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Selection for pentobarbital withdrawal severity: correlated differences in withdrawal from other sedative drugs
Brain Research 1009 (2004) 17 – 25 www.elsevier.com/locate/brainres
Research report
Selection for pentobarbital withdrawal severity: correlated differences in withdrawal from other sedative drugs Christopher L. Kliethermes *, Pamela Metten, John K. Belknap, Kari J. Buck, John C. Crabbe Department of Behavioral Neuroscience, Oregon Health & Science University and the Portland Alcohol Research Center, Veterans Affairs Medical Center, c/o Portland VA Medical Center, R&D 12, 3710 SW US Veterans Hospital Road, Portland, OR 97239, USA Accepted 21 February 2004
Abstract In mice, withdrawal from agents that depress central nervous system function, such as barbiturates and benzodiazepines, results in the production of a withdrawal syndrome, one feature of which is increased severity of handling induced convulsions (HICs). High and Low Pentobarbital Withdrawal mice (HPW and LPW) were selectively bred to display severe and mild pentobarbital withdrawal HICs, respectively. These mice provide a valuable means to assess genetic correlations between withdrawal from pentobarbital and other sedative agents. We tested HPW and LPW mice for severity of HICs elicited during withdrawal from ethanol, diazepam, and zolpidem, and measured consumption of and preference for pentobarbital solutions in HPW and LPW mice. HPW mice displayed greater HICs than LPW mice during ethanol and zolpidem withdrawal, but differed less robustly during diazepam withdrawal. LPW mice consumed more pentobarbital in a solution of a moderate concentration than did HPW mice, but did not consume more pentobarbital at a higher or lower concentration. These results indicate that some of the same genes that affect the severity of withdrawal from pentobarbital also influence ethanol and zolpidem withdrawal, but that diazepam withdrawal may be less influenced by these genes. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Drugs of abuse: alcohol, barbiturates, and benzodiazepines Keywords: Pentobarbital withdrawal; Barbiturate; Benzodiazepine; Genetics
1. Introduction A primary feature of physical dependence on drugs that depress central nervous system activity in both humans and rodents is the presence of a withdrawal syndrome following cessation of drug administration. In general, the withdrawal syndrome is characterized acutely by anxiety, nausea, tremor, and convulsions, among other symptoms [21]. Similarities among the withdrawal syndromes seen from various sedative drugs (including alcohol, barbiturates, and benzodiazepines) imply that dependence on and withdrawal from these drugs involves similar genetic and neurobiological mechanisms. Clinically, the suggestion of shared neurobiology is supported by the frequent prescrip* Corresponding author. Tel.: +1-503-220-8262x56675 (lab), +1-503220-8262x54392 (office); fax: +1-503-721-1029. E-mail address: [email protected] (C.L. Kliethermes). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.040
tion of benzodiazepines during the acute stages of alcohol withdrawal [21,26]. This treatment is particularly efficacious in reducing both anxiety and incidences of convulsion in withdrawing individuals, and suggests that a primary mechanism of withdrawal from alcohol involves the gamma-aminobutyric-acid A (GABAA) system. Preclinically, the development of tolerance to a given sedative agent can confer cross-tolerance to other sedatives (see Ref. [16]). However, cross-tolerance does not necessarily extend among all classes of sedative drugs or members of a given class of sedatives [16 – 18,20]. These findings suggest that even though physical dependence displays certain similarities across sedatives, some differences in the mechanisms of tolerance and/or dependence specific to each drug or type of drug may exist. Historically, the primary means of quantifying physical dependence in rodent models has been to follow the withdrawal symptoms following termination of sedative
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administration. A particularly robust characteristic of withdrawal in mice is the handling-induced convulsion (HIC) [14,15]. HICs occur normally during routine handling, but are exacerbated during withdrawal from sedatives. As with clinical symptoms of withdrawal, benzodiazepine administration during withdrawal from alcohol reduces the severity of HICs [8], which suggests that the HIC is a valid index of withdrawal severity from ethanol. Inbred strains of mice differ markedly in the expression of HICs during withdrawal from ethanol, and the severity among inbred strains correlates with those seen in withdrawal from diazepam and pentobarbital, which suggests that withdrawal from the three drugs share some common genetic mechanisms [22,23]. This suggestion is further supported by the observation that mice selectively bred to be either sensitive or resistant to HICs during withdrawal from chronic ethanol administration display correlated differences in withdrawal from barbiturates and diazepam [1,2,10]. In a more recent study, mice selected for High and Low acute Alcohol Withdrawal severity (HAW and LAW, respectively) after acute ethanol administration also differed in severity of acute pentobarbital, diazepam, zolpidem, and nitrous oxide withdrawal [24]. These instances of genetic correlation among severity of withdrawal from ethanol, pentobarbital, and diazepam suggest that some genes act to enhance or reduce more than one of these withdrawal responses, a condition called pleiotropy. The HAW/LAW selective breeding project was performed to refine regions within quantitative trait loci (QTL) influencing alcohol withdrawal [6]. Each QTL is a site on a chromosome harboring a gene or genes affecting the trait. Some QTLs originally shown to affect alcohol withdrawal severity were subsequently shown to influence withdrawal from pentobarbital [7,13]. While the specific gene or genes remain unidentified, on the whole, it appears likely that withdrawal from pentobarbital, diazepam, and ethanol involve similar mechanisms regulated in part by a common set of genes (QTLs). In a meta-analysis of studies using many mouse genetic models, a substantial negative genetic correlation was seen between ethanol withdrawal severity and ethanol consumption [25], indicating that some genes that regulate withdrawal severity exert an opposite effect on the voluntary ingestion of ethanol. This inverse relationship has also been demonstrated in mice lacking the adenosine A2A receptor, in that these mice exhibit increased ethanol consumption [27] and decreased HICs during ethanol withdrawal [12] relative to wildtypes. However, in other experiments with mice lacking either a specific potassium channel subtype [3] or one of two GABAA receptor subunits [4], no relationship between ethanol preference and severity of withdrawal was observed when comparing either null mutant mouse model to the respective wildtype. Combined, these previous results provide direct evidence for the role of specific genes in both ethanol preference and withdrawal. Furthermore, while these studies indicate that some genes influence both phenotypes, they also suggest that not all individual genes
regulating ethanol withdrawal are identical to those regulating preference for ethanol. High and Low Pentobarbital Withdrawal mice (HPW and LPW) mice were selected for severe and mild severity of convulsions, respectively, elicited during withdrawal from an acute injection of pentobarbital [7]. Similar to the HAW and LAW selection, HPW and LPW mice were developed to reduce the size of QTL regions associated with pentobarbital withdrawal and as an aid to identifying the genes responsible [6,7]. After one generation of selective breeding, HPW mice exhibited significantly higher pentobarbital withdrawal HICs than LPW mice. By the fourth generation of selection, HPW mice exhibited more than three-fold greater withdrawal severity than LPW mice [7]. Because of these profound genetic differences in withdrawal from pentobarbital, these mice provide an alternative means to evaluate genetic correlations seen with the lines of mice selected for divergence in ethanol withdrawal. If HPW and LPW mice display correlated differences in withdrawal from ethanol and benzodiazepines, this observation would further support the idea that the mechanisms of barbiturate dependence and withdrawal are genetically similar to those seen with other sedative drugs. To address this question, we tested HPW and LPW mice for severity of HICs elicited following administration of ethanol, diazepam, and zolpidem. In addition, pentobarbital consumption and preference were measured in these selected lines of mice in order to determine if the negative genetic correlation between ethanol withdrawal severity and ethanol consumption seen by Metten et al. [25] would extend to pentobarbital.
