Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice have been selectively bred to have severe (WSP) or mild (WSR) handling-induced convulsions (HICs) after withdrawal from chronic ethanol. By the 11th selected generation, withdrawal from exposure to ethanol vapor for 72 h, measured by the increase in HICs after removal from the inhalation chambers, was more than 10-fold greater in the WSP versus the WSR lines (Crabbe et al., 1985). Even though both lines were exposed to equivalent ethanol exposure, HIC scores in the ethanol-withdrawing WSR mice were negligible. This enhanced sensitivity of WSP mice to withdrawal induced by chronic ethanol generalized to withdrawal produced by chronic exposure to barbiturates, diazepam, and nitrous oxide (Kosobud and Crabbe, 1995; Metten and Crabbe, 1996). WSP mice also experience significant elevations in withdrawal HIC scores several hours after a single injection of various compounds with sedative effects on the central nervous system (i.e., acute withdrawal). When given convulsant or anticonvulsant treatments at the peak of acute ethanol withdrawal, WSP mice were more sensitive to N-methyl-D-aspartate (NMDA) and dizocilpine, and were marginally more sensitive to pentylenetetrazol (PTZ) and kainic acid, than nonwithdrawing WSP mice (Crabbe et al., 1993). Interestingly, naive WSP and WSR mice do not differ in sensitivity to ethanol-induced hypnosis, hypothermia, or locomotor activation, nor do they differ in tolerance to ethanol-induced hypothermia. These results suggest that largely independent genetic factors affect ethanol sensitivity, tolerance, and dependence.

It is unlikely that the WSP and WSR lines have been selected for large general differences in central nervous system excitability. Even though the WSP line exhibits basal HICs and has slightly greater threshold sensitivity than WSR mice to several convulsants (Crabbe and Kosobud, 1990), WSR mice are more sensitive than WSP to NMDA-induced convulsions when the drug is administered by tail vein (Kosobud and Crabbe, 1993). In addition, the lines do not differ in the rate of development of kindled seizures induced by repeated PTZ injections. Therefore, an investigation into the mechanisms responsible for the differential seizure susceptibility between the lines during ethanol withdrawal would provide insights into the mechanisms underlying ethanol withdrawal.

Exposure to chronic ethanol produces well documented ion channel adaptations (Crews et al., 1996). Briefly, -aminobutyric acid (i.e., GABAergic) inhibitory neurotransmission is reduced, whereas glutamatergic excitatory transmission is increased following chronic ethanol administration (Morrow, 1995; Tabakoff and Hoffman, 1996). An increase in the number of voltage-sensitive calcium channels after chronic ethanol administration also increases neuronal excitability (Little, 1991). Overall, the resultant changes in ion channel sensitivity or number lead to decreased inhibitory and increased excitatory receptor function during withdrawal from chronic ethanol exposure.

The present studies were conducted to investigate further the seizure susceptibility differences between WSP and WSR mice during ethanol withdrawal after chronic ethanol exposure. It was hypothesized that the genetically based difference in ethanol withdrawal severity between WSP and WSR mice would be manifest as a specific pattern of changes in seizure susceptibility to some, but not all, convulsants. That is, the purpose of the present studies was to determine whether any specific set of drugs eliciting convulsions via neurochemical pathways active during ethanol withdrawal would show large line differences in sensitivity (i.e., WSP > WSR), which might approach the magnitude of the selected line difference in HIC severity observed during withdrawal from chronic ethanol. Due to the demonstrated changes in sensitivity of -aminobutyric acid_A (GABA_A) and NMDA receptors after exposure to chronic ethanol, the present studies tested ethanol-withdrawing WSP and WSR mice for sensitivity to convulsants active at GABA_A and excitatory amino acid (EAA) receptors. We also tested the lines for sensitivity to strychnine, as sensitivity to this convulsant was recently reported to be unaltered during ethanol withdrawal (Gonzalez, 1993), and its mechanism of action does not involve GABA_A or EAA systems (Curtis et al., 1971). Mice were tested after exposure to ethanol vapor for 24 or 72 h to determine whether varying the duration of ethanol exposure resulted in different changes in sensitivity to convulsive stimuli.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Subjects. Two genetically independent WSP (WSP-1 and -2) and WSR (WSR-1 and -2) lines have been bred from a genetically heterogeneous stock of known composition (i.e., HS/ibg) and proceeded by within-family, bidirectional selection with replicate and control lines. The genetic selection pressure used in developing these lines has been described (Crabbe et al., 1985). Male mice from all four lines were used in the experiments. The animals were bred in the Veterinary Medical Unit at the Veterans Affairs Medical Center (Portland, Oregon). All animals were naive at the time of testing. Mice were maintained in groups of four in individually ventilated cages (Thorens) with ad libitum access to food and water under a 12:12 h light/dark cycle at 26 ± 1°C. At the time of testing, the mice were from selected generation 26 (filial generations 50-69) and were 70 to 104 days old. All procedures complied with the United States Public Health Service-National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the two local Institutional Animal Care and Use Committees.

