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Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice
European Journal of Pharmacology, 157 (1988) 147-154
147
Elsevier EJP 50533
Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice D a n i e l J. F e l l e r
1,2,.,
R. A d r o n H a r r i s 3 a n d J o h n C. C r a b b e 1,2
1 Research Service, VA Medical Center, Portland, OR 97201, : Departments of Medical Psychology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201, and 3 Research Service, VA Medical Center and Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, U.S.A.
Received 25 April 1988, revised MS received 3 August 1988, accepted 30 August 1988
Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice~ have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [3Ss]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [ 3H]flunitrazepam and the potency of "t-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities. Picrotoxin; Bicuculline; Pentylenetetrazole; Convulsions (handling-induced); [35S]TBPS; [3H]Flunitrazepam; (Withdrawal seizure-prone and withdrawal seizure-resistant lines, Selected lines, Pharmacogenetics)
1. Introduction Seizures are a c o m m o n sign of withdrawal behavior after chronic ethanol exposure (Friedman, 1980). Mice chronically intoxicated with ethanol and then withdrawn have increased susceptibility to handling-induced convulsions (Goldstein, 1972). F o u r lines of mice have been genetically selected based on the severity of handling-induced convulsions observed during withdrawal from chronic ethanol exposure (Crabbe et al., 1985). T w o lines of withdrawal seizure prone (WSP-1 and WSP-2)
* To whom all correspondence should be addressed: Research Service (151-W), VA Medical Center, Portland, OR 97201, U.S.A.
and two lines of withdrawal seizure resistant (WSR-1 and WSR-2) mice have been developed which differ by at least 10-fold in withdrawal handling-induced convulsion severity. Both W S P lines also exhibit more severe tremors than both W S R lines while undergoing withdrawal (Kosobud and Crabbe, 1986). These lines should provide a useful experimental model for studying the mechanism by which convulsant activity develops during withdrawal, and the relationship between withdrawal behavior and alcohol dependence. Since neither the two W S P lines nor the two W S R lines differ from each other on any relevant variables, these lines will henceforth be referred to as W S P and WSR, respectively. We considered the possibility that the difference in the handling-induced convulsion scores
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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of WSP compared to WSR mice simply reflects a non-specific difference in CNS excitability. However, the EDs0 of strychnine, flurothyl, GABA antagonists, and the CAs0 of electroconvulsive shock to produce a maximum seizure response did not differ significantly between the lines (McSwigan et al., 1984). It is also possible that eliciting a maximum seizure response may not be a sensitive enough approach for comparing the general seizure susceptibility of WSP and WSR mice to treatments other than ethanol withdrawal. The handling-induced convulsion behavior is likely a more subtle measure of CNS excitability. The initial experiments described in this paper were designed to test the hypothesis that WSP mice were more sensitive than WSR mice to the effect of subconvulsant doses of the GABA antagonists picrotoxin, bicuculline and pentylenetetrazole to exacerbate handling-induced convulsions. Acute and chronic ethanol treatment have been reported to alter brain GABA concentrations and to affect the GABA-benzodiazepine-picrotoxin receptor ionophore system at multiple sites (for review see Hunt, 1983; 1985; Allan and Harris, 1987). GABA agonists and inhibitors of GABA degradation can attenuate some signs of ethanol withdrawal behavior, while GABA antagonists potentiate withdrawal signs (Goldstein, 1973; Hillbom, 1975; Cooper et al., 1979; Frye et al., 1983a,b). [35S]t_Butylbicyclophosphorothionate (TBPS), a cage convulsant, binds to the GABAchloride channel complex at the same site as picrotoxin and its binding is modulated by GABA agonists, depressant and convulsant drugs (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984). It has been proposed that pentylenetetrazole's mechanism of action to cause convulsions involves the picrotoxin-TBPS binding site (Ramanjaneyulu and Ticku, 1984; Maksay and Ticku, 1984). Bicuculline specifically acts to block the binding of GABA to its receptor site. The GABA-chloride channel complex also contains a site which binds benzodiazepines and is allosterically coupled to the G A B A site. Benzodiazepines enhance GABA mediated chloride flux; conversely, GABA increases the affinity of [3H]flunitrazepam. Other sedative-hypnotic drugs which interact with this system can also cause
withdrawal seizure behavior in WSP and WSR mice: significantly higher handling-induced convulsion scores measured during withdrawal were observed in WSP compared to WSR mice after they were made physically dependent on either phenobarbital or diazepam (Belknap et al., 1988; in press). The reported experiments were designed to see whether WSP and WSR mice