Genetic analysis of the corticosterone response to ethanol in BXD recombinant inbred mice

Acknowledgement: This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA08621, National Institute on Drug Abuse Grant DA05496, and the Department of Veteran Affairs. We thank Steve Mitchell for valuable statistical contributions.

C57BL/6 (B6) and DBA/2 (D2) inbred mice are among the most studied mouse strains in ethanol (EtOH) research. This is undoubtedly because of their divergent responses to EtOH. For example, B6 mice will drink EtOH whereas D2 mice avoid it (Crabbe, Kosobud, Young, & Janowsky, 1983; McClearn & Rodgers, 1959; Phillips, Crabbe, Metten, & Belknap, 1994). D2 mice are sensitive to the stimulating effects of EtOH and the development of sensitized locomotor activation, whereas B6 mice are more resistant to these effects (Crabbe, Johnson, Gray, Kosobud, & Young, 1982; Cunningham, Niehus, Malott, & Prather, 1992; Dudek, Phillips, & Hahn, 1991; Phillips, Dickinson, & Burkhart-Kasch, 1994; Tabakoff & Kiianmaa, 1982). D2 mice have also been shown to express more severe withdrawal handling-induced convulsions than B6 mice following chronic (Crabbe et al., 1983; Goldstein & Kakihana, 1974; Griffiths & Littleton, 1977) as well as acute (Roberts, Crabbe, & Keith, 1992) exposure to EtOH.

D2 mice have a greater stress response than B6 mice to EtOH (Kakihana, Butte, & Noble, 1968; Roberts et al., 1992). For example, they have higher plasma adrenocorticotropin (ACTH) for at least 90 min and higher corticosterone levels for about 3 hr following a single 4 g/kg EtOH injection (Roberts et al., 1992). In addition, D2 mice have been shown to have higher plasma corticosterone levels between 6 hr and 8 hr following a single injection of EtOH at times corresponding to acute withdrawal (Roberts et al., 1992).

Evidence is accumulating that supports a role for stress hormones that include corticosterone in modulating EtOH-related behaviors. For example, adrenalectomy decreases and corticosterone replacement reinstates EtOH self-administration in rats (Fahlke, Engel, Eriksson, Hard, & Söderpalm, 1994; Morin & Forger, 1982). It is not clear whether these effects are due to alterations in taste reactivity or alterations in the reinforcing effects of EtOH. Corticosterone may modulate reward pathways as it has been shown to possess reinforcing properties itself (Deroche, Piazza, Deminiere, Le Moal, & Simon, 1993; Piazza et al., 1993). Chronic stress and corticosterone administration have been shown to produce sensitized locomotor responsiveness to amphetamine (Antelman, Eichler, Black, & Kocan, 1980; Badiani, Cabib, & Puglisi-Allegra, 1992; Deroche, Piazza, Maccari, Le Moal, & Simon, 1992; Pauly, Robinson, & Collins, 1993). Preliminary evidence suggests that stress may also produce sensitization to the locomotor stimulant effect of EtOH (Roberts, Lessov, & Phillips, in press). Adrenalectomy significantly decreased the acute locomotor stimulant effects of 1.5 g/kg EtOH in C3H mice (Wallis, Anton, & Randall, 1984). Adrenalectomy decreased and corticosterone replacement normalized the severity of EtOH withdrawal convulsions in mice (Sze, 1977; Sze, Yanai, & Ginsburg, 1974). Furthermore, increases in corticosterone were shown to exacerbate acute EtOH withdrawal handling-induced convulsions in withdrawal-seizure-prone mice (Roberts, Chu, Crabbe, & Keith, 1991; Roberts, Crabbe, & Keith, 1994).