Chronic Ethanol Administration. Drug-naive male WSP and WSR mice from both replicate lines were exposed to ethanol vapor or air for 24 or 72 h using a standard method for inducing ethanol dependence developed by Crabbe and colleagues (Terdal and Crabbe, 1994). The goal was to expose mice to a chronic blood ethanol concentration (BEC) in the 1.0 to 1.5 mg/ml range for the 24-h exposure and in the 1.5 to 2.0 mg/ml range for the 72-h exposure. During the experiment, the animals were housed in stainless steel 1/4-inch hardware cloth cages inside a large Plexiglas chamber. A closed food hopper and water bottle were freely available. Chamber temperatures ranged from 28-30°C.

24-h Inhalation. Mice were given an i.p. injection of saline or ethanol (2.0 g/kg for WSP-1, 2.2 g/kg for WSP-2, and 2.4 g/kg for WSR-1 and WSR-2) and placed into the inhalation chambers for 24 h. The ethanol vapor concentration was 12 to 14 mg of ethanol/liter of air for WSP and 14 to 16 mg of ethanol/liter of air for WSR mice. The differential initial dosing and chronic vapor concentrations used for the lines is required by small differences in metabolic rate for ethanol; these differences accumulate during the 24-h exposure (Terdal and Crabbe, 1994). For the saline-injected mice, the chamber contained only air.

72-h Inhalation. These animals were treated similarly to those in the 24-h paradigm, with the exception that these studies utilized the alcohol dehydrogenase inhibitor pyrazole hydrochloride (Sigma Chemical Co., St. Louis, MO) to stabilize BEC during the longer exposure to ethanol vapor (Terdal and Crabbe, 1994). On day 1, mice in the ethanol groups were weighed, injected i.p. with a priming dose of ethanol (1.75 g/kg for WSP-2, 1.5 g/kg for WSP-1, WSR-1 and WSR-2) and pyrazole (1.2 mmol/kg for WSP-2, 1.0 mmol/kg for WSP-1, WSR-1 and WSR-2), and exposed to ethanol vapor (8-9 mg/liter) inside the inhalation chamber. Lower chamber ethanol concentrations are required during inhibition of alcohol dehydrogenase to achieve similar chronic BEC to that of the mice in the 24-h exposure experiments. At 24 and 48 h, the animals were briefly removed from the chambers, weighed, reinjected with pyrazole and placed back into the chamber. Tail blood samples were taken from a subset of the animals each day to monitor BEC. Air-exposed animals were treated similarly, with the exception that half the animals were injected with saline (air-saline group) and the rest with pyrazole (air-pyrazole group).

BEC Determination. A 20-µl sample of blood from the tip of the tail was added to 50 µl of chilled 5% ZnSO₄ and stored on ice. Fifty microliters of 0.3 N Ba(OH)₂ and 300 µl of distilled water were added to each sample. The samples were shaken for 5 s and centrifuged for 5 min at 12,000 rpm. The supernatant was transferred to crimp-top glass vials and analyzed for ethanol concentration by gas chromatography. Four pairs of external standards of known ethanol concentration (0.5-4.0 mg/ml) were run before the samples.

