AFT to loss of righting response, or hypothermia, indicating different mechanisms or neuronal systems mediate genetic influences on these measures. Voluntary ethanol consumption was low in both of the replicate lines, but HAFT lines consumed greater amounts of ethanol than LAFT lines. The HAFT and LAFT lines developed AFT to pentobarbital-induced loss of balance, however, there were no line differences in rates or extent of the AFT development. These results show that genetic regulation of AFT development is drug- as well as response-specific.

MK-801-induced locomotor activity in long-sleep x short-sleep recombinant inbred mouse strains: correlational analysis with low-dose ethanol and provisional quantitative trait loci.

BACKGROUND: Low doses of the N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine) or ethanol increase locomotor activity to a lesser extent in long-sleep (LS), than in short-sleep (SS), mice. LS mice also have fewer brain [3H]MK-801 binding sites than SS mice. In this study, LSXSS recombinant inbred (RI) mice were used to investigate whether different NMDAR densities contribute to differential MK-801 activation and whether common genes are involved in initial sensitivity to MK-801-and ethanol-induced activation. METHODS: Locomotor activity was measured for 90 min after saline or MK-801 injection. Quantitative autoradiographic analysis of [3H]MK-801 binding was used to measure densities of NMDARs in seven brain regions. The ethanol (1-2 g/kg) activation scores from Erwin and colleagues (1997) were used for correlational analysis, as was their method for quantitative trait loci (QTL) analysis. RESULTS: Both saline and MK-801 (0.3 mg/kg, given intraperitoneally) induced a continuum of locomotor responses across the LSXSS RI strains. There was a 4-fold range of MK-801 difference scores (MK-801 score-saline baseline), with the RI 9 and RI 4 strains representing low and high responders, respectively. Dose-response experiments with these two strains confirmed that 0.3 mg/kg MK-801 produced significant activation, similar to previous results with LS and SS mice. However, unlike previous LS/SS results, lower densities of NMDARs were not observed in the RI 9 than in the RI 4 mouse brains. No significant genetic correlations were observed between MK-801-induced and ethanol-induced responses in the LSXSS RI mice. Two provisional MK-801 activation QTLs were identified (p < 0.01) on chromosomes 11 and 19, neither in common with those mapped for ethanol activation. CONCLUSIONS: Different densities of brain NMDARs are unlikely to account for the differential activation of LSXSS RI mice by MK-801. Additionally, in the RI mice either separate sets of genes regulate low dose MK-801- and ethanol-induced locomotor responses or the overlapping subset of genes controlling these two behaviors is small (< or =10%).

Common quantitative trait loci for alcohol-related behaviors and CNS neurotensin measures: voluntary ethanol consumption.

The C57BL/6, DBA/2, and recombinant inbred (RI) strains derived from them (BxD RIs) are the most frequently studied mouse strains with regard to genetic regulation of voluntary ethanol consumption (YEC). We have studied VEC in an alternate genetic model provided by the LSxSS RIs. These RI strains exhibit phenotypic extremes in VEC comparable to the C57BL/6 and DBA/2 mice and genotype-dependent sex differences in drinking behavior. A correlational analysis between various ethanol-related behaviors suggests genetic independence of VEC from high-dose neurosensitivity (sleep time), acute ethanol tolerance, hypothermia, and low-dose locomotor activity. A search for quantitative trait loci identified a number of putative quantitative trait loci (QTL), three of which are identical to those previously reported for 10% ethanol drinking in the BxD RIs. We also find a significant correlation between low-affinity neurotensin receptor densities (NTRL) in the frontal cortex and VEC, and more common QTL between these two phenotypes than expected by chance. This suggests a role for frontal cortex NTRL in regulating voluntary ethanol intake.

N-glycosylation at the conserved sites ensures the expression of properly folded functional ACh receptors.

The role of the conserved carbohydrate moiety in the expression of complete acetylcholine receptor (AChR), alpha2 beta gamma delta was re-investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subunits were stably expressed and appeared to associate with other normal subunits; however, removal of carbohydrate on the alpha subunit inhibited the formation of 125I-alpha-bungarotoxin (alpha-BuTX) binding sites and functional ACh-gated ion channels. 125I-alpha-BuTX binding to AChRs was also significantly reduced by removal of the conserved carbohydrate on the gamma or delta subunits. Immunoprecipitation with monoclonal antibodies that recognize the two distinct alpha-BuTX sites on the AChR indicated that the mutant gamma subunit did not interfere with efficient formation of the alpha-BuTX binding site at the alpha/delta interface, but loss of the carbohydrate did interfere with formation of the alpha-BuTX binding site at the alpha/mutant gamma interface. A similar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated efficiently into the mature (correct tertiary conformation capable of alpha-BuTX binding) alpha beta delta or alpha beta gamma complexes, respectively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these results suggest that the formation of the two alpha-BuTX binding sites requires correct folding of the alpha gamma and alpha delta complexes, which is aided by the conserved carbohydrate on the gamma and delta subunits. Electrophysiological experiments demonstrated that functional receptors containing mutant subunits were produced, but the functional properties of the mutant receptors were differentially altered, depending on the subunit mutated. Together, our results suggest that N-glycosylation of AChR subunits ensures the correct folding of important functional domains and expression of proper functional receptors in the plasma membrane.