The involvement of corticosterone in EtOH-related behaviors and the differences in corticosterone response to EtOH in B6 and D2 mice suggest a possible relationship between these two traits. It is possible that the larger corticosterone response in D2 mice is partially responsible for the more severe EtOH withdrawal in this mouse strain. In order to more closely examine the relationship between corticosterone responses and behavioral responses to EtOH, we have used BXD recombinant inbred (RI) strains developed from B6 and D2 progenitors. BXD RI are the inbred descendants of an F2 intercross between B6 and D2 inbred strains. Because of chance recombinations of progenitor chromosomes, each BXD RI strain contains different patterns of B6 and D2 alleles in the homozygous state (Taylor, 1978).

Traits examined in BXD RI can be compared with others that have been collected and stored in a database to determine genetic correlations. Significant genetic correlations indicate shared underlying genetic influences (Crabbe, Phillips, Kosobud, & Belknap, 1990). In addition, the data derived from BXD RI can be used for quantitative trait locus (QTL) mapping (Gora-Maslak et al., 1991; Plomin, McClearn, Gora-Maslak, & Neiderhiser, 1991). Quantitative traits are continuously distributed as a result of polygenic (>1 gene) determination. A QTL is a chromosomal locus contributing to this quantitative trait. Over 800 marker loci have been mapped throughout the mouse genome which are polymorphic in B6 and D2 mice. Strain means are determined for traits in BXD RI and correlated with the strain distribution patterns for each marker locus. Significant correlations suggest that a gene (a QTL) in the vicinity of the marker may influence the trait of interest. The current status of QTL mapping of drug-related traits in BXD RI has been recently reviewed (Crabbe, Belknap, & Buck, 1994).

In the present experiments, corticosterone levels were determined at several time points following acute EtOH and vehicle (0.9% NaCl) administration in 19–23 BXD RI and their B6 and D2 progenitor strains. The 1-hr time point was chosen as an index of the acute stress response to EtOH, whereas the 6-and 7-hr time points were chosen because acute withdrawal handling-induced convulsions are highest at these times. D2 mice were previously shown to have higher corticosterone levels than B6 mice at each of these times following 4 g/kg EtOH (Roberts, Crabbe, & Keith, 1992). In one experiment, mice were scored for handling-induced convulsions several times after EtOH in order to examine possible effects of convulsion scoring on subsequent corticosterone determinations. Strain means were used to examine genetic correlations between corticosterone measures and several other EtOH-related behaviors and to perform QTL analyses.

Male BXD/Ty RI mice and their progenitor strains, C57BL/6J (B6) and DBA/2J (D2), were used in these experiments. Ages of mice ranged from 65 to 115 days. Animals within each BXD RI strain are considered identical at more than 99.9% of their genes. These mice were born and reared at the Portland Veterans Administration Animal Research Facility from breeding stock originally obtained from Jackson Labs (Bar Harbor, ME). Mice were housed in isosexual groups following weaning, at 21 ± 2 days, and were given ad libitum access to food and water. Lights were on a 12-hr light–dark cycle with lights on at 6 a.m. Mice used in each experiment could not be tested in a single pass because of limited availability of some strains; however, in most cases both B6 and D2 mice were included in each cohort. The responses of these progenitor strains did not vary significantly over testing sessions. In addition, each BXD strain was tested in 3 or more cohorts. Animal care and use were in compliance with the National Institutes of Health's guidelines.

Corticosterone levels were determined from either tail blood or trunk blood. To obtain tail blood, tails were nicked approximately 2 mm from the tip, and 20 μl blood was collected into heparinized capillary tubes. Blood was obtained in this manner for corticosterone levels 1 hr and 6 hr following acute EtOH and saline. To obtain trunk blood, mice were rapidly decapitated and trunk blood was collected into glass test tubes containing ethylenediamine tetraacetic acid (EDTA). Blood was obtained in this manner for corticosterone levels 7 hr following acute EtOH in mice that had been scored for handling-induced convulsions at several time points before blood sampling. All samples were taken within 2 min of cage disturbance in order to avoid artifactually high hormone measurements (Keith, Winslow, & Reynolds, 1978).

