Introduction

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Sex steroids were reported to influence brain excitability as long ago as 1942 (Seyle, 1942). However, it was not until 1984 that the synthetic steroid alphaxalone (3-hydroxy-5-pregnan-11,20-dione) was electrophysiologically demonstrated to potentiate GABA-gated chloride conductance (Harrison and Simmonds, 1984). Subsequent studies determined that the progesterone metabolite 3,5-THP and the deoxycorticosterone metabolite 3,5-THDOC were potent GABA-agonist modulators of the GRC via a stereospecific interaction at a unique steroid recognition site associated with the GRC (for reviews, see Belelli et al., 1990; Paul and Purdy, 1992; Lambert et al., 1995). Both 3,5-THP and 3,5-THDOC enhance GABA-stimulated chloride uptake in rat brain synaptoneurosomes at nanomolar concentrations (Morrow et al., 1987) and interact with the known sites on the GRC in a noncompetitive manner (for review, see Belelli et al., 1990). These in vitro demonstrations provide evidence that some steroid metabolites have rapid membrane actions that are distinct from the genomic action of "classical" steroid hormones.

Consistent with their GABA-agonist pharmacological profiles, exogenous administration of 3,5-THP or 3,5-THDOC produces anesthetic (Mok et al., 1991), hypnotic (Mendelson et al., 1987), anticonvulsant (Belelli et al., 1989; Finn and Gee, 1994) and anxiolytic (Crawley et al., 1986; Bitran et al., 1991; Weiland et al., 1995) effects. Both 3,5-THP and 3,5-THDOC, as well as the synthetic steroid anesthetic alphaxalone, were potent anxiolytics in several animal models of anxiety, i.e., the light/dark transition test (Crawley et al., 1986; Weiland et al., 1995), the open field test (Weiland et al., 1991), the conflict test (Crawley et al., 1986; Britton et al., 1991; Weiland et al., 1995) and the elevated plus maze (Bitran et al., 1991; Britton et al., 1991). In addition, 3,5-THP was anticonvulsant against PTZ-, (+)-bicuculline- and picrotoxin-induced seizures, with maximum potency against (+)-bicuculline-induced convulsions (Belelli et al., 1989). These behavioral responses closely follow the anticipated pharmacological patterns based on in vitro evidence.

Recent work has demonstrated that endogenous 3,5-THP can reach pharmacologically relevant concentrations (Paul and Purdy, 1992). After swim stress in male rats and during the estrus cycle in female rats, brain 3,5-THP levels increased to approximately 10 to 30 nM. Plasma 3,5-THP levels also reached 100 nM during the third trimester of pregnancy. These concentrations achieved in vivo have been shown to potentiate the action of GABA by in vitro studies. Therefore, the available evidence suggests, but does not prove, that fluctuations in endogenous GABAergic steroids can modify the functioning of central GABA_A receptors in vivo.

Based on recent results from our laboratory, we believed that one way to provide support for the hypothesis that 3,5-THP represented a physiologically significant endogenous neuromodulator would be to demonstrate that lower endogenous levels of, or decreased sensitivity to, 3,5-THP were correlated with genetic differences in basal or ethanol withdrawal hyperexcitability. The B6 and D2 inbred strains differ in a number of behaviors, including locomotor and exploratory activity (Lhotellier et al., 1993), learning and memory (Fordyce and Wehner, 1993; Rossi-Arnaud and Ammassari-Teule, 1994), anxiety (Trullas and Skolnick, 1993) and seizure susceptibility (Kosobud and Crabbe, 1990; Ferraro et al., 1995). When tested for susceptibility to a number of convulsants, B6 mice are generally very seizure resistant, in comparison with other inbred strains, whereas D2 animals are relatively seizure prone (Kosobud and Crabbe, 1990). B6 and D2 mice also differ markedly in many ethanol-related behaviors, of which ethanol preference, ethanol-induced locomotor activation and ethanol withdrawal severity are the most notable (for review, see Phillips and Crabbe, 1991). D2 mice exhibit more severe handling-induced convulsions than do B6 animals after withdrawal from both acute (Roberts et al., 1992) and chronic (Crabbe et al., 1983) ethanol administration. Therefore, B6 and D2 mice represent useful animal models to test the hypothesis that genetic differences in the modulatory effects of 3,5-THP on ethanol withdrawal severity might result from differences in 3,5-THP sensitivity or biosynthesis.