Convulsion Scoring

HIC. Scoring for HIC was done according to a previously published scale (Crabbe et al., 1991). This procedure involved lifting the animal by the tail, gently spinning it 180° if necessary, and observing convulsions. HIC scores ranging from 1 to 3 required the gentle spin to elicit a tonic or clonic convulsion, whereas convulsions elicited by merely lifting the mouse by the tail were scored as 4 to 6 (Table 1).

Timed Tail Vein Infusion. The apparatus and procedure for tail vein infusion have been described in detail (Kosobud et al., 1992). Mice were administered the convulsants via a lateral tail vein. The convulsants and concentration infused were: PTZ (5 mg/ml in saline), NMDA (8 mg/ml in dH₂0), (+)bicuculline (0.06 mg/ml in acidified saline), kainic acid (5 mg/ml in saline), and strychnine (0.05 mg/ml in saline). The infusion rate was 0.5 ml/min for all convulsants except (+)bicuculline and strychnine (0.25 ml/min). The infusion was terminated once the animals had exhibited tonic hindlimb extension (THE). 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), based on infusion rate, body weight, and latency.

Statistical Analysis. For some experiments, there were differences between WSP and WSR mice in the BEC at removal from the ethanol chamber after the 24- or 72-h exposure paradigms. Because the purpose of these studies was to test seizure susceptibility in WSP and WSR mice that were matched for ethanol exposure, animals with BEC at the time of withdrawal <0.1 mg/ml and >3.0 mg/ml (24-h exposure) and <0.9 mg/ml and >2.5 mg/ml (72-h exposure) were eliminated from the statistical analyses.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

24-h Ethanol Inhalation. We initially measured seizure susceptibility to PTZ and NMDA in WSP and WSR mice exposed to a shorter chronic ethanol exposure. After the 24-h inhalation paradigm, ethanol-exposed WSP mice exhibited significant withdrawal, which was measured by the increase in hourly HIC. Peak HIC scores in WSP were attained at 3 h postremoval from the chamber, and remained at this level through the measurement at h 6 (Fig. 1). In contrast, HIC scores in the ethanol-exposed WSR did not increase after removal from the inhalation chamber. Mean BEC in the animals that were scored for HIC did not differ significantly (1.23 ± 0.34 for WSP and 1.04 ± 0.34 for WSR). Therefore, separate groups of ethanol- and air-exposed mice were tested for seizure susceptibility between 6.5 and 8 h post removal from the chambers (i.e., during peak withdrawal).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   HIC scores in WSP and WSR mice after exposure to 24 h of ethanol vapor. Values represent the mean ± S.E.M. for eight animals per selected line. The absence of error columns indicates that the S.E.M. was contained within the symbol.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   PTZ seizure susceptibility after exposure to 24 h of ethanol vapor or air. During peak withdrawal, mice were administered PTZ and observed for onset to MC twitch (A), FF clonus (B), RB clonus (C), and THE (D). Values represent the mean ± S.E.M. for 18 to 20 mice per selected line for air-exposed and 25 to 34 for ethanol-exposed animals. Note that the y-axis ranges from 0 to 40 mg/kg PTZ for MC twitch and FF clonus and from 0 to 125 mg/kg PTZ for RB clonus and THE.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   NMDA seizure susceptibility after exposure to 24 h of ethanol vapor or air. WSP and WSR mice were administered NMDA during peak withdrawal and observed for onset to RB clonus (A) and THE (B). Values represent the mean ± S.E.M. for 11 mice per selected line for air-exposed and 15 to 16 for ethanol-exposed animals. +p < .005 versus air-exposed WSP; **p < .01 versus air-exposed WSR

72-h Ethanol Inhalation. For the next series of experiments, we increased the duration of ethanol or air exposure from 24 to 72 h. Separate groups of mice were tested for sensitivity to two convulsants active at GABA_A (i.e., PTZ and (+)bicuculline) and EAA (i.e., NMDA and kainic acid) receptors in addition to the glycine antagonist, strychnine.