Following centrifugation (1,000 × g, 20 min, 4 °C), 5 μl plasma was removed and diluted with deionized water. Samples were immersed in boiling water for 5 min in order to denature corticosterone binding globulin, which would compete with the antibody for binding to corticosterone. Corticosterone radioimmunoassay was adapted from a previously reported procedure (Keith et al., 1978) and used antibody from Ventrex (Portland, ME) and [125I]corticosterone from ICN Biomedicals (Costa Mesa, CA). Interassay and intraassay variabilities were both less than 10%.

Corticosterone data were analyzed using analysis of variance with the between-subjects factor mouse strain. Strain means were used in correlational and QTL analyses described below.

EtOH (Pharmco Products Inc., Norwalk, CT) was diluted in 0.9% NaCl to a final concentration of 20% v/v. This solution was administered intraperitoneally at a dose of 4 g/kg. This dose of EtOH is within the range shown previously to produce a mild state of physical dependence as defined by measurable withdrawal convulsions induced by handling (Goldstein, 1972; Kosobud & Crabbe, 1986). Injections occurred between 8 a.m. and 9 a.m.

Handling-induced convulsion scoring is a sensitive procedure for detecting drug withdrawal hyperexcitability (Goldstein, 1973). This procedure involves lifting the mouse by the tail, gently twisting the tail so that the mouse rotates, and observing convulsions. Convulsions are scored on the basis of stimulation required to produce a convulsion and the severity of the convulsion elicited. The scale, which ranges from 0 to 7, has been published previously (Crabbe, Merrill, & Belknap, 1991). Typical scores range from 0 to 5: Scores of 1 to 3 require a gentle spin, whereas scores of 4 and 5 only require tail lift to elicit tonic or tonic-clonic convulsions.

The QTL mapping procedure followed that described previously (Gora-Maslak et al., 1991). Differences in marker loci alleles in the BXD RI strains were correlated with variability in corticosterone measurements. Each BXD strain has been typed for whether it has the B6 or D2 allele at most marker loci. B6 alleles have been given arbitrarily the numerical value of 0 and D2 alleles have been given the numerical value of 1. Therefore, means obtained from behavioral tests can be correlated with allelic patterns across all BXD RI tested. A significant positive correlation between a trait and a marker locus indicates an association between the D2 allele and high levels of the trait, whereas a significant negative correlation indicates an association between the B6 allele and high levels of the trait.

Because each behavioral trait was compared with each marker locus, a large number of correlations were performed. This greatly increases the possibility of Type I errors. However, as this analysis is considered a screening process, we wanted to avoid ruling out QTL that may be important (Type II errors). Therefore, markers that correlated with a corticosterone measure were listed if they were significant at the p < .01 level and occasionally at the p < .05 level if they were also significantly correlated with another corticosterone measure. The identified QTL should be regarded as provisional until tested in a second genetic model (e.g., B6D2 F2; Crabbe et al., 1994).

The total amount of genetic variance accounted for by the significant QTL associations for each trait was estimated by multiple regression analysis. This analysis takes into account intercorrelations between marker loci to more accurately estimate the percentage of the genetic variance accounted for by all identified QTLs. In this way, this procedure provides a more accurate estimate of the number of QTL contributing to the genetic variation for which the marker set accounts.

Mice in this study were used simultaneously to examine acute EtOH withdrawal severity in addition to corticosterone levels. The withdrawal data have been published (Belknap et al., 1993). Before EtOH or saline was administered, mice were scored for handling-induced convulsions. Mice were then injected intraperitoneally with either 4 g/kg EtOH or saline and were immediately returned to their home cages and left undisturbed for 1 hr. At this time, tail blood was sampled for corticosterone determinations. A total of 195 mice were sampled for corticosterone levels following EtOH (5 to 15 mice per strain; average = 9), and 115 mice were sampled following saline (3 to 9 mice per strain; average = 5). Nineteen BXD RI strains plus B6 and D2 mice were tested in this experiment. Data were collected over six experiments.