Common quantitative trait loci for alcohol-related behaviors and central nervous system neurotensin measures: hypnotic and hypothermic effects.

Genetic correlations were found between high-affinity neurotensin receptor (NTR(H)) densities and NT-immunoreactivity (NT-ir) levels in specific brain regions and sensitivity to hypnotic and hypothermic effects of ethanol in LSXSS recombinant inbred strains of mice. Simple sequence length polymorphisms were used to identify quantitative trait loci (QTL) influencing hypnotic and hypothermic sensitivity to ethanol, NTR(H) and low-affinity neurotensin receptor densities and NT-ir levels in LSXSS recombinant inbred strains. Common QTL for NTR(H) receptor densities, NT-ir levels and these ethanol actions were identified. One of the QTL (chromosome 2, 80 cM) for NTR(H) density and hypnotic sensitivity is linked to the NTR(H) gene, Ntsr. Also, QTL for NTR(H) density were found in common with confirmed QTL for hypnotic sensitivity on chromosomes 1 (43 cM), 11 (57 cM) and 15 (56 cM) and with an unconfirmed QTL on chromosome 3 (19 cM). Two common QTL for NT-ir levels, but not NTR(H) or low-affinity neurotensin receptor receptors, and ethanol-induced hypothermia were observed on chromosomes 4 (43 cM) and 6 (41 cM). Two common QTL for NT-ir levels and sleep time were identified on chromosomes 3 (19 cM) and 9 (55 cM). Common QTL indicate that genes regulating NT receptor and/or NT-ir expression may be the same as those regulating sensitivity to ethanol.

Common quantitative trait loci for alcohol-related behaviors and central nervous system neurotensin measures: locomotor activation.

We have analyzed LSXSS recumbinant inbred for ethanol-induced activity using 2.0 g/kg ethanol and a new method we call ethanol activation slope. The ethanol activation slope provides a robust dose-response measure of ethanol activation, independent of both activity after saline and the inhibitory effects of ethanol on locomotor activity. These behavioral data were used in a quantitative trait locus analysis to map chromosomal loci involved in ethanol-induced locomotor activity. We tentatively identified seven loci that mediate the low-dose stimulatory effect of ethanol and six loci involved in locomotion after 2.0 g/kg ethanol. Only one of the loci are in common between the two behaviors. We also compared the behavioral quantitative trait locus to those previously identified that are involved in regulating central nervous system neurotensin levels and neurotensin receptor densities. Six chromosomal regions were identified that regulate at least one central nervous system neurotensin measure and an ethanol-induced locomotor behavior. The identification of loci controlling both central nervous system neurotensin levels or neurotensin receptor densities and ethanol-induced locomotor activity strengthens the proposal that neurotensin regulates, in part, ethanol-induced behaviors and central nervous system sensitivity to ethanol.

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes.

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the gamma subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the gamma subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the gamma subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the gamma subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.

Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.

Site-directed mutagenesis of the conserved N-glycosylation site on the nicotinic acetylcholine receptor subunits.

The role of the conserved N-glycosylation site on each subunit of the Torpedo acetylcholine receptor (AChR) in the biogenesis and function of the receptor was examined by expressing site-directed mutant subunits in Xenopus oocytes. Different mutant subunits caused different effects. The most striking effect was seen with the mutant gamma subunit which, when co-expressed with alpha, beta, and delta subunits, caused degradation of all the subunits. N-Glycosylation of the other subunits appears to contribute to stability of the subunits and/or efficient insertion of the receptor into the plasma membrane, but is not required for assembly. The AChRs containing the mutant alpha subunit formed functional ion channels in the plasma membrane and their activity could be blocked by alpha-bungarotoxin (alpha-BuTX). Thus, attachment of a carbohydrate moiety at the conserved N-glycosylation site is not an absolute requirement for the formation of ACh and alpha-BuTX binding sites.