HIC Scores. Mean BEC in the animals which were scored for HIC after removal from the inhalation chambers was 1.47 ± 0.12 mg/ml for WSP and 1.44 ± 0.10 mg/ml for WSR. As with the 24-h paradigm, withdrawal from 72-h chronic ethanol significantly increased HIC scores in WSP, but not in WSR, mice (Fig. 4). Peak withdrawal in WSP mice was achieved at the 5-h measurement and remained at this level through the 8-h measurement. Therefore, separate groups of mice were tested for seizure susceptibility from 6.5 to 8 h after removal from the inhalation chambers (i.e., during peak withdrawal).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   HIC scores in WSP and WSR mice after exposure to 72 h of ethanol vapor or air. Values represent the mean ± S.E.M. for 15 to 16 animals per selected line and treatment. The absence of error columns indicates that the S.E.M. was contained within the symbol.

GABA_A Receptor Sensitivity. Mean ± S.E.M. BEC upon removal from the inhalation chamber was 1.40 ± 0.16 mg/ml for WSP and 1.16 ± 0.06 mg/ml for WSR mice that were subsequently tested for sensitivity to PTZ during peak withdrawal. These BEC values did not differ statistically. Withdrawal from chronic ethanol exposure significantly increased sensitivity to PTZ versus the respective control animals (Fig. 5). The threshold doses for onset to MC twitch and FF clonus were both significantly reduced by ethanol treatment [F_(1,94)  5.0, p < .03]. For these two convulsion measures, there was a significant effect of line [F_(1,94)  3.9, p < .05], with WSR mice more sensitive to PTZ than WSP mice. Both RB clonus and THE were reduced significantly by ethanol treatment [F_(1,91) > 26.02, p < .0001]. THE dose was significantly affected by line [F_(1,91) = 3.86, p = .05], but for this convulsion measure the WSP mice were more sensitive than the WSR mice. The interaction between treatment and line was not significant for any of the four convulsion measures. These results indicate that withdrawal from exposure to 72-h ethanol vapor produced similar increases in sensitivity to PTZ in WSP and WSR mice. This finding is similar to that observed after shorter ethanol exposure (i.e., 24 h).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   PTZ seizure susceptibility after exposure to 72 h of ethanol vapor or air. Mice were administered PTZ during peak withdrawal and observed for onset to MC twitch (A), FF clonus (B), RB clonus (C), and THE (D). Values represent the mean ± S.E.M. for 31 to 32 mice per selected line for the collapsed air-exposed and 15 to 16 for the ethanol-exposed animals. Note that the y-axis ranges from 0 to 40 mg/kg PTZ for MC twitch and FF clonus and from 0 to 125 mg/kg PTZ for RB clonus and THE.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   (+)Bicuculline seizure susceptibility after exposure to 72 h of ethanol vapor or air. Mice were administered (+)bicuculline during peak withdrawal and observed for onset to MC twitch (A), FF clonus (B), RB clonus (C), and THE (D). Values represent the mean ± S.E.M. for 23 to 29 mice per selected line for the air-exposed and 14 to 15 for the ethanol-exposed animals. Note that the y-axis ranges from 0 to 1 mg/kg (+)bicuculline for MC twitch and FF clonus and from 0 to 2 mg/kg (+)bicuculline for RB clonus and THE.

EAA Receptor Sensitivity. WSP and WSR mice that were matched for ethanol exposure (mean ± S.E.M. BEC = 1.96 ± 0.07 mg/ml for WSP and 1.97 ± 0.05 mg/ml for WSR) were administered NMDA during peak withdrawal (Fig. 7). For both RB clonus and THE threshold doses, WSR mice were more sensitive than WSP to NMDA [F_(1,160) > 10.12, p < .002], but there was no significant main effect of treatment. However, the interaction between main effects was significant for both RB clonus and THE [F_(1,160)  4.62, p  .04]. Subsequent analyses indicated that there was a significant effect of line in the air groups (p < .0001 for both convulsion measures), and a significant effect of treatment in the WSR mice (p < .05 for RB clonus and p < .01 for THE). As with the 24-h ethanol exposure, these results indicate that the lines differ in basal sensitivity to NMDA (i.e., WSR > WSP) and that ethanol withdrawal decreased sensitivity to NMDA in the WSR, but not in WSP, mice.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   NMDA seizure susceptibility after exposure to 72 h of ethanol vapor or air. Mice were administered NMDA during peak withdrawal and observed for onset to RB clonus (A) and THE (B). Values represent the mean ± S.E.M. for 56 to 58 mice per line for air-exposed and 20 to 27 for ethanol-exposed animals. +p < .005 versus respective air-exposed WSP; *p < .05, **p < .01 versus air-exposed WSR