The results of this experiment are shown in Figure 1. There was a significant effect of strain on plasma corticosterone levels 1 hr following EtOH, F(20, 174) = 2.79, p = .0002. The top panel shows the strain distribution for this measure in ascending order with respect to plasma corticosterone levels. There was not a significant effect of strain on plasma corticosterone levels following saline, F(20, 94) = 1.12, p = 0.34. The bottom panel shows the strain distribution pattern, ordered as in the top panel, for this measure. It should be noted that even if strains with sample sizes less than 5 were excluded from the analysis, the effect of strain was not significant. A nonsignificant effect of strain suggests that the genetic influence over this trait in these mice is negligible; therefore, no further genetic analyses were performed on these data. As is apparent from inspection of Figure 1, there was no significant correlation between corticosterone levels following EtOH and saline.

Examination of the top panel of Figure 1 suggests that corticosterone levels 1 hr following EtOH may be bimodally distributed. A statistical approach was used, therefore, to more fully examine the distributions of these traits (Belknap et al., 1992). K-means cluster analysis (SYSTAT) was used to assign strains to two clusters, maximizing between-cluster and minimizing within-cluster variability. The criteria for accepting each cluster as a mode reflecting an allele at a major gene are as follows: (a) The B6 and D2 strains must be in separate modes, and (b) the number of strains in each cluster must be approximately equal. These criteria were met for corticosterone levels 1 hr following EtOH, but not corticosterone levels 1 hr following saline. Furthermore, mean (Y-intercept) estimates from the regression of each cluster were significantly different (t = 7.84, p < .005, df = 19), suggesting that they were drawn from two different distributions (bimodal) rather than one (unimodal). This further supports the suggestion that a single gene accounts for most of the genetic variability in the corticosterone response measured 1 hr following 4 g/kg EtOH.

In this experiment, the same mice were used to obtain corticosterone levels following EtOH and 1 week later, saline. This allowed us to analyze the corticosterone response to EtOH by subtracting the corticosterone response to saline. We predicted that this may give us a more precise index of response to EtOH by taking into account possible differences in baseline corticosterone levels and the effects of handling and injecting associated with the experiment. All mice received an injection of 4 g/kg EtOH and were returned to their home cages for 6 hr. At this time tail blood was collected for corticosterone determinations. Mice were then allowed to recover undisturbed except for routine husbandry for 1 week. Saline was then administered to all of the mice, and again tail blood was taken at 6 hr.

A total of 179 mice were used in this experiment. Twenty-one BXD RI strains plus B6 and D2 mice were tested. Data were collected over three experimental passes. The results are shown in Figure 2. There was a significant effect of strain on corticosterone levels 6 hr following EtOH, F(22, 156) = 2.7, p < .001. These data are shown by strain in ascending order in the top panel of Figure 2. There was also a significant effect of strain on corticosterone response 6 hr following saline, F(22, 156) = 2.4, p < .001. These data are shown in the bottom of Figure 2. The line graph shows adjusted means for the corticosterone response to EtOH using each animal's corticosterone response to EtOH minus its corticosterone response to saline. There was also a significant strain effect on these adjusted scores, F(22, 156) = 2.4, p < .01. Corticosterone levels following EtOH and saline were not significantly correlated; however, the corticosterone response to EtOH and the adjusted response to EtOH were, r = 0.8, p < .001. This suggests that the impact of the handling and injection 6 hr previously was not great enough to significantly impact the corticosterone response to EtOH. Distributions of these traits (not shown) are unimodal, indicating that they are polygenically determined.

In this experiment, mice were scored for baseline handling-induced convulsions and injected with 4 g/kg EtOH. At 2, 4, 5, 6, and 7 hr following EtOH all mice were again scored for handling-induced convulsions. Immediately following the final round of scoring, mice were rapidly decapitated and trunk blood was collected. Blood was collected in this manner in order to assay another hormone (not reported here). The 7-hr time point was chosen (as opposed to 6 hr as in the previous experiment) in order to ensure that peak handling-induced convulsions were scored prior to blood sampling.