Preliminary results suggested that B6 and D2 mice do not differ in biosynthesis of 3,5-THP after 24-hr exposure to ethanol vapor (Finn et al., 1995a). In both B6 and D2 mice, plasma 3,5-THP levels were unchanged during peak ethanol withdrawal and were increased in only the animals that were scored hourly for withdrawal. Therefore, the present studies were conducted to test the hypothesis that differences in 3,5-THP sensitivity may contribute to ethanol withdrawal hyperexcitability differences in B6 and D2 animals (i.e., B6 and D2 mice would differ in sensitivity to 3,5-THP in a manner that was consistent with their genetic difference in ethanol withdrawal severity). Because ethanol withdrawal is characterized by multiple behavioral changes in addition to convulsions, a number of other behaviors were evaluated, i.e., anxiolytic effects and locomotor activity (measured on an elevated plus maze), ataxic effects (measured by Rotarod performance), muscle relaxation (measured in a test of grip strength) and anticonvulsant effects (measured by tail vein infusion of PTZ), to determine whether a difference between B6 and D2 mice in sensitivity to 3,5-THP could be generalized to all pharmacological properties of 3,5-THP.

	Methods

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Subjects

Drug-naive male B6 and D2 mice were used in all experiments. The animals were purchased from The Jackson Laboratory (Bar Harbor, ME) at 5 to 6 weeks of age, housed four per cage with ad libitum access to food and water and acclimated to a 12/12-hr light/dark cycle for a minimum of 1 week before experimentation. All procedures adhered to the United States Public Health Service/National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by two local institutional Animal Care and Use Committees.

Experiment 1: Anticonvulsant Sensitivity to 3,5-THP

The anticonvulsant effect of 3,5-THP was measured by administering 3,5-THP (5 or 10 mg/kg) or an equivalent volume of vehicle (20% w/v 2-hydroxypropyl--cyclodextrin; Research Biochemicals International, Natick, MA) by i.p. injection 15 min before timed tail vein infusion of PTZ (5 mg/ml in saline, 0.5 ml/min; Sigma Chemical Co., St. Louis, MO). The 3,5-THP was prepared as a 0.5 or 1.0 mg/ml solution in 20% 2-hydroxypropyl--cyclodextrin and injected in a volume of 0.01 ml/g body weight. The apparatus and procedure for tail vein infusion have been described in detail (Kosobud and Crabbe, 1990). The infusion was terminated when the animals exhibited THE. The animals were euthanized by decapitation, and trunk blood was collected for subsequent analysis of plasma 3,5-THP concentration by RIA.

The convulsion end-points are described in detail elsewhere (Kosobud and Crabbe, 1990). Briefly, clonus indicates rapid rhythmic movements due to alternating contraction and relaxation of muscles, whereas tonus indicates rigidity due to contraction of muscles. Four convulsion signs, which occur in progression, characterize PTZ-induced convulsions, i.e., MC twitch (sudden involuntary muscle jerks), FF clonus (rapid writhing movements of the head and neck and forelimb clonus), RB clonus (violent whole-body clonus, including running and explosive jumps) and THE (extreme rigidity, with forelimbs and hindlimbs extended caudally). Latency to each sign was recorded in seconds and subsequently converted to threshold convulsant dosage (i.e., milligrams of drug per kilogram of body weight).