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Kainic acid seizure susceptibility after exposure to 72 h of ethanol vapor or air. Mice were administered kainic acid during peak withdrawal and observed for onset to RB clonus (A) and THE (B). Values represent the mean ± S.E.M. for 34 to 39 per selected line for air-exposed and 14 to 17 for ethanol-exposed animals.

Strychnine Sensitivity. WSP and WSR mice were matched for ethanol exposure (mean ± S.E.M. BEC = 1.73 ± 0.09 mg/ml for WSPs and 1.79 ± 0.10 mg/ml for WSRs) and were administered strychnine during peak withdrawal (Fig. 9). There was no effect of treatment on the threshold dose for onset to MC twitch. However, RB clonus and THE threshold doses were significantly increased during ethanol withdrawal [F_(1,86)  26.08, p = .0001]. The lines did not differ in sensitivity to strychnine, nor was there a significant interaction between line and treatment. These results suggest that ethanol-withdrawing WSP and WSR mice have decreased sensitivity to strychnine, as measured by the threshold dose for onset to RB clonus and THE.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Strychnine seizure susceptibility after exposure to 72 h of ethanol vapor or air. Mice were administered strychnine during peak withdrawal and observed for onset to MC twitch (A), RB clonus (B), and THE (C). Values represent the mean ± S.E.M. for 28 to 32 mice per selected line for the collapsed air-exposed and 11 to 16 per line for ethanol-exposed animals for RB clonus and THE. All animals did not experience MC twitch, so the n/line was 21 to 25 for the air-exposed and 6 to 12 for the ethanol-exposed for this dependent variable.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The present findings are consistent with the hypothesis that the genetically based difference in ethanol withdrawal severity between WSP and WSR mice would reveal a specific pattern of changes in sensitivity to some, but not all convulsants. The results are summarized in Table 2. Overall, ethanol withdrawal increased sensitivity to both convulsants active at GABA_A receptors and decreased sensitivity to glycine antagonist seizures to a similar extent in WSP and WSR mice. In contrast, sensitivity to convulsants active at EAA receptors was affected differentially in the lines. These results are generally in agreement with the notion that there is a genetic relationship between ethanol withdrawal severity and sensitivity to convulsants at EAA receptors.

View this table: [in this window] [in a new window]   TABLE 2 Change in convulsant sensitivity during ethanol withdrawal Change in sensitivity (i.e., ethanol-exposed versus air-exposed) to the various convulsants administered from 6.5 and 8 h after removal from the inhalation chambers is summarized. BEC upon removal from the inhalation chambers is depicted for each study to demonstrate that the ethanol exposure was similar for the two lines. The genetic comparisons were similar for all convulsion signs, but the effects of treatment were generally greater for RB clonus and THE. For this reason, and because all drugs elicited RB clonus and THE, the interpretation summarized here is based on these two convulsion signs. Even though MC twitch and FF clonus convulsions have a physiological basis distinct from RB clonus and THE, we saw no indication of dissociation between them by either genotype or ethanol withdrawal. Therefore, we characterized the withdrawal-related effects as "seizure susceptibility" without differentiation. A significant decrease in the threshold dose for onset to convulsion in ethanol-withdrawing versus respective air controls is interpreted as an increase in sensitivity to the convulsant (depicted as ). An increase in convulsant threshold dose in ethanol- versus air-exposed mice is interpreted as a decrease in sensitivity to the convulsant (depicted as ). The presence of two arrows indicates that the magnitude of change in sensitivity for that drug was greater in the WSP than the WSR mice. The lack of change in sensitivity in ethanol- versus air-exposed mice is depicted ().

In the present studies, sensitivity to convulsants active at GABA_A receptors significantly increased during ethanol withdrawal in both WSP and WSR mice (Table 2). These results are consistent with previous findings that indicated that sensitivity of GABA_A receptors to agonist ligands was decreased, whereas sensitivity to antagonist ligands was increased (reviewed in Morrow, 1995). After chronic exposure to an ethanol-containing liquid diet, increased sensitivity to (+)bicuculline was demonstrated in ethanol-withdrawing rats versus animals that consumed control diet (Devaud et al., 1995, 1996). Increased sensitivity to PTZ has also been demonstrated in rats receiving chronic intermittent ethanol versus controls (Kokka et al., 1993). Therefore, the similar increase in sensitivity to convulsants active at GABA_A receptors in both the ethanol-withdrawing WSP and WSR lines suggests that alterations in GABA_A receptor sensitivity or function appear to be important for alcohol withdrawal, but may not be involved in those aspects of withdrawal convulsion severity which is determined by genetic factors.

It is curious that withdrawal from chronic ethanol exposure did not reveal any differences between the lines in sensitivity to PTZ or (+)bicuculline, because microsacs prepared from ethanol-dependent WSP and WSR mice have been reported to differ in sensitivity of GABA_A receptors to benzodiazepine-inverse agonists (Buck et al., 1991b). Ethanol-dependent as well as ethanol-naive WSP and WSR mice also differ in the expression of specific GABA_A receptor subunit mRNAs (Buck et al., 1991a; Keir and Morrow, 1994). It is noteworthy that a decrease in expression of the 1 subunit mRNA, which was reported in WSP but not WSR mice, also has been consistently demonstrated in mice, rats, and neuronal cell cultures that were exposed to chronic ethanol (Morrow, 1995; reviewed in Crews et al., 1996). This finding suggests the possibility of a genetic link between subsequent alcohol withdrawal severity and decreased level of the 1 subunit mRNA.

Although the supposition of a genetic link between expression of GABA_A receptor subunit mRNAs and ethanol withdrawal severity does not seem consistent with the present results, it is possible that the time course for genetic differences in ethanol withdrawal-related neurochemical changes and behavioral manifestations of excitability may differ. That is, the documented differences between WSP and WSR in GABA_A receptor function or expression of the 1 subunit mRNA utilized animals that were intoxicated (i.e., at the initiation of withdrawal; Buck et al., 1991a,b), whereas the present studies were conducted on animals during peak withdrawal, when BEC was near zero. After exposure to chronic ethanol (Sanna et al., 1993), changes in receptor binding and GABA-stimulated chloride uptake were observed at 1 to 3 h, whereas symptoms of withdrawal were observed at 12 to 23 h, consistent with the notion that the time course for neurochemical and behavioral changes during withdrawal may differ.

Sensitivity to convulsants active at EAA receptors was differentially affected by ethanol withdrawal in WSP and WSR mice (Table 2). Therefore, the difference between WSP and WSR mice in sensitivity to convulsants active at EAA receptors after exposure to chronic ethanol is consistent with the selected line difference in ethanol withdrawal severity (i.e., decreased susceptibility to NMDA in WSR mice and increased susceptibility to kainic acid in WSP mice).

Chronic ethanol exposure has been reported to increase the number of binding sites for several NMDA receptor ligands (reviewed in Crews et al., 1996; Tabakoff and Hoffman, 1996). Initial studies found that ethanol-naive WSP mice had a greater number of binding sites for the NMDA receptor ion channel blocker MK-801 versus WSR (Valverius et al., 1990). After exposure to chronic ethanol, there was a significant increase in the number of binding sites in hippocampus, but not cortex, of both lines. In contrast, other studies which utilized a different chronic ethanol paradigm and equilibrium binding suggested that the characteristics of the NMDA receptor complex did not differ in hippocampus or cortex prepared from ethanol-naive and ethanol-withdrawing WSP and WSR mice (Carter et al., 1995). Recent work in rats exposed to several different chronic ethanol paradigms also did not observe robust increases in NMDA receptor binding (Rudolph et al., 1997). Therefore, it is unclear at the present time if the differential change in sensitivity to glutamatergic convulsants in ethanol-withdrawing WSP and WSR mice is paralleled by functional changes at excitatory glutamatergic receptors.