A total of 255 mice of 23 BXD strains plus B6 and D2 progenitors were tested in this experiment. These data were collected over six experiments. The results are shown in Figure 3. There was a significant effect of strain on corticosterone levels 7 hr following EtOH in mice scored for handling-induced convulsions, F(24, 230) = 4.9, p < .0001. These data are shown in ascending order with respect to plasma corticosterone levels in the top panel of Figure 3. This trait was also continuously distributed, indicating polygenic influences.

The bottom panel of Figure 3 shows 7-hr handling-induced convulsion scores (using the same strain order as the top panel). There was a significant effect of strain on this measure, F(24, 230) = 12.0, p < .0001. The handling-induced convulsion time course following 4 g/kg EtOH in B6 and D2 mice has been previously published (Roberts et al., 1992), and handling-induced convulsions following EtOH in BXD mice have been more closely examined elsewhere (Belknap et al., 1993); therefore, the data are not presented in detail. However, it is noteworthy that the present 7-hr convulsion data correlated significantly with peak acute EtOH withdrawal handling-induced convulsion scores reported previously (r = 0.75, p < .001).

Mean corticosterone levels obtained 7-hr post-EtOH did not correlate significantly with the 6-hr post-EtOH data. This suggests that these traits do not share a significant degree of common genetic determination. Although this result seems rather odd, it may be accounted for by the differences between experimental designs. For example, the mice used for the 7-hr corticosterone determinations were handled repeatedly by the tail, producing convulsions in a subset of strains. This handling is likely to alter plasma corticosterone levels. In fact, corticosterone levels during withdrawal are generally higher in animals experiencing repeated handling-induced convulsions versus unhandled animals. This difference is apparent only in strains that display convulsions (D. A. Finn, personal communication). In addition, there was an hour between the sampling times in the two experiments. Although both of these time points are associated with acute withdrawal symptoms and elevated corticosterone levels, it is possible that the strains differ with regard to the time course of withdrawal corticosterone levels.

Correlational analyses using Pearson's r statistic were performed between the traits analyzed presently and traits associated with EtOH drinking, acute and chronic EtOH effects on locomotion, and EtOH withdrawal severity. Significant genetic correlations are listed in Table 1. Although correlations that were hypothesized to be significant, such as those between corticosterone levels during acute withdrawal and withdrawal convulsion severity, were not significant, several are quite interesting. Because many correlations were examined, the following should be seen as suggestive. For example, the acute stress response to EtOH (1-hr post-EtOH) is negatively correlated with consumption of 3% EtOH. This suggests that a lesser stress response to EtOH is associated with increased consumption of this drug. Corticosterone levels (and adjusted corticosterone levels) 6-hr post-EtOH correlated with the locomotor response to 2 g/kg EtOH. This implies that a relationship between the stress and locomotor responses to this drug exists; however, the differences between these experiments in time following EtOH and dose of EtOH make an interpretation of this relationship difficult. It is interesting that corticosterone levels 6 hr following saline, probably reflecting baseline stress axis activity, correlated with several measures. The corticosterone response 7 hr following EtOH did not correlate significantly with any of the EtOH-related measures used in these analyses.

Marker loci significantly correlated with each trait are listed in Table 2. The most significantly correlated marker within each 10 centimorgan (cM) region is shown, with the exception of a few additional significant markers that were found to be associated with other EtOH-related traits. Only a small region of chromosome 5 significantly correlated with corticosterone levels 1 hr following EtOH. This is consistent with the notion of a major single gene effect as suggested by the bimodal frequency distribution. Corticosterone levels 1 hr following saline were not used in QTL analysis because there was no significant effect of strain on this trait.