2. Materials and methods 2.1. Subjects HPW and LPW mice were selectively bred for severity of pentobarbital withdrawal HICs as previously described [7]. These mice were selected from an F2 cross of the inbred strains C57BL/6J, which exhibit mild withdrawal from several sedatives, and DBA/2J, which demonstrate robust withdrawal from the same sedatives [22,23]. Mice demonstrating the highest withdrawal convulsions following an acute administration of pentobarbital were bred together to form the HPW line, while those exhibiting the lowest formed the LPW line. For the present experiments, naı¨ve mice from the second and fourth selection generations (S2 and S4) were tested. All mice were housed at the Portland Veterans Affairs Medical Center Veterinary Medical Unit with water and food available continuously. Mice were housed two to four per cage with corncob bedding on a standard 12:12-light/dark cycle (lights on at 0600), and were aged 50– 80 days at the time of testing. Equal numbers of HPW and LPW mice were tested for correlated differences in acute withdrawal from ethanol (4 g/kg), diazepam (20 mg/kg), and zolpidem (20 mg/kg). Due to limitations in the
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number and sex of mice available for testing, the experiments with ethanol and diazepam used male and female mice, while zolpidem withdrawal was measured in female mice only.
dem withdrawal HICs were measured every 15 min after a single injection up to 120 min post injection, and then at 150 and 180 min.
2.2. Acute withdrawal
2.3. Pentobarbital drinking
Withdrawal was assessed using the HIC scale [9], which is a modified version of the scale used by Goldstein and Pal [15]. The procedure and HIC scale used for the present experiments were identical to that used for the selection of HPW and LPW mice [7]. Briefly, a score from 0 (no convulsion) to 7 (spontaneous, lethal tonic – clonic convulsion) is assigned depending on the severity of the convulsion elicited after the mouse is picked up by the tail and if necessary, gently spun in a 180j arc. Following two baseline HIC assessments, withdrawal was measured at multiple time points following a single injection of either vehicle or drug.
We assessed oral consumption of three concentrations of pentobarbital in naı¨ve female HPW and LPW mice of the S4 generation (n=15 each) using a two bottle choice procedure. All mice were singly housed and allowed to drink from two tubes containing tap water for 4 days prior to the introduction of pentobarbital to one of the tubes. The mice were then exposed for 4 days each to three ascending concentrations of pentobarbital (250, 500, and 1000 mg/l pentobarbital, all in a 2 g/l saccharin solution) in one of the tubes, which was alternated between the left and right side every 2 days. All fluids were available constantly and volumes consumed were recorded daily. The total amount of pentobarbital consumed at each concentration (mg/kg/day) and average daily preference for pentobarbital relative to water [ml pentobarbital/(ml pentobarbital+ml water)] were calculated based on the volumes consumed and mouse weight, determined prior to the introduction of each of the three concentrations of pentobarbital.
2.2.1. Ethanol Male and female HPW and LPW mice (n=14/line/sex) of the S2 generation were tested for acute withdrawal following intraperitoneal injection of 4 g/kg ethanol (20% v/v). HICs were scored at 2 h, and then every hour from 4 to 10 h after injection. 2.2.2. Diazepam Diazepam, a relatively long-lasting drug, suppresses HIC acutely, but does not result in a subsequent waxing and waning pattern of HIC exacerbation after injection [8]. However, if the drug is antagonized at the receptor by injection with flumazenil, a brief, relatively intense withdrawal reaction is precipitated, lasting for several minutes after flumazenil injection [22,23]. In both the S2 and S4 generations, diazepam withdrawal was precipitated by an injection of 10 mg/kg flumazenil 60 min after injection of 20 mg/kg diazepam or vehicle in male and female mice. Both diazepam and flumazenil were prepared in saline to which 1 drop of Tween 80 per 5 ml total volume was added, and the vehicle was prepared identically except for the addition of the drug. For both experiments, HICs were measured at 30 and 55 min after the diazepam injection to establish that diazepam had exerted its anticonvulsant effect on HICs. Five additional HIC assessments were then made at 1, 3, 5, 8, and 12 min following flumazenil injection [22 – 24]. In the S2 generation, 14 –16 mice per line (7– 8 of each sex) received diazepam, while 5 – 7 per line and sex were injected with vehicle. When reassessed in generation S4, 11 HPW and 10 LPW (4 –7 per sex) received diazepam, and 4 – 6 mice per line per sex received a vehicle injection. The vehicle-injected group was included as a control for the mild anticonvulsant effects of flumazenil [23]. 2.2.3. Zolpidem Acute withdrawal from zolpidem, which does not require precipitation by an antagonist [24], was measured in female HPW and LPW mice from generation S4 (n=8/line). Zolpi-
2.4. Statistical analyses In all HIC experiments, the two baseline HICs in all experiments were averaged to serve as an index of basal HIC susceptibility, and peak withdrawal and corrected peak withdrawal after the injection of a drug are reported. For the ethanol and zolpidem experiments, peak withdrawal was defined as the average of the three highest consecutive HIC scores, and corrected peak withdrawal was calculated as the difference between the peak withdrawal score and the average baseline for each subject. For the diazepam experiments, a peak withdrawal score was calculated as the average of the three highest consecutive HIC scores [14], from which the average score of the vehicle-treatment group over the same time points was subtracted [23,24]. When they occurred, negative individual corrected peak withdrawal scores (corrected either for baseline or vehicle group, as appropriate) were converted to zero. In the ethanol and zolpidem experiments, area under the withdrawal curve (AUC) was calculated as the difference between baseline HIC and the sum of all HICs elicited during withdrawal, corrected for differences in time intervals between each assessment. AUC for diazepam withdrawal was calculated similarly, except that the withdrawal scores were corrected for the vehicle response over the time course of withdrawal. Selected line differences in average baseline and peak withdrawal were analyzed by analysis of variance (ANOVA), with line as a factor in all experiments, and sex included as a factor where appropriate. Significant interactions were analyzed by Tukey’s HSD post hoc test. Differences in pentobarbital consumption and preference
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were analyzed by repeated measures analysis of variance. Significant main effects and interactions in this experiment were analyzed by simple main effects analysis. All results were considered significant at p<0.05. In cases where significance was not attained, exact p values are given to provide some indication of the strength of the trends observed. Based on the literature reviewed above, we hypothesized that pentobarbital withdrawal severity would be positively correlated with the withdrawal severities of the other drugs tested. Therefore, we used one-tailed tests for these comparisons. Pentobarbital consumption was analyzed using twotailed tests. We also report the proportion of the total variance due to the selection line differences in order to better compare magnitudes of correlated responses across the drugs tested. This was quantified as R2 from a one-way ANOVA by line.
3. Results With the exception of the zolpidem withdrawal experiment, using AUC as the dependent variable produced equivalent statistical outcomes to those obtained using peak withdrawal. Therefore, only peak and corrected peak withdrawal scores are shown for the other drugs for ease of comparison with previous studies that used these same drugs and indices [22 –24]. 3.1. Ethanol No sex differences or line by sex interactions were observed for either average baseline or peak withdrawal from ethanol, so the data presented are collapsed on sex. As shown in Fig. 1A, the lines did not differ in baseline HIC and both lines responded to ethanol with complete suppression of the HIC at hour 2. Large differences in HIC scores between HPW and LPW mice were apparent by hour 6 that persisted to hour 10, with average peak withdrawal occur-
ring at hour 8. Interestingly, LPW mice appeared to return to baseline HIC levels by hour 8, but tended towards lower scores at hours 9 and 10. At peak withdrawal, HPW demonstrated three-fold greater HIC severity than LPW mice ( F1,54=35.45; p<0.0001). This difference was still present when corrected for baseline HIC ( F1,54=37.95; p<0.0001; Fig. 1B). This corrected line difference for EtOH withdrawal accounted for 41% of the total variation observed in the S2 generation. 3.2. Diazepam Diazepam withdrawal was first measured in the S2 generation. In this experiment, HPW mice demonstrated higher basal HICs than LPW mice ( F1,50=5.67; p<0.05; data not shown), and male mice of both genotypes exhibited significantly higher basal HICs than did female mice ( F1,50=4.80; p<0.05; data not shown). A significant interaction between selected line and sex was also observed ( F1,50=1.73; p<0.05), in that HPW male mice exhibited significantly higher basal HICs than HPW females, and both LPW male and female mice ( p<0.05; Tukey’s post hoc). Diazepam reduced HIC scores at 30 and 55 min after injection, and when precipitated by flumazenil, peak withdrawal in both lines occurred 1 min later. However, no line differences were observed for peak withdrawal ( F1,26=2.49; p=0.67) or peak withdrawal corrected for the vehicle group ( F1,26=0.10; p=0.38). The corrected line difference for DZ withdrawal accounted for less than 1% of the total variation seen in the S2. In addition, no sex differences or line by sex interactions were observed for either variable (all F’s<1.87; p>0.05). When reassessed in generation S4, no sex difference in basal HIC ( F1,32=0.08; p>0.05), or line by sex interaction for basal HIC were observed ( F1,32=1.65; p>0.05). As in generation S2, HPW mice exhibited higher basal HICs than did LPW mice ( F1,32=17.04; p<0.001; Fig. 2A), and HIC
Fig. 1. Acute withdrawal from ethanol in HPW and LPW mice of the S2 selection generation. (A) Time course of ethanol withdrawal handling induced convulsion (HIC) scores. (B) Peak withdrawal and corrected peak withdrawal from ethanol. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
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Fig. 2. Acute withdrawal from diazepam in HPW and LPW mice in generation S4. Group identification is shown in the legend. Flumazenil injection occurred at the 60-min time point (indicated by arrow). (A) Time course of diazepam withdrawal HIC scores. Minutes 61 – 72 represent minutes 1 – 12 after flumazenil injection. The x-axis scale is compressed up to 55 min to better demonstrate the withdrawal curve. (B) Peak and corrected peak withdrawal from diazepam in the S2 and S4 generations. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
scores for all mice were suppressed at 30 and 55 min after injection of diazepam. HIC scores of either HPW or LPW mice treated with vehicle followed by flumazenil did not change appreciably over the course of the experiment. Peak withdrawal in HPW mice occurred at 3 min, while LPW mice demonstrated highest HIC scores at 1 min after flumazenil injection. No sex differences or line by sex interactions were observed for peak or corrected peak withdrawal (all F’s<2.47; p’s>0.05), so for further analysis we collapsed on sex. Fig. 2B shows peak and corrected peak diazepam withdrawal severity in the S2 and S4 generations. When uncorrected for basal differences in HICs or the vehicle group response, peak withdrawal was higher in HPW than LPW mice in the S4 generation ( F1,19=9.40; p<0.01). When corrected for the response seen in vehicleinjected animals, a difference in peak withdrawal was observed that closely approached statistical significance ( F1,19=2.84; p=0.054). The corrected line difference for
DZ withdrawal accounted for 13% of the total variation observed in the S4 generation. 3.3. Zolpidem No basal differences in average baseline HIC were observed between the lines ( F1,14<1.0; Fig. 3A). After the initial suppression of HIC scores by zolpidem at 15, 30, and 45 min, HPW and LPW mice returned to basal levels of HIC susceptibility by 60 min. Peak withdrawal in HPW mice occurred at 120 min, and in LPW mice at 60 min, although the scores of LPW mice did not change appreciably after recovering to baseline over the entire course of withdrawal. HPW and LPW mice did not differ significantly in peak withdrawal ( F1,14=1.54; p=0.12), or peak withdrawal corrected for baseline HICs ( F1,14=0.95; p=0.36; Fig. 3B). The corrected line difference for zolpidem withdrawal accounted for 6% of the total variation seen in the S4. However, as
Fig. 3. Acute withdrawal from zolpidem in HPW and LPW mice of generation S4. (A) Time course of zolpidem withdrawal HIC scores. (B) Peak withdrawal and corrected peak withdrawal from zolpidem. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
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indicated by Fig. 3A, it appears that HPW and LPW mice may differ in withdrawal from zolpidem, but that this difference is obscured by high variation due to the small number of mice available, and the use of peak withdrawal to index the severity of withdrawal (see Discussion). Analysis of AUCs corrected for baseline HICs indicated that HPW mice exhibited higher withdrawal severity than LPW mice (HPW 6.5F2.3, LPW 1.6F1.0; F1,14=3.85; p<0.05; data not shown). This line difference accounted for 22% of the variation in AUC for zolpidem.
differ in pentobarbital consumption at either the 250 or 1000 mg/l concentrations (both F’s<1.08; p>0.05). Although the pattern of line differences in preference for pentobarbital shown in Fig. 4B appears similar to that seen in consumption of pentobarbital, these differences between HPW and LPW mice did not reach statistical significance ( F1,28=1.42; p=0.12). As concentrations of pentobarbital increased over the course of the experiment, preference declined ( F2,56= 23.73; p<0.0001), and this declining preference did not interact with line ( F2,56=2.13; p=0.13).
3.4. Pentobarbital drinking 4. Discussion Pentobarbital consumption changed as a function of concentration ( F2,56=6.07; p<0.01), and was greater in LPW than in HPW mice ( F1,28=5.71; p<0.05; Fig. 4A). The line difference for consumption accounted for 17% of the total variation in seen in the S4. A significant interaction between concentration of pentobarbital and line was observed ( F2,56=3.18; p<0.05), in that LPW mice consumed more of the 500 mg/l concentration than did HPW mice ( F1,28=10.07; p<0.01), but HPW and LPW mice did not
Fig. 4. Voluntary daily consumption of three concentrations of pentobarbital in HPW and LPW mice of generation S4. (A) Total consumption (mg/kg) of pentobarbital over four successive days at each concentration of pentobarbital. (B) Average daily preference for each concentration of pentobarbital.
Sedative drugs have complex effects on the central nervous system. For instance, administration of ethanol results in alterations in the function of many neurotransmitter systems, including glutamate, GABA, endogenous opioids, dopamine, and serotonin [11,19,28]. Chronic ethanol administration exacerbates these changes, and results in ethanol dependence [19]. As a result, severe HICs during ethanol withdrawal could result from alterations in any of a number of neurotransmitter systems, while resistance to withdrawal HICs could result from changes in either the same or different brain systems, each of which in turn is regulated by different underlying genotypes. Acute pentobarbital administration results in multiple changes in neurotransmission as well, although as with ethanol, the primary mechanism of pentobarbital’s sedative action is thought to be at the GABAA receptor (see Ref. [16]). Artificial selection for severity of withdrawal from either pentobarbital or ethanol could therefore result in changes in multiple brain systems that may or may not be similar between the two drugs. Other than two inbred strain experiments [22,23], all of the previous data examining genetic correlations between ethanol and barbiturate withdrawal were obtained from mice selectively bred for high and low severity of ethanol withdrawal. The present results are thus the first demonstration that artificial selection for withdrawal severity from pentobarbital results in genetically correlated differences in withdrawal from ethanol. HPW and LPW differed three-fold in peak withdrawal from ethanol, and over five-fold when corrected for average baseline HICs. These differences in withdrawal severity are comparable to those observed during selection for pentobarbital withdrawal (see Ref. [7]), and strongly suggest that the genes involved in severity of withdrawal from pentobarbital are similar to those implicated in ethanol withdrawal. In the S2 generation, HPW and LPW mice did not differ in corrected or uncorrected peak withdrawal from diazepam. At this generation of selection, significant differences between HPW and LPW mice were apparent in pentobarbital [7] and ethanol withdrawal severity (current results). Two generations later, the divergence in pentobarbital withdrawal severity was more striking, and HPW mice exhibited significantly higher peak diazepam withdrawal than did
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LPW mice, although not when corrected for the response of the control groups to flumazenil. In both the S2 and S4 generations, then, the magnitude of the line difference in diazepam withdrawal severity was smaller than that seen for ethanol, suggesting either that the genetic mechanism influencing diazepam withdrawal severity is not identical to that involved in ethanol and pentobarbital withdrawal, or that we did not have sufficient statistical power in these experiments to detect a difference that was real. A larger experiment might have produced a more clear result, but the availability of animals limited the size of the experiments to those reported here. Buck et al. [6] identified QTLs on mouse chromosomes 1, 4, and 11 that influence the severity of ethanol withdrawal as measured by HICs, and these QTLs were subsequently shown to influence pentobarbital withdrawal severity [5,7]. Using finer mapping techniques, Fehr et al. [13] narrowed the QTL region on chromosome 4, and identified Mpdz as a candidate gene that reduced the severity of ethanol and pentobarbital withdrawal in congenic mice. The congenic mice developed for this experiment express a segment of chromosome 4 from the withdrawal seizure resistant C57BL/6J strain against a seizure susceptible DBA/2J background. Since mice that possess this chromosomal interval exhibited less severe withdrawal HICs than the background DBA/2J strain, it is likely that the introgressed chromosomal interval contained either one or multiple gene variants that reduce the severity of ethanol withdrawal. We have begun comparing the chromosome 4 congenic mice developed by Fehr et al. [13] to the DBA/2J background strain, in order to test whether a pleiotropic QTL that also affects diazepam withdrawal exists there. Though preliminary, it does not appear that these congenic mice display reduced severity of diazepam withdrawal compared to the DBA/2J background strain. This result, combined with those reported here, suggests that withdrawal from diazepam is somewhat genetically distinct from that produced by ethanol or pentobarbital. This suggestion contrasts with data obtained from inbred strains and the lines selected for high and low ethanol withdrawal severity, which have demonstrated that withdrawal from all three sedatives is genetically similar [1,2,10,22 – 24]. However, many genes are likely to be responsible for any proposed pleiotropic effect, and selection may simply not have advanced far enough by S4 to capture the genes that also affect diazepam withdrawal. Indeed, congenic mice that harbor a segment on chromosome 1 that reduces ethanol withdrawal severity display reduced diazepam withdrawal severity (unpublished data), suggesting that at least some of the QTLs influencing ethanol and pentobarbital withdrawal also influence diazepam withdrawal. A barrier to interpretation of the diazepam experiments is that withdrawal scores (both corrected and uncorrected) were confounded with differences between HPW and LPW mice in basal HIC susceptibility in both experiments. This was an unexpected finding based on the selection
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method used for these lines of mice. HPW and LPW mice were selected on the basis of either a large positive or negative residual, respectively, from the regression of peak pentobarbital withdrawal HIC on the average baseline HIC [7]. By definition, this residual score has a correlation of zero with the baseline HIC. The mice produced from this selection could still demonstrate correlated differences in basal HIC, but this difference would be strictly due to a chance fixation of genes, and should be observed in most experiments with these mice. A correlated difference in basal HICs between HPW and LPW mice was not reported by Buck et al. [7], and the basal difference in HICs was not observed in the current experiments with zolpidem or ethanol. Considering that the same technician conducted all of the withdrawal studies presented here, the reason for correlated differences in baseline HICs in the two studies with diazepam is unclear. It is apparent that both selected lines experienced withdrawal from diazepam; however, the role that differences in general HIC susceptibility between HPW and LPW mice had in this withdrawal is less apparent. A tentative explanation of the results with diazepam could be that due to the unexpected correlated differences in basal HIC, insufficient statistical power was present to detect a difference between HPW and LPW mice. Unfortunately, these lines of mice are no longer maintained, so retesting HPW and LPW mice for correlated differences in diazepam withdrawal is impossible. However, it is clear that HPW and LPW mice differed more dramatically in severity of withdrawal from both pentobarbital and ethanol than from diazepam. This conclusion is also supported by the proportion of the variance due to the line differences, which was very much higher with ethanol (S2, 41%) than with diazepam (S2, <1%; S4, 13%). This indicates that pentobarbital and ethanol withdrawal have greater commonality in their genetic influences than do pentobarbital and diazepam withdrawal. Zolpidem has been previously shown to induce withdrawal reactions in mice over the same time course tested in the present experiments, and mice selected to differ in severity of ethanol withdrawal also differ in withdrawal from zolpidem [24]. Similar to diazepam, HPW and LPW mice did not differ in peak withdrawal severity from zolpidem. However, peak withdrawal from zolpidem, as described in the methods section, occurred at 60 min in LPW mice, but not until 120 min after injection in HPW mice. After the initial anticonvulsant effects of zolpidem, LPW mice had returned to basal HIC levels by 60 min after injection, and did not significantly change over the next 120 min. Therefore, LPW mice never displayed significant ‘‘withdrawal’’ from zolpidem (as indexed by HICs elevated over baseline), so the selection of a particular time point as being indicative of their peak withdrawal may have been misleading. Indeed, differences in AUCs between HPW and LPW mice attained statistical significance ( p=0.04, onetailed test), indicating that withdrawal from zolpidem may differ between the selected lines. Further, comparison of
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HPW and LPW mice at 120 min after injection, when HPW mice were experiencing peak withdrawal, suggests that correlated differences in zolpidem withdrawal may exist between HPW and LPW mice, but that the correlation is hidden in the high variability observed early in the withdrawal curve of LPW mice. In any case, the proportion of the variance due to line differences was much higher with ethanol (S2, 41%) than zolpidem (S4, either 6% or 22% depending on the method of analysis), indicating that pentobarbital and ethanol withdrawal have greater commonality in their genetic influences than do pentobarbital and zolpidem withdrawal. A meta-analysis of data collected in inbred strains and lines of mice selected for either ethanol withdrawal severity or ethanol preference indicates that a negative genetic correlation exists between the amount of ethanol voluntarily consumed and the severity of withdrawal [25]. That is, genes that increase the amount of ethanol a mouse will drink decrease the severity of the withdrawal reaction experienced by the mouse. Our results with HPW and LPW mice suggest that this inverse relationship exists with pentobarbital as well, since LPW mice voluntarily consumed more pentobarbital than did HPW mice. This finding implies that the genes influencing pentobarbital withdrawal are similar to those affecting consumption, and, since HPW and LPW mice display correlated differences in ethanol withdrawal, those genes that affect pentobarbital consumption and withdrawal severity may be similar to those influencing ethanol consumption and severity of withdrawal. Identification of the specific genes involved in this correlation would provide a clinically relevant target for therapies aimed at reducing the initiation of drinking, as well as novel therapeutics for the treatment of withdrawal. Interestingly, neither HPW nor LPW mice displayed an absolute preference (i.e., >50%) for any of the three concentrations of pentobarbital tested. This observation makes it unlikely that the mice were consuming the pentobarbital solutions merely for the taste of saccharin, a preferred taste, relative to water. It should be noted that preference for the drug solution declined with increasing concentrations of pentobarbital, raising the possibility that saccharin was less able to mask the taste of pentobarbital with increasing concentrations of the drug. In the absence of a saccharin control, it is difficult to determine which of these possibilities is correct. Some additional unpublished analyses from our laboratory indicate that across inbred strains, pentobarbital withdrawal severity was not correlated with saccharin consumption. In addition, in the current experiments, pentobarbital consumption (in mg/kg) tended to be higher with increasing concentrations of the drug. Since decreases in pentobarbital preference occurred at the higher solution concentrations, it is possible that the mice were attempting to titrate the total amount of pentobarbital consumed. The experiments presented here demonstrate a clear genetic correlation between severity of withdrawal from ethanol and pentobarbital, a finding consistent with previous
research [10,22 –24]. However, these correlated differences were not as strong for withdrawal from diazepam in HPW and LPW mice. The experiment with zolpidem suggests that a genetic correlation between pentobarbital and zolpidem withdrawal severity exists when an index is used that takes into account the entire course of withdrawal. Finally, the pentobarbital consumption experiment extends previous data demonstrating a negative correlation between ethanol withdrawal severity and consumption to include pentobarbital, by showing that LPW mice voluntarily consume more of the drug than do HPW mice. Further refinements of ethanol, pentobarbital, and diazepam withdrawal QTLs will elucidate the specific genes involved in the observed genetic correlations.
Acknowledgements The authors would like to thank Cathy Merrill and Charlotte Wenger for expert technical assistance. This research was supported by NIH grants AA10760, AA05828, AD05228, and AD06243, and by the Department of Veterans Affairs.
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[20] A.D. Leˆ, J.M. Khanna, H. Kalant, Effects of chronic treatment with ethanol on the development of cross-tolerance to other alcohols and pentobarbital, J. Pharmacol. Exp. Ther. 263 (1992) 480 – 485. [21] M.F. Mayo-Smith, Pharmacological management of alcohol withdrawal. A meta-analysis and evidence-based practice guideline. American Society of Addiction Medicine Working Group on Pharmacological Management of Alcohol Withdrawal, JAMA 278 (1997) 144 – 151. [22] P. Metten, J.C. Crabbe, Common genetic determinants of severity of acute withdrawal from ethanol, pentobarbital and diazepam in inbred mice, Behav. Pharmacol. 5 (1994) 533 – 547. [23] P. Metten, J.C. Crabbe, Genetic determinants of severity of acute withdrawal from diazepam in mice: commonality with ethanol and pentobarbital, Pharmacol. Biochem. Behav. 63 (1999) 473 – 479. [24] P. Metten, J.K. Belknap, J.C. Crabbe, Drug withdrawal convulsions and susceptibility to convulsants after short-term selective breeding for acute ethanol withdrawal, Behav. Brain Res. 95 (1998) 113 – 122. [25] P. Metten, T.J. Phillips, J.C. Crabbe, L.M. Tarantino, G.E. McClearn, R. Plomin, V.G. Erwin, J.K. Belknap, High genetic susceptibility to ethanol withdrawal predicts low ethanol consumption, Mamm. Genome 9 (1998) 983 – 990. [26] H. Myrick, K.T. Brady, R. Malcolm, New developments in the pharmacotherapy of alcohol dependence, Am. J. Addict. 10 (2001) 3 – 15 (Suppl.). [27] M. Naassila, C. Ledent, M. Daoust, Low ethanol sensitivity and increased ethanol consumption in mice lacking adenosine A2A receptors, J. Neurosci. 22 (2002) 10487 – 10493. [28] I. Nevo, M. Hamon, Neurotransmitter and neuromodulatory mechanisms involved in alcohol abuse and alcoholism, Neurochem. Int. 26 (1995) 305 – 336 (discussion 337-42).
Research report
Selection for pentobarbital withdrawal severity: correlated differences in withdrawal from other sedative drugs Christopher L. Kliethermes *, Pamela Metten, John K. Belknap, Kari J. Buck, John C. Crabbe Department of Behavioral Neuroscience, Oregon Health & Science University and the Portland Alcohol Research Center, Veterans Affairs Medical Center, c/o Portland VA Medical Center, R&D 12, 3710 SW US Veterans Hospital Road, Portland, OR 97239, USA Accepted 21 February 2004
Abstract In mice, withdrawal from agents that depress central nervous system function, such as barbiturates and benzodiazepines, results in the production of a withdrawal syndrome, one feature of which is increased severity of handling induced convulsions (HICs). High and Low Pentobarbital Withdrawal mice (HPW and LPW) were selectively bred to display severe and mild pentobarbital withdrawal HICs, respectively. These mice provide a valuable means to assess genetic correlations between withdrawal from pentobarbital and other sedative agents. We tested HPW and LPW mice for severity of HICs elicited during withdrawal from ethanol, diazepam, and zolpidem, and measured consumption of and preference for pentobarbital solutions in HPW and LPW mice. HPW mice displayed greater HICs than LPW mice during ethanol and zolpidem withdrawal, but differed less robustly during diazepam withdrawal. LPW mice consumed more pentobarbital in a solution of a moderate concentration than did HPW mice, but did not consume more pentobarbital at a higher or lower concentration. These results indicate that some of the same genes that affect the severity of withdrawal from pentobarbital also influence ethanol and zolpidem withdrawal, but that diazepam withdrawal may be less influenced by these genes. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Drugs of abuse: alcohol, barbiturates, and benzodiazepines Keywords: Pentobarbital withdrawal; Barbiturate; Benzodiazepine; Genetics
1. Introduction A primary feature of physical dependence on drugs that depress central nervous system activity in both humans and rodents is the presence of a withdrawal syndrome following cessation of drug administration. In general, the withdrawal syndrome is characterized acutely by anxiety, nausea, tremor, and convulsions, among other symptoms [21]. Similarities among the withdrawal syndromes seen from various sedative drugs (including alcohol, barbiturates, and benzodiazepines) imply that dependence on and withdrawal from these drugs involves similar genetic and neurobiological mechanisms. Clinically, the suggestion of shared neurobiology is supported by the frequent prescrip* Corresponding author. Tel.: +1-503-220-8262x56675 (lab), +1-503220-8262x54392 (office); fax: +1-503-721-1029. E-mail address: [email protected] (C.L. Kliethermes). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.040
tion of benzodiazepines during the acute stages of alcohol withdrawal [21,26]. This treatment is particularly efficacious in reducing both anxiety and incidences of convulsion in withdrawing individuals, and suggests that a primary mechanism of withdrawal from alcohol involves the gamma-aminobutyric-acid A (GABAA) system. Preclinically, the development of tolerance to a given sedative agent can confer cross-tolerance to other sedatives (see Ref. [16]). However, cross-tolerance does not necessarily extend among all classes of sedative drugs or members of a given class of sedatives [16 – 18,20]. These findings suggest that even though physical dependence displays certain similarities across sedatives, some differences in the mechanisms of tolerance and/or dependence specific to each drug or type of drug may exist. Historically, the primary means of quantifying physical dependence in rodent models has been to follow the withdrawal symptoms following termination of sedative
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administration. A particularly robust characteristic of withdrawal in mice is the handling-induced convulsion (HIC) [14,15]. HICs occur normally during routine handling, but are exacerbated during withdrawal from sedatives. As with clinical symptoms of withdrawal, benzodiazepine administration during withdrawal from alcohol reduces the severity of HICs [8], which suggests that the HIC is a valid index of withdrawal severity from ethanol. Inbred strains of mice differ markedly in the expression of HICs during withdrawal from ethanol, and the severity among inbred strains correlates with those seen in withdrawal from diazepam and pentobarbital, which suggests that withdrawal from the three drugs share some common genetic mechanisms [22,23]. This suggestion is further supported by the observation that mice selectively bred to be either sensitive or resistant to HICs during withdrawal from chronic ethanol administration display correlated differences in withdrawal from barbiturates and diazepam [1,2,10]. In a more recent study, mice selected for High and Low acute Alcohol Withdrawal severity (HAW and LAW, respectively) after acute ethanol administration also differed in severity of acute pentobarbital, diazepam, zolpidem, and nitrous oxide withdrawal [24]. These instances of genetic correlation among severity of withdrawal from ethanol, pentobarbital, and diazepam suggest that some genes act to enhance or reduce more than one of these withdrawal responses, a condition called pleiotropy. The HAW/LAW selective breeding project was performed to refine regions within quantitative trait loci (QTL) influencing alcohol withdrawal [6]. Each QTL is a site on a chromosome harboring a gene or genes affecting the trait. Some QTLs originally shown to affect alcohol withdrawal severity were subsequently shown to influence withdrawal from pentobarbital [7,13]. While the specific gene or genes remain unidentified, on the whole, it appears likely that withdrawal from pentobarbital, diazepam, and ethanol involve similar mechanisms regulated in part by a common set of genes (QTLs). In a meta-analysis of studies using many mouse genetic models, a substantial negative genetic correlation was seen between ethanol withdrawal severity and ethanol consumption [25], indicating that some genes that regulate withdrawal severity exert an opposite effect on the voluntary ingestion of ethanol. This inverse relationship has also been demonstrated in mice lacking the adenosine A2A receptor, in that these mice exhibit increased ethanol consumption [27] and decreased HICs during ethanol withdrawal [12] relative to wildtypes. However, in other experiments with mice lacking either a specific potassium channel subtype [3] or one of two GABAA receptor subunits [4], no relationship between ethanol preference and severity of withdrawal was observed when comparing either null mutant mouse model to the respective wildtype. Combined, these previous results provide direct evidence for the role of specific genes in both ethanol preference and withdrawal. Furthermore, while these studies indicate that some genes influence both phenotypes, they also suggest that not all individual genes
regulating ethanol withdrawal are identical to those regulating preference for ethanol. High and Low Pentobarbital Withdrawal mice (HPW and LPW) mice were selected for severe and mild severity of convulsions, respectively, elicited during withdrawal from an acute injection of pentobarbital [7]. Similar to the HAW and LAW selection, HPW and LPW mice were developed to reduce the size of QTL regions associated with pentobarbital withdrawal and as an aid to identifying the genes responsible [6,7]. After one generation of selective breeding, HPW mice exhibited significantly higher pentobarbital withdrawal HICs than LPW mice. By the fourth generation of selection, HPW mice exhibited more than three-fold greater withdrawal severity than LPW mice [7]. Because of these profound genetic differences in withdrawal from pentobarbital, these mice provide an alternative means to evaluate genetic correlations seen with the lines of mice selected for divergence in ethanol withdrawal. If HPW and LPW mice display correlated differences in withdrawal from ethanol and benzodiazepines, this observation would further support the idea that the mechanisms of barbiturate dependence and withdrawal are genetically similar to those seen with other sedative drugs. To address this question, we tested HPW and LPW mice for severity of HICs elicited following administration of ethanol, diazepam, and zolpidem. In addition, pentobarbital consumption and preference were measured in these selected lines of mice in order to determine if the negative genetic correlation between ethanol withdrawal severity and ethanol consumption seen by Metten et al. [25] would extend to pentobarbital.
2. Materials and methods 2.1. Subjects HPW and LPW mice were selectively bred for severity of pentobarbital withdrawal HICs as previously described [7]. These mice were selected from an F2 cross of the inbred strains C57BL/6J, which exhibit mild withdrawal from several sedatives, and DBA/2J, which demonstrate robust withdrawal from the same sedatives [22,23]. Mice demonstrating the highest withdrawal convulsions following an acute administration of pentobarbital were bred together to form the HPW line, while those exhibiting the lowest formed the LPW line. For the present experiments, naı¨ve mice from the second and fourth selection generations (S2 and S4) were tested. All mice were housed at the Portland Veterans Affairs Medical Center Veterinary Medical Unit with water and food available continuously. Mice were housed two to four per cage with corncob bedding on a standard 12:12-light/dark cycle (lights on at 0600), and were aged 50– 80 days at the time of testing. Equal numbers of HPW and LPW mice were tested for correlated differences in acute withdrawal from ethanol (4 g/kg), diazepam (20 mg/kg), and zolpidem (20 mg/kg). Due to limitations in the
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number and sex of mice available for testing, the experiments with ethanol and diazepam used male and female mice, while zolpidem withdrawal was measured in female mice only.
dem withdrawal HICs were measured every 15 min after a single injection up to 120 min post injection, and then at 150 and 180 min.
2.2. Acute withdrawal
2.3. Pentobarbital drinking
Withdrawal was assessed using the HIC scale [9], which is a modified version of the scale used by Goldstein and Pal [15]. The procedure and HIC scale used for the present experiments were identical to that used for the selection of HPW and LPW mice [7]. Briefly, a score from 0 (no convulsion) to 7 (spontaneous, lethal tonic – clonic convulsion) is assigned depending on the severity of the convulsion elicited after the mouse is picked up by the tail and if necessary, gently spun in a 180j arc. Following two baseline HIC assessments, withdrawal was measured at multiple time points following a single injection of either vehicle or drug.
We assessed oral consumption of three concentrations of pentobarbital in naı¨ve female HPW and LPW mice of the S4 generation (n=15 each) using a two bottle choice procedure. All mice were singly housed and allowed to drink from two tubes containing tap water for 4 days prior to the introduction of pentobarbital to one of the tubes. The mice were then exposed for 4 days each to three ascending concentrations of pentobarbital (250, 500, and 1000 mg/l pentobarbital, all in a 2 g/l saccharin solution) in one of the tubes, which was alternated between the left and right side every 2 days. All fluids were available constantly and volumes consumed were recorded daily. The total amount of pentobarbital consumed at each concentration (mg/kg/day) and average daily preference for pentobarbital relative to water [ml pentobarbital/(ml pentobarbital+ml water)] were calculated based on the volumes consumed and mouse weight, determined prior to the introduction of each of the three concentrations of pentobarbital.
2.2.1. Ethanol Male and female HPW and LPW mice (n=14/line/sex) of the S2 generation were tested for acute withdrawal following intraperitoneal injection of 4 g/kg ethanol (20% v/v). HICs were scored at 2 h, and then every hour from 4 to 10 h after injection. 2.2.2. Diazepam Diazepam, a relatively long-lasting drug, suppresses HIC acutely, but does not result in a subsequent waxing and waning pattern of HIC exacerbation after injection [8]. However, if the drug is antagonized at the receptor by injection with flumazenil, a brief, relatively intense withdrawal reaction is precipitated, lasting for several minutes after flumazenil injection [22,23]. In both the S2 and S4 generations, diazepam withdrawal was precipitated by an injection of 10 mg/kg flumazenil 60 min after injection of 20 mg/kg diazepam or vehicle in male and female mice. Both diazepam and flumazenil were prepared in saline to which 1 drop of Tween 80 per 5 ml total volume was added, and the vehicle was prepared identically except for the addition of the drug. For both experiments, HICs were measured at 30 and 55 min after the diazepam injection to establish that diazepam had exerted its anticonvulsant effect on HICs. Five additional HIC assessments were then made at 1, 3, 5, 8, and 12 min following flumazenil injection [22 – 24]. In the S2 generation, 14 –16 mice per line (7– 8 of each sex) received diazepam, while 5 – 7 per line and sex were injected with vehicle. When reassessed in generation S4, 11 HPW and 10 LPW (4 –7 per sex) received diazepam, and 4 – 6 mice per line per sex received a vehicle injection. The vehicle-injected group was included as a control for the mild anticonvulsant effects of flumazenil [23]. 2.2.3. Zolpidem Acute withdrawal from zolpidem, which does not require precipitation by an antagonist [24], was measured in female HPW and LPW mice from generation S4 (n=8/line). Zolpi-
2.4. Statistical analyses In all HIC experiments, the two baseline HICs in all experiments were averaged to serve as an index of basal HIC susceptibility, and peak withdrawal and corrected peak withdrawal after the injection of a drug are reported. For the ethanol and zolpidem experiments, peak withdrawal was defined as the average of the three highest consecutive HIC scores, and corrected peak withdrawal was calculated as the difference between the peak withdrawal score and the average baseline for each subject. For the diazepam experiments, a peak withdrawal score was calculated as the average of the three highest consecutive HIC scores [14], from which the average score of the vehicle-treatment group over the same time points was subtracted [23,24]. When they occurred, negative individual corrected peak withdrawal scores (corrected either for baseline or vehicle group, as appropriate) were converted to zero. In the ethanol and zolpidem experiments, area under the withdrawal curve (AUC) was calculated as the difference between baseline HIC and the sum of all HICs elicited during withdrawal, corrected for differences in time intervals between each assessment. AUC for diazepam withdrawal was calculated similarly, except that the withdrawal scores were corrected for the vehicle response over the time course of withdrawal. Selected line differences in average baseline and peak withdrawal were analyzed by analysis of variance (ANOVA), with line as a factor in all experiments, and sex included as a factor where appropriate. Significant interactions were analyzed by Tukey’s HSD post hoc test. Differences in pentobarbital consumption and preference
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were analyzed by repeated measures analysis of variance. Significant main effects and interactions in this experiment were analyzed by simple main effects analysis. All results were considered significant at p<0.05. In cases where significance was not attained, exact p values are given to provide some indication of the strength of the trends observed. Based on the literature reviewed above, we hypothesized that pentobarbital withdrawal severity would be positively correlated with the withdrawal severities of the other drugs tested. Therefore, we used one-tailed tests for these comparisons. Pentobarbital consumption was analyzed using twotailed tests. We also report the proportion of the total variance due to the selection line differences in order to better compare magnitudes of correlated responses across the drugs tested. This was quantified as R2 from a one-way ANOVA by line.
3. Results With the exception of the zolpidem withdrawal experiment, using AUC as the dependent variable produced equivalent statistical outcomes to those obtained using peak withdrawal. Therefore, only peak and corrected peak withdrawal scores are shown for the other drugs for ease of comparison with previous studies that used these same drugs and indices [22 –24]. 3.1. Ethanol No sex differences or line by sex interactions were observed for either average baseline or peak withdrawal from ethanol, so the data presented are collapsed on sex. As shown in Fig. 1A, the lines did not differ in baseline HIC and both lines responded to ethanol with complete suppression of the HIC at hour 2. Large differences in HIC scores between HPW and LPW mice were apparent by hour 6 that persisted to hour 10, with average peak withdrawal occur-
ring at hour 8. Interestingly, LPW mice appeared to return to baseline HIC levels by hour 8, but tended towards lower scores at hours 9 and 10. At peak withdrawal, HPW demonstrated three-fold greater HIC severity than LPW mice ( F1,54=35.45; p<0.0001). This difference was still present when corrected for baseline HIC ( F1,54=37.95; p<0.0001; Fig. 1B). This corrected line difference for EtOH withdrawal accounted for 41% of the total variation observed in the S2 generation. 3.2. Diazepam Diazepam withdrawal was first measured in the S2 generation. In this experiment, HPW mice demonstrated higher basal HICs than LPW mice ( F1,50=5.67; p<0.05; data not shown), and male mice of both genotypes exhibited significantly higher basal HICs than did female mice ( F1,50=4.80; p<0.05; data not shown). A significant interaction between selected line and sex was also observed ( F1,50=1.73; p<0.05), in that HPW male mice exhibited significantly higher basal HICs than HPW females, and both LPW male and female mice ( p<0.05; Tukey’s post hoc). Diazepam reduced HIC scores at 30 and 55 min after injection, and when precipitated by flumazenil, peak withdrawal in both lines occurred 1 min later. However, no line differences were observed for peak withdrawal ( F1,26=2.49; p=0.67) or peak withdrawal corrected for the vehicle group ( F1,26=0.10; p=0.38). The corrected line difference for DZ withdrawal accounted for less than 1% of the total variation seen in the S2. In addition, no sex differences or line by sex interactions were observed for either variable (all F’s<1.87; p>0.05). When reassessed in generation S4, no sex difference in basal HIC ( F1,32=0.08; p>0.05), or line by sex interaction for basal HIC were observed ( F1,32=1.65; p>0.05). As in generation S2, HPW mice exhibited higher basal HICs than did LPW mice ( F1,32=17.04; p<0.001; Fig. 2A), and HIC
Fig. 1. Acute withdrawal from ethanol in HPW and LPW mice of the S2 selection generation. (A) Time course of ethanol withdrawal handling induced convulsion (HIC) scores. (B) Peak withdrawal and corrected peak withdrawal from ethanol. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
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Fig. 2. Acute withdrawal from diazepam in HPW and LPW mice in generation S4. Group identification is shown in the legend. Flumazenil injection occurred at the 60-min time point (indicated by arrow). (A) Time course of diazepam withdrawal HIC scores. Minutes 61 – 72 represent minutes 1 – 12 after flumazenil injection. The x-axis scale is compressed up to 55 min to better demonstrate the withdrawal curve. (B) Peak and corrected peak withdrawal from diazepam in the S2 and S4 generations. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
scores for all mice were suppressed at 30 and 55 min after injection of diazepam. HIC scores of either HPW or LPW mice treated with vehicle followed by flumazenil did not change appreciably over the course of the experiment. Peak withdrawal in HPW mice occurred at 3 min, while LPW mice demonstrated highest HIC scores at 1 min after flumazenil injection. No sex differences or line by sex interactions were observed for peak or corrected peak withdrawal (all F’s<2.47; p’s>0.05), so for further analysis we collapsed on sex. Fig. 2B shows peak and corrected peak diazepam withdrawal severity in the S2 and S4 generations. When uncorrected for basal differences in HICs or the vehicle group response, peak withdrawal was higher in HPW than LPW mice in the S4 generation ( F1,19=9.40; p<0.01). When corrected for the response seen in vehicleinjected animals, a difference in peak withdrawal was observed that closely approached statistical significance ( F1,19=2.84; p=0.054). The corrected line difference for
DZ withdrawal accounted for 13% of the total variation observed in the S4 generation. 3.3. Zolpidem No basal differences in average baseline HIC were observed between the lines ( F1,14<1.0; Fig. 3A). After the initial suppression of HIC scores by zolpidem at 15, 30, and 45 min, HPW and LPW mice returned to basal levels of HIC susceptibility by 60 min. Peak withdrawal in HPW mice occurred at 120 min, and in LPW mice at 60 min, although the scores of LPW mice did not change appreciably after recovering to baseline over the entire course of withdrawal. HPW and LPW mice did not differ significantly in peak withdrawal ( F1,14=1.54; p=0.12), or peak withdrawal corrected for baseline HICs ( F1,14=0.95; p=0.36; Fig. 3B). The corrected line difference for zolpidem withdrawal accounted for 6% of the total variation seen in the S4. However, as
Fig. 3. Acute withdrawal from zolpidem in HPW and LPW mice of generation S4. (A) Time course of zolpidem withdrawal HIC scores. (B) Peak withdrawal and corrected peak withdrawal from zolpidem. Values shown represent meanFS.E.M. AVB refers to average baseline HIC.
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indicated by Fig. 3A, it appears that HPW and LPW mice may differ in withdrawal from zolpidem, but that this difference is obscured by high variation due to the small number of mice available, and the use of peak withdrawal to index the severity of withdrawal (see Discussion). Analysis of AUCs corrected for baseline HICs indicated that HPW mice exhibited higher withdrawal severity than LPW mice (HPW 6.5F2.3, LPW 1.6F1.0; F1,14=3.85; p<0.05; data not shown). This line difference accounted for 22% of the variation in AUC for zolpidem.
differ in pentobarbital consumption at either the 250 or 1000 mg/l concentrations (both F’s<1.08; p>0.05). Although the pattern of line differences in preference for pentobarbital shown in Fig. 4B appears similar to that seen in consumption of pentobarbital, these differences between HPW and LPW mice did not reach statistical significance ( F1,28=1.42; p=0.12). As concentrations of pentobarbital increased over the course of the experiment, preference declined ( F2,56= 23.73; p<0.0001), and this declining preference did not interact with line ( F2,56=2.13; p=0.13).
3.4. Pentobarbital drinking 4. Discussion Pentobarbital consumption changed as a function of concentration ( F2,56=6.07; p<0.01), and was greater in LPW than in HPW mice ( F1,28=5.71; p<0.05; Fig. 4A). The line difference for consumption accounted for 17% of the total variation in seen in the S4. A significant interaction between concentration of pentobarbital and line was observed ( F2,56=3.18; p<0.05), in that LPW mice consumed more of the 500 mg/l concentration than did HPW mice ( F1,28=10.07; p<0.01), but HPW and LPW mice did not
Fig. 4. Voluntary daily consumption of three concentrations of pentobarbital in HPW and LPW mice of generation S4. (A) Total consumption (mg/kg) of pentobarbital over four successive days at each concentration of pentobarbital. (B) Average daily preference for each concentration of pentobarbital.
Sedative drugs have complex effects on the central nervous system. For instance, administration of ethanol results in alterations in the function of many neurotransmitter systems, including glutamate, GABA, endogenous opioids, dopamine, and serotonin [11,19,28]. Chronic ethanol administration exacerbates these changes, and results in ethanol dependence [19]. As a result, severe HICs during ethanol withdrawal could result from alterations in any of a number of neurotransmitter systems, while resistance to withdrawal HICs could result from changes in either the same or different brain systems, each of which in turn is regulated by different underlying genotypes. Acute pentobarbital administration results in multiple changes in neurotransmission as well, although as with ethanol, the primary mechanism of pentobarbital’s sedative action is thought to be at the GABAA receptor (see Ref. [16]). Artificial selection for severity of withdrawal from either pentobarbital or ethanol could therefore result in changes in multiple brain systems that may or may not be similar between the two drugs. Other than two inbred strain experiments [22,23], all of the previous data examining genetic correlations between ethanol and barbiturate withdrawal were obtained from mice selectively bred for high and low severity of ethanol withdrawal. The present results are thus the first demonstration that artificial selection for withdrawal severity from pentobarbital results in genetically correlated differences in withdrawal from ethanol. HPW and LPW differed three-fold in peak withdrawal from ethanol, and over five-fold when corrected for average baseline HICs. These differences in withdrawal severity are comparable to those observed during selection for pentobarbital withdrawal (see Ref. [7]), and strongly suggest that the genes involved in severity of withdrawal from pentobarbital are similar to those implicated in ethanol withdrawal. In the S2 generation, HPW and LPW mice did not differ in corrected or uncorrected peak withdrawal from diazepam. At this generation of selection, significant differences between HPW and LPW mice were apparent in pentobarbital [7] and ethanol withdrawal severity (current results). Two generations later, the divergence in pentobarbital withdrawal severity was more striking, and HPW mice exhibited significantly higher peak diazepam withdrawal than did
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LPW mice, although not when corrected for the response of the control groups to flumazenil. In both the S2 and S4 generations, then, the magnitude of the line difference in diazepam withdrawal severity was smaller than that seen for ethanol, suggesting either that the genetic mechanism influencing diazepam withdrawal severity is not identical to that involved in ethanol and pentobarbital withdrawal, or that we did not have sufficient statistical power in these experiments to detect a difference that was real. A larger experiment might have produced a more clear result, but the availability of animals limited the size of the experiments to those reported here. Buck et al. [6] identified QTLs on mouse chromosomes 1, 4, and 11 that influence the severity of ethanol withdrawal as measured by HICs, and these QTLs were subsequently shown to influence pentobarbital withdrawal severity [5,7]. Using finer mapping techniques, Fehr et al. [13] narrowed the QTL region on chromosome 4, and identified Mpdz as a candidate gene that reduced the severity of ethanol and pentobarbital withdrawal in congenic mice. The congenic mice developed for this experiment express a segment of chromosome 4 from the withdrawal seizure resistant C57BL/6J strain against a seizure susceptible DBA/2J background. Since mice that possess this chromosomal interval exhibited less severe withdrawal HICs than the background DBA/2J strain, it is likely that the introgressed chromosomal interval contained either one or multiple gene variants that reduce the severity of ethanol withdrawal. We have begun comparing the chromosome 4 congenic mice developed by Fehr et al. [13] to the DBA/2J background strain, in order to test whether a pleiotropic QTL that also affects diazepam withdrawal exists there. Though preliminary, it does not appear that these congenic mice display reduced severity of diazepam withdrawal compared to the DBA/2J background strain. This result, combined with those reported here, suggests that withdrawal from diazepam is somewhat genetically distinct from that produced by ethanol or pentobarbital. This suggestion contrasts with data obtained from inbred strains and the lines selected for high and low ethanol withdrawal severity, which have demonstrated that withdrawal from all three sedatives is genetically similar [1,2,10,22 – 24]. However, many genes are likely to be responsible for any proposed pleiotropic effect, and selection may simply not have advanced far enough by S4 to capture the genes that also affect diazepam withdrawal. Indeed, congenic mice that harbor a segment on chromosome 1 that reduces ethanol withdrawal severity display reduced diazepam withdrawal severity (unpublished data), suggesting that at least some of the QTLs influencing ethanol and pentobarbital withdrawal also influence diazepam withdrawal. A barrier to interpretation of the diazepam experiments is that withdrawal scores (both corrected and uncorrected) were confounded with differences between HPW and LPW mice in basal HIC susceptibility in both experiments. This was an unexpected finding based on the selection
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method used for these lines of mice. HPW and LPW mice were selected on the basis of either a large positive or negative residual, respectively, from the regression of peak pentobarbital withdrawal HIC on the average baseline HIC [7]. By definition, this residual score has a correlation of zero with the baseline HIC. The mice produced from this selection could still demonstrate correlated differences in basal HIC, but this difference would be strictly due to a chance fixation of genes, and should be observed in most experiments with these mice. A correlated difference in basal HICs between HPW and LPW mice was not reported by Buck et al. [7], and the basal difference in HICs was not observed in the current experiments with zolpidem or ethanol. Considering that the same technician conducted all of the withdrawal studies presented here, the reason for correlated differences in baseline HICs in the two studies with diazepam is unclear. It is apparent that both selected lines experienced withdrawal from diazepam; however, the role that differences in general HIC susceptibility between HPW and LPW mice had in this withdrawal is less apparent. A tentative explanation of the results with diazepam could be that due to the unexpected correlated differences in basal HIC, insufficient statistical power was present to detect a difference between HPW and LPW mice. Unfortunately, these lines of mice are no longer maintained, so retesting HPW and LPW mice for correlated differences in diazepam withdrawal is impossible. However, it is clear that HPW and LPW mice differed more dramatically in severity of withdrawal from both pentobarbital and ethanol than from diazepam. This conclusion is also supported by the proportion of the variance due to the line differences, which was very much higher with ethanol (S2, 41%) than with diazepam (S2, <1%; S4, 13%). This indicates that pentobarbital and ethanol withdrawal have greater commonality in their genetic influences than do pentobarbital and diazepam withdrawal. Zolpidem has been previously shown to induce withdrawal reactions in mice over the same time course tested in the present experiments, and mice selected to differ in severity of ethanol withdrawal also differ in withdrawal from zolpidem [24]. Similar to diazepam, HPW and LPW mice did not differ in peak withdrawal severity from zolpidem. However, peak withdrawal from zolpidem, as described in the methods section, occurred at 60 min in LPW mice, but not until 120 min after injection in HPW mice. After the initial anticonvulsant effects of zolpidem, LPW mice had returned to basal HIC levels by 60 min after injection, and did not significantly change over the next 120 min. Therefore, LPW mice never displayed significant ‘‘withdrawal’’ from zolpidem (as indexed by HICs elevated over baseline), so the selection of a particular time point as being indicative of their peak withdrawal may have been misleading. Indeed, differences in AUCs between HPW and LPW mice attained statistical significance ( p=0.04, onetailed test), indicating that withdrawal from zolpidem may differ between the selected lines. Further, comparison of
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HPW and LPW mice at 120 min after injection, when HPW mice were experiencing peak withdrawal, suggests that correlated differences in zolpidem withdrawal may exist between HPW and LPW mice, but that the correlation is hidden in the high variability observed early in the withdrawal curve of LPW mice. In any case, the proportion of the variance due to line differences was much higher with ethanol (S2, 41%) than zolpidem (S4, either 6% or 22% depending on the method of analysis), indicating that pentobarbital and ethanol withdrawal have greater commonality in their genetic influences than do pentobarbital and zolpidem withdrawal. A meta-analysis of data collected in inbred strains and lines of mice selected for either ethanol withdrawal severity or ethanol preference indicates that a negative genetic correlation exists between the amount of ethanol voluntarily consumed and the severity of withdrawal [25]. That is, genes that increase the amount of ethanol a mouse will drink decrease the severity of the withdrawal reaction experienced by the mouse. Our results with HPW and LPW mice suggest that this inverse relationship exists with pentobarbital as well, since LPW mice voluntarily consumed more pentobarbital than did HPW mice. This finding implies that the genes influencing pentobarbital withdrawal are similar to those affecting consumption, and, since HPW and LPW mice display correlated differences in ethanol withdrawal, those genes that affect pentobarbital consumption and withdrawal severity may be similar to those influencing ethanol consumption and severity of withdrawal. Identification of the specific genes involved in this correlation would provide a clinically relevant target for therapies aimed at reducing the initiation of drinking, as well as novel therapeutics for the treatment of withdrawal. Interestingly, neither HPW nor LPW mice displayed an absolute preference (i.e., >50%) for any of the three concentrations of pentobarbital tested. This observation makes it unlikely that the mice were consuming the pentobarbital solutions merely for the taste of saccharin, a preferred taste, relative to water. It should be noted that preference for the drug solution declined with increasing concentrations of pentobarbital, raising the possibility that saccharin was less able to mask the taste of pentobarbital with increasing concentrations of the drug. In the absence of a saccharin control, it is difficult to determine which of these possibilities is correct. Some additional unpublished analyses from our laboratory indicate that across inbred strains, pentobarbital withdrawal severity was not correlated with saccharin consumption. In addition, in the current experiments, pentobarbital consumption (in mg/kg) tended to be higher with increasing concentrations of the drug. Since decreases in pentobarbital preference occurred at the higher solution concentrations, it is possible that the mice were attempting to titrate the total amount of pentobarbital consumed. The experiments presented here demonstrate a clear genetic correlation between severity of withdrawal from ethanol and pentobarbital, a finding consistent with previous
research [10,22 –24]. However, these correlated differences were not as strong for withdrawal from diazepam in HPW and LPW mice. The experiment with zolpidem suggests that a genetic correlation between pentobarbital and zolpidem withdrawal severity exists when an index is used that takes into account the entire course of withdrawal. Finally, the pentobarbital consumption experiment extends previous data demonstrating a negative correlation between ethanol withdrawal severity and consumption to include pentobarbital, by showing that LPW mice voluntarily consume more of the drug than do HPW mice. Further refinements of ethanol, pentobarbital, and diazepam withdrawal QTLs will elucidate the specific genes involved in the observed genetic correlations.
Acknowledgements The authors would like to thank Cathy Merrill and Charlotte Wenger for expert technical assistance. This research was supported by NIH grants AA10760, AA05828, AD05228, and AD06243, and by the Department of Veterans Affairs.
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