Consistent with previous findings, ethanol-naive WSR mice were more sensitive to NMDA-induced convulsions than WSP mice (Kosobud and Crabbe, 1993). There was also a trend for a line difference in basal sensitivity to kainic acid, with the WSR line more sensitive than WSP. Therefore, the increased sensitivity to convulsants active at EAA receptors in ethanol-naive WSR versus WSP mice provides additional evidence that the animals have not been selected for general differences in central nervous system excitability because naive WSP are slightly more sensitive than WSR mice to most other convulsants.

In contrast to previous findings that ethanol-withdrawing rats did not differ in sensitivity to strychnine-induced convulsions versus controls (Gonzalez, 1993), the present results indicate that sensitivity to strychnine was decreased in both WSP and WSR mice exposed to chronic ethanol. It is unlikely that the decreased sensitivity was due to residual BEC in the animals because the BEC at 6.5 h in a group of mice treated similar to those in the present studies was less than 0.25 mg/ml, and our unpublished studies indicated that blood and brain ethanol concentrations  0.5 mg/ml were not anticonvulsant versus NMDA. Therefore, the decreased sensitivity to strychnine in ethanol-withdrawing WSP and WSR mice and decreased sensitivity to NMDA in ethanol-exposed WSR mice may result from a differential time course for adaptation to chronic ethanol in different neurochemical systems. This notion is consistent with recent work which found that the ethanol withdrawal-related alterations in response to a variety of convulsive stimuli differed in both direction and in time course (Sanna et al., 1993; Watson and Little, 1995).

The present findings contrast with recent work that measured the time course for changes in sensitivity to convulsive stimuli during ethanol withdrawal after a liquid diet in mice (Watson and Little, 1995). The authors reported a nonsignificant decrease in sensitivity to (+)bicuculline and the benzodiazepine receptor inverse-agonist methyl-6,7-dimethoxy-4-ethyl--carboline-3-carboxylate at 4 and 8 h into the withdrawal period. Even though the change in sensitivity to (+)bicuculline and methyl-6,7-dimethoxy-4-ethyl--carboline-3-carboxylate did not reach statistical significance in this study, the pattern of change in sensitivity to GABAergic convulsants during ethanol withdrawal (i.e., increased sensitivity) was similar to that observed in the present and previous studies (Kokka et al., 1993; Devaud et al., 1995, 1996). In addition, sensitivity to NMDA was significantly increased at 16 h into the withdrawal period, whereas sensitivity to kainic acid was unchanged between 4 and 24 h of withdrawal (Watson and Little, 1995). These differences between studies in the change in sensitivity to convulsants during withdrawal may be due to the different chronic ethanol paradigms which were utilized, the time points during withdrawal which were studied, the sensitivity of the methods, or the genotypes utilized.

The change in sensitivity to PTZ and NMDA was similar in ethanol-withdrawing WSP and WSR mice exposed to ethanol vapor for either 24 or 72 h. This demonstrates that pyrazole, which was used in the 72-h paradigm, is not an important factor in the results. More important, the similar changes in sensitivity to PTZ and NMDA suggest that the chronic ethanol-induced neurochemical adaptations in GABA_A or NMDA receptors, which underlie these differences in seizure susceptibility, are similar in the two durations of ethanol exposure and are occurring as early as 24-h exposure to chronic ethanol.

Overall, the present results suggest that there is a genetic relationship between ethanol withdrawal severity and sensitivity to convulsants active at EAA receptors. In addition, the opposing changes in seizure susceptibility during ethanol withdrawal (i.e., increases, decreases, or no change) to convulsants with different mechanisms of action suggest a different time course for adaptation of the many neurochemical systems that are influenced by exposure to chronic ethanol. Clearly, the pattern of neurochemical changes produced after exposure to chronic ethanol are complex. Additional studies are necessary to determine whether changes in a particular neurochemical system contribute to one specific symptom of ethanol withdrawal or whether alterations in one neurochemical system generate a cascade of neurochemical changes that manifest as the withdrawal syndrome.