Corticosterone levels 6 hr following EtOH provisionally mapped to loci found on chromosomes 3, 7, 17, and 18. The marker on chromosome 7 (D7Mit7) was shown to be significantly correlated with the degree of taste aversion developed to 4 g/kg EtOH (Crabbe et al., 1994). In addition, the location of the marker on chromosome 18 is within a region shown to correlate with place preference and locomotor activity produced by 2 g/kg EtOH (Cunningham, 1995). Perhaps genes in these regions have a modulatory role in EtOH's reinforcing effects. The loci on chromosomes 7 and 17 were positively correlated with corticosterone levels 6-hr postsaline. Genes in these regions may be involved in responsiveness to various stressors. In addition to the marker loci on chromosomes 7 and 17, one locus on chromosome 1 correlated significantly with the corticosterone response 6 hr following saline. The adjusted means for 6-hr corticosterone levels provisionally mapped to markers on chromosomes 9, 17, and 18. The marker on chromosome 9 is in the same region found in association with both acute and chronic EtOH withdrawal severity (Crabbe et al., 1994; Crabbe et al., 1983), and the markers on chromosomes 17 and 18 were the same as were mapped by QTL analyses of acute EtOH-stimulated locomotor activity (Phillips et al., 1995). Although there was not a significant genetic correlation between 6-hr adjusted corticosterone levels and EtOH withdrawal severity, there does appear to be overlapping genetic influences on these traits.

Corticosterone levels determined 7 hr following EtOH provisionally mapped to a large region of chromosome 1 as well as markers on chromosomes 3, 4, and 5. The region of chromosome 1 spans about 35 cM and thus potentially contains more than 1 gene contributing to the trait. Only one marker within each 10 cM span and few others of special interest are shown. In addition, markers within this region were found to be associated with acute EtOH withdrawal severity (DOByu26, D1Byu7, and D1Byu3). A region at approximately 90 cM on chromosome 1 (including D1Byu3 and D1Byu8) is correlated with chronic EtOH withdrawal severity (Crabbe et al., 1994; Crabbe et al., 1983). This suggests that although the genetic correlation between corticosterone levels during acute withdrawal and withdrawal severity did not significantly correlate, they may share minor genes in common.

The results of multiple regression analyses are shown in Table 3. Table 3 shows percentages of genetic variation accounted for by the significant gene markers (from Table 2), estimated number of QTL accounting for this variation, and coefficients of genetic determination. Coefficients of genetic determination are derived from sums of squares data provided in the analysis of variance performed for each trait. It appears that 24–34% of the variation in these traits can be explained by genetic differences. Furthermore, these results indicate that 43 to 78% of this genetic variation can be accounted for by the marker loci obtained from the QTL analyses. In fact, 1 to 3 QTL appear to explain this genetic variation.

The goal of the present experiments was to determine corticosterone responses to acute EtOH using a genetic animal model that permits identification of regions of chromosomes that potentially contain genes involved in determining trait variation. Furthermore, we hypothesized that the possible importance of corticosterone in modulating EtOH-related traits would result in overlap in genetic determination between corticosterone responses and other responses to EtOH. Here we have reported plasma corticosterone levels 1, 6, and 7 hr (with handling-induced convulsions scored) following acute EtOH and levels 1 hr and 6 hr following saline in 19–23 BXD RI strains. In addition, genetic correlations and QTL analyses have been performed. Provisional QTLs are specific to differences between D2 and B6 mouse strains and therefore do not necessarily account for all of genetic influence over these traits.

A single gene locus may account for a large amount of the genetic variability in corticosterone levels measured 1 hr following 4 g/kg EtOH. This is supported by the apparent bimodal nature of the distribution of this trait along with the localization of the B6 and D2 progenitor strains in separate modes. In addition, a single 5 cM region of chromosome 5 was mapped for this trait, suggesting that a gene in this region accounts for a significant proportion of the genetic trait variability. The results of the multiple regression analysis for this trait indicated that 43% of the variation between the strains examined can be accounted for by 1 QTL. This percentage is lower than would be expected for a single major gene effect; however, it is possible that the modulating gene is some distance away from the marker locus.

The other traits presently measured were found to be polygenic in nature, suggested by their continuous distributions (data not shown). Several QTLs were identified for each of these traits. Multiple regression analysis, which tests for significant changes in the explained variance with different combinations of the significant marker loci, predicted that 1 to 3 QTLs accounted for 43 to 78% of the genetic variability in these traits. These gene number estimates may be low with respect to the total number of genes influencing trait variability. Important genes are potentially located in chromosomal regions that have not yet been extensively mapped.