Experiment 2: Behavioral Sensitivity to 3,5-THP

We recently established a procedure for measuring a series of behaviors, taking advantage of our experience with the individual behavioral protocols and validating the accuracy of repeated testing of individual animals (E. J. Gallaher, unpublished observations). The anticonvulsant effect of 3,5-THP was reexamined in this study for two reasons; 1) an expanded dose range was evaluated and 2) the timed tail vein infusion of PTZ occurred at the end of this series of behavioral tests, so that comparisons between experiments would yield a measure of the reliability of repeated behavioral testing in a single animal.

Each mouse was pretested for forelimb grip strength (three trials; time 20 min) and Rotarod baseline performance (three trials; time 10 min) and then weighed and injected with 3,5-THP (0, 1, 3.2, 10, 17 or 32 mg/kg in 20% 2-hydroxypropyl--cyclodextrin i.p.) at time 0. At time +20 min the mouse was placed on the plus maze for a 5-min trial. Total arm entries (locomotor activation) and percent open arm entries (anxiolysis) were recorded. At time +30 min the mouse was tested for forelimb grip strength (compared with base- line). At time +40 min the mouse was tested for impairment on the accelerating Rotarod (compared with baseline). At time +50 min the mouse was infused, via a lateral tail vein, with the convulsant PTZ and was observed for latency to MC twitch, FF clonus, RB clonus and THE. The threshold dose of PTZ for each sign was calculated from the infusion rate, body weight and latency. The infusion was terminated when the animals exhibited THE or at 4 min (in cases where the mice were protected by 3,5-THP). The animal was then euthanized by decapitation, and trunk blood was collected for subsequent analysis of plasma 3,5-THP concentration by RIA.

Behavioral Assessments

Muscle relaxation. In this test, mice are placed on a tray, allowed to grasp a horizontal bar connected to a strain gauge and then pulled gently until they lose their grip. For comparative purposes, grip strength decreases in a dose-dependent manner after graded doses of chlordiazepoxide (3, 9 and 27 mg/kg) and phenobarbital (20, 40 and 80 mg/kg). The lowest doses produce a 10 to 20% decrease in grip strength and the higher doses a 50 to 60% decrease (Meyer et al., 1979).

Ataxia. The Rotarod test for ataxia was first described by Dunham and Miya (1957). The Rotarod is a horizontal rotating dowel (5 cm in diameter) suspended 60 cm above a bed of sawdust. It is divided into six segments (10.2-cm wide) by means of opaque white disks 28 cm in diameter. The surface of the dowel is covered with 320-grit, wet-dry sandpaper to ensure a uniform surface. Typically, mice are placed on the Rotarod and observed for ability to remain on the dowel as it turns. In the present experiment, each mouse was placed on a stationary Rotarod, which began to accelerate linearly (20 rpm/min) until the mouse fell off. The latency to fall was then used to calculate the speed (rpm) at which the mouse could no longer remain on the Rotarod. This procedure allowed each mouse to be tested for baseline ability. The drug effect was then expressed as a change from baseline ability.

Anxiolysis. The animal model of anxiety that was used was the elevated plus maze method (Pellow et al., 1985; Lister, 1987; Trullas and Skolnick, 1993; Cole and Rodgers, 1994), which is based on a rodent's natural avoidance of open elevated alleys (Montgomery, 1958). The elevated plus maze consists of two open and two enclosed horizontal perpendicular arms extending from a central platform (5 × 5 cm), 50 cm above the floor. Each mouse was placed on the central platform and allowed to explore freely for 5 min. During the 5-min test period, the number of entries into the open and closed arms and the amount of time spent in the open and closed arms were measured. For an arm entry to be measured, all four paws had to be within the arm. Mice normally prefer the closed arms of the plus maze. Anxiolytic drugs typically increase the proportion of open arm entries and the time spent on the open arm (anxiolysis). The elevated plus maze is able to detect both anxiolytic and anxiogenic agents in mice (Lister, 1987).

Low-dose locomotor activation. Sedative/hypnotic drugs commonly cause locomotor activation after low doses. This has been ascribed to an anxiolytic effect, because locomotion may be an expression of increased exploration in the absence of anxiety. The information obtained from elevated plus maze testing (i.e., total number of arm entries) was used as the estimate of locomotor activation.

Seizure protection. Mice were administered the convulsant PTZ (5 mg/ml in saline) via timed tail vein infusion into a lateral vein (0.5 ml/min infusion rate). Latencies to each convulsion measure were recorded in seconds and subsequently converted to threshold convulsant dosage (i.e., milligram of drug per kilogram of body weight). This method allowed for observation and qualitative analysis of several different endpoints (i.e., MC twitch, FF clonus, RB clonus and THE). Drugs that alter seizure threshold can be tested for pro- or anticonvulsant activity by pretreating mice and observing the effect on PTZ seizure threshold. A decreased PTZ seizure threshold indicates proconvulsant activity, whereas an increased PTZ threshold suggests anticonvulsant activity. This simple method has been used to demonstrate the anticonvulsant efficacy of 3,5-THP (Finn and Gee, 1994; Finn et al., 1995a,b).

RIA

The RIA for 3,5-THP was adapted from the method of Purdy et al. (1990) and is described in detail elsewhere (Finn and Gee, 1994). The RIA used a polyclonal antiserum, which was kindly provided by CoCensys, Inc. (Irvine, CA), and [³H]3,5-THP (54 Ci/mmol; New England Nuclear, Boston, MA). Counts per minute were normalized and fit to a least-squares-fit regression equation produced by log-logit transformation of the standards. The mass of samples was calculated by interpolation of the standards and correction for recovery. The minimum detectable limit in the present assay was 25 pg. The intraassay coefficient of variation averaged 14%, and the interassay coefficient of variation in seven assays averaged 15%.

Data Analysis

The data are expressed as the mean ± S.E. Analysis of variance was used to assess strain and dose effects on the dependent variables muscle relaxation (change in baseline forelimb grip strength), ataxia (change in baseline Rotarod performance), locomotor activity (total arm entries on the elevated plus maze), anxiolysis (percent open arm entries on the plus maze), seizure protection (threshold dose for onset to MC twitch, FF clonus, RB clonus and THE) and plasma 3,5-THP concentration. When appropriate, simple main-effects analyses followed by post hoc comparisons were used to examine significant dose effects within each strain. Because the results of experiment 1 indicated that B6 and D2 mice differed in sensitivity to 3,5-THP, statistical analyses for experiment 2 were conducted on each strain separately.

	Results

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Experiment 1: anticonvulsant sensitivity to 3,5-THP. To evaluate whether differences in sensitivity to an endogenous anticonvulsant steroid (i.e., 3,5-THP) contribute to the genetic differences in ethanol withdrawal severity found in B6 and D2 mice, it was important to first investigate anticonvulsant sensitivity to 3,5-THP in ethanol-naive animals. The results indicated that vehicle-treated D2 mice were more sensitive to PTZ than B6 animals, as illustrated in figure 1 for all convulsion measures (compare with vehicle-treated animals in fig. 1). More importantly, B6 mice were more sensitive than D2 mice to the anticonvulsant effect of 3,5-THP, as measured by the increase in PTZ seizure threshold for onset to MC twitch (fig. 1A) and FF clonus (fig. 1B). There were significant main effects of strain [F(1,54) > 94.3, P < .0001] and dose [F(2,54) > 28.7, P < .0001], with significant interactions between strain and dose [F(2,54) > 6.5, P < .005]. Post hoc tests indicated that both doses of 3,5-THP significantly increased PTZ seizure threshold in B6 mice, whereas only the 10 mg/kg dose significantly increased seizure threshold in D2 mice. Analysis of the RB clonus (fig. 1C) and THE (fig. 1D) results indicated that there were significant main effects of strain [F(1,52) > 9.5, P < .005] and dose [F(2,52) > 21.7, P < .0001]. The interaction between main effects was a nonsignificant trend for RB clonus [F(2,52) = 2.73, P = .07] and was not significant for THE [F(2,52) = 1.18], suggesting that the anticonvulsant effects of 3,5-THP against PTZ-induced RB clonus and THE were similar in B6 and D2 mice.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Anticonvulsant effects of 3,5-THP against PTZ-induced MC twitch (A), FF clonus (B), RB clonus (C) and THE (D) in B6 and D2 mice. Administration of 3,5-THP 15 min before tail vein infusion of PTZ significantly increased the threshold dose of PTZ for onset for all four convulsion measures. Values represent the mean ± S.E. for 8 to 10 animals per dose and strain. In cases where the S.E. is not visible, it is contained within the symbol. *P < .05, **P < .01, ***P < .001 vs. respective vehicle-treated group.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Plasma 3,5-THP levels in B6 and D2 mice. Plasma samples were taken upon completion of the behavioral testing (i.e., approximately 30 min after injection of 3,5-THP). Administration of 3,5-THP significantly increased plasma 3,5-THP concentrations in a dose-dependent manner. Values represent the mean ± S.E. for the animals depicted in figure 1.

Experiment 2: behavioral sensitivity to 3,5-THP. The results of experiment 1 indicated that B6 mice were more sensitive than D2 animals to the anticonvulsant effect of 3,5-THP and that this difference in sensitivity was not pharmacokinetic. Subsequent studies evaluated whether this strain difference in sensitivity to the anticonvulsant effect of 3,5-THP was generalized across several of the pharmacological effects of 3,5-THP.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Locomotor stimulant (A) and anxiolytic (B) effects of 3,5-THP in B6 and D2 mice. Mice were administered 3,5-THP 20 min before testing on the elevated plus maze. The total number of entries was used as an estimate of locomotor activity, and the percent open arm entries was used as a measure of anxiolysis. Values represent the mean ± S.E. for five to eight animals per strain and dose, except for D2 mice injected with 32 mg/kg 3,5-THP (n = 3). *P < .05, **P < .01 vs. respective vehicle-treated mice.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Anticonvulsant effects of 3,5-THP against PTZ-induced MC twitch (A), FF clonus (B), RB clonus (C) and THE (D) in B6 and D2 mice. 3,5-THP was administered 50 min before the timed tail vein infusion of PTZ and significantly increased the threshold dose of PTZ required for onset for all four convulsion measures. Values represent the mean ± S.E. for the animals depicted in figure 3. *P < .05, **P < .01, ***P < .001 vs. respective vehicle-treated mice.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of 3,5-THP on forelimb grip strength (A) and Rotarod performance (B) in B6 and D2 mice. Animals were injected with 3,5-THP and then tested for forelimb grip strength (compared with baseline) at 30 min after injection and tested for performance on the accelerating Rotarod (compared with baseline) at 40 min after injection. Values represent the mean ± S.E. (percent change from baseline) for the animals depicted in figures 3 and 4. *P < .05, **P < .01, ***P < .001 vs. respective vehicle-treated group.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Plasma 3,5-THP levels in B6 and D2 mice. Plasma samples were taken upon completion of the behavioral testing (i.e., approximately 60 min after injection of 3,5-THP). Values represent the mean ± S.E. for the animals depicted in figures 3, 4, 5. See "Results" for details regarding group differences.

	Discussion

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The results of the present studies suggest that B6 mice were more sensitive than D2 animals to the anxiolytic, locomotor stimulant and anticonvulsant effects of 3,5-THP. In contrast, D2 mice appeared to be more sensitive than B6 mice to the muscle relaxation and ataxia produced by 3,5-THP. Although these results do not indicate that increased sensitivity to endogenous 3,5-THP in B6 mice is the factor responsible for their decreased seizure susceptibility, relative to D2 animals, the enhanced sensitivity to the anticonvulsant effect of exogenous 3,5-THP in B6 vs. D2 mice is consistent with their genetic differences in seizure susceptibility and ethanol withdrawal severity (i.e., decreased seizure susceptibility and ethanol withdrawal severity in B6 vs. D2 mice). More important, the genetic differences in sensitivity to the pharmacological effects of 3,5-THP were not pharmacokinetic, because plasma 3,5-THP levels did not differ in B6 and D2 mice after injection of this neuroactive steroid.

The conclusion that B6 mice were more sensitive than D2 mice to the anxiolytic and locomotor stimulant effects of 3,5-THP was based on the results obtained from testing on the elevated plus maze (i.e., percent open arm entries and total entries). Recent work has indicated that additional behavioral measures, collectively referred to as "risk assessment," may enhance the sensitivity of the elevated plus maze as an animal model of anxiety (Cole and Rodgers, 1994). Although these behavioral assessments were not evaluated in the present study, the 3,5-THP-induced anxiolysis in B6 and D2 mice is consistent with published results, which have used several animal models of anxiety (Bitran et al., 1991; Weiland et al., 1991, 1995). Nonetheless, future studies that incorporate these risk assessment behaviors may yield more information about differences between genotypes in 3,5-THP-induced anxiolysis.

Administration of 3,5-THP significantly increased plasma 3,5-THP concentrations in both inbred strains. Depending on the experiment, doses of 3,5-THP of 5 mg/kg produced a significant increase in plasma 3,5-THP levels, compared with the respective vehicle-treated animals. Even though the highest dose of 3,5-THP administered (i.e., 32 mg/kg) produced significant ataxia and plasma concentrations ranging from 450 to 480 ng/ml, these levels are considerably lower than the anesthetic levels recently reported for mice (Mok et al., 1991). Brain 3,5-THP levels were not measured in the present study. However, the data reported by Mok et al. (1991) indicated that i.v. administration of 3,5-THP produced peak brain levels within 1 min of injection. Because the animals in the present studies were euthanized for determination of plasma 3,5-THP concentrations at approximately 30 or 60 min after injection of 3,5-THP, the plasma levels should reflect brain 3,5-THP levels. Therefore, it is unlikely that differences in brain 3,5-THP levels between B6 and D2 mice administered 3,5-THP contributed to the differences in sensitivity to the pharmacological effects of 3,5-THP.

Comparisons between endogenous 3,5-THP levels and plasma 3,5-THP concentrations after injection suggest that exogenous administration of low doses of 3,5-THP may lead to physiologically relevant concentrations. Doses of 3,5-THP ranging from 3.2 to 32 mg/kg were significantly anticonvulsant in B6 mice, with doses of 3.2 and 10 mg/kg producing significant anxiolysis in these animals. Although data are limited, endogenous plasma 3,5-THP levels of 30 ng/ml or 100 nM have been reported to occur under some conditions (Paul and Purdy, 1992). Therefore, the 3.2 mg/kg dose of 3,5-THP, which produced a 34 ng/ml plasma level in B6 mice and was anxiolytic and anticonvulsant in these animals, might be physiologically relevant. Administration of the 5 or 10 mg/kg doses produced plasma 3,5-THP concentrations that were at least 2- to 3-fold higher than published endogenous levels measured after swim stress or during pregnancy (Paul and Purdy, 1992). Because recent work demonstrated that endogenous 3,5-THP levels were higher in brain than in plasma (Corpechot et al., 1993), the possibility exists that the plasma 3,5-THP concentrations achieved after exogenous administration of 5 or 10 mg/kg 3,5-THP in the present studies may more closely reflect values attainable in the brain under some circumstances.

The strain differences in sensitivity to the anticonvulsant effect of 3,5-THP were similar in the two experiments, in which animals were injected 15 min (experiment 1) vs. 50 min (experiment 2) before infusion of PTZ. Although there was a slight decrease in the protection provided by 3,5-THP in the animals tested at 50 min after injection, the dose-dependent increase in PTZ threshold was still evident in B6 mice and lacking in D2 mice. This time-dependent decrease in sensitivity to the anticonvulsant effect of the same dose of 3,5-THP in experiment 2 vs. experiment 1 is exemplified by the differences between the two experiments in plasma 3,5-THP concentrations after the same dose of 3,5-THP (e.g., 10 mg/kg). These results also are consistent with recent work, which determined that protection by 3,5-THP against PTZ-induced convulsions peaked at 15 min after injection of 3,5-THP, had decreased to approximately 60% by 60 min after injection and was no longer apparent at 180 min after injection (Kokate et al., 1994). More importantly, there was no difference in the PTZ thresholds of vehicle-treated animals tested at 15 or 50 min after injection. This emphasizes that the series of behavioral tests did not alter basal seizure susceptibility to PTZ.

The strain difference in sensitivity to the anticonvulsant effect of 3,5-THP was not generalized across all seizure measures. B6 mice were more sensitive than D2 mice to the 3,5-THP-induced increase in the threshold dose of PTZ for onset to MC twitch and FF clonus. The two strains had similar increases in the threshold dose for onset to PTZ-induced RB clonus and THE after administration of 3,5-THP. These strain differences in anticonvulsant sensitivity to 3,5-THP, which vary with the seizure measure, may be related to the different anatomical systems that are believed to underlie the two major types of convulsions. Specifically, forebrain substrates (predominantly limbic) appear to be important in mediating MC twitch and FF clonus, whereas brainstem circuitry (e.g., caudal midbrain and pontine reticular formation) appears to be important for RB clonus and THE (for review, see Gale, 1988). Because there are brain regional variations in the distribution of GABA_A receptor subunit mRNAs (Vicini, 1991; Laurie et al., 1992; Wisden et al., 1992) and in the potency of 3,5-THP as a modulator of the GRC (Gee and Lan, 1991; Sapp et al., 1992), it is possible that the potency of 3,5-THP at its site on the GRC in these two strains may differ within specific brain regions. In other words, increased sensitivity of GABA_A receptors in the hippocampus, amygdala and other limbic structures to 3,5-THP in B6 vs. D2 mice would be consistent with the increased sensitivity to 3,5-THP-induced anxiolysis and anticonvulsant effects, as measured by the threshold dose for onset to MC twitch and FF clonus, in B6 vs. D2 mice. Likewise, the similar increase in threshold dose for onset to RB clonus and THE in B6 and D2 mice suggests that the sensitivities of GABA_A receptors to 3,5-THP in brainstem circuitry are similar in these animals.

It is noteworthy that administration of the 10 mg/kg dose of 3,5-THP to B6 mice was anxiolytic and anticonvulsant and stimulated activity but did not produce muscle relaxation or ataxia. This result is consistent with recent findings in both genetically heterogeneous and inbred mouse strains (Weiland et al., 1995) and suggests that the pharmacological effects of 3,5-THP can be dissociated in some genotypes. However, in the present studies D2 animals were sensitive to the anxiolytic and anticonvulsant effects of 3,5-THP only at doses that also produced muscle relaxation and ataxia.

Overall, the present results suggest that there are genetic differences in sensitivity to 3,5-THP, which vary, depending on the pharmacological effect. Coupled with recent results obtained in the selectively bred Withdrawal Seizure-Prone and -Resistant mice, indicating that chronic ethanol treatment significantly decreased endogenous 3,5-THP levels only in Withdrawl Seizure-Prone mice (Finn et al., 1994), these results are consistent with the hypothesis that genetic differences in neuroactive steroid sensitivity and biosynthesis may contribute to ethanol withdrawal severity. In addition, the differences in sensitivity to 3,5-THP found in B6 and D2 mice can be further evaluated, because these animals are the progenitor strains from which 26 recombinant inbred strains have been derived by F2 crosses (i.e., BXD Recombinant Inbred strains). Therefore, future studies can use this genetic animal model to determine genetic correlations between measures of 3,5-THP sensitivity and ethanol-related traits, including withdrawal severity.