The markers that were associated with these traits are not closely located to any neurotransmitter receptor gene or other candidate genes yet mapped. In the future it may be possible to repeat QTL analyses and identify new markers and possibly candidate genes accounting for a greater proportion of the genetic variability. Corticosterone's classical effects are mediated by way of binding to intracellular receptors, translocation of the activated receptor to the nucleus, and binding of specific response elements on DNA, leading to alterations in the transcription of genes (Carson-Jurica, Schrader, & O'Malley, 1990; O'Malley & Tsai, 1992). The QTL analysis technique offers a vehicle by which genes under the control of corticosterone may be discovered.

There were several interesting genetic correlations involving corticosterone responses and other EtOH-related traits. For example, there appeared to be a negative association between the corticosterone response to a high dose of EtOH and consumption of EtOH (Phillips et al., 1994). This suggests that a large stress response to EtOH in a particular individual may be associated with avoidance of EtOH. Corticosterone responses 6 hr following EtOH were correlated with locomotor activity following EtOH. Although there is evidence in the literature suggesting a modulatory role for corticosterone in open field activity (Veldhuis, de Kloet, Van Zoest, & Bohus, 1982), it should be noted that 4 g/kg EtOH was administered in the present experiments and 2 g/kg EtOH was used for the studies of locomotion (Phillips et al., 1995). This lower dose of EtOH results in locomotor activation in many mouse strains, whereas the higher dose used presently produces marked sedation. Several traits were genetically correlated with corticosterone levels 6 hr postsaline. This suggests that the tone of the animal, reflected by these presumably basal levels of corticosterone, is partly responsible for the nature of drug responses.

There were several instances of overlapping QTLs between the traits measured herein and other EtOH-related traits. QTLs identified for the corticosterone response 6 hr post-EtOH overlapped with those correlating with high-dose EtOH-induced taste aversion and low-dose EtOH-induced place preference and increased locomotor activity. Difference scores mapped to markers overlapping those found in association with acute and chronic EtOH withdrawal severity and acute EtOH locomotor stimulation. Taste aversion, place preference, and locomotor activation are believed to measure hedonic drug effects; therefore, it is possible that genes in the regions of these QTL are involved in the reinforcing effect of EtOH. The corticosterone response to EtOH may modulate the hedonic properties of this drug. In addition, the overlap with chronic EtOH withdrawal severity suggests a possible relationship between corticosterone levels during withdrawal and the severity of withdrawal convulsions.

QTLs identified for corticosterone responses 7 hr following EtOH in mice tested for handling-induced convulsions also were shown to be associated with acute and chronic EtOH withdrawal severity. This suggests that a relationship exists between corticosterone levels during withdrawal and withdrawal severity. This is consistent with previously published data showing that decreasing endogenous corticosterone levels during withdrawal decreases withdrawal severity (Roberts et al., 1991; Sze, 1977; Sze et al., 1974), whereas increased corticosterone levels are associated with exacerbated withdrawal convulsions (Roberts et al., 1991; Roberts et al., 1994). There is also accumulating evidence for a relationship between corticosterone levels and susceptibility to other types of convulsions (Roberts & Keith, 1994).

The results of these experiments indicate that there are significant genetic influences on stress responsiveness. Genetic correlations and overlapping QTL with EtOH-related traits suggest that the corticosterone response to EtOH may play a modulatory role in this drug's effects. One of the compelling characteristics of genetic analyses of traits using RI strains is their cumulative and integrative nature (Gora-Maslak et al., 1991). Data collected at different locations and at different times can be compared with those existing, so that the possibility of discovering new commonalities in genetic determinants continues. In addition, as marker loci accumulate and locations of genes with known function are mapped, insight may be gained into the mechanisms of corticosterone effects on EtOH-related as well as other traits.

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Submitted: October 13, 1994 Revised: March 23, 1995 Accepted: June 12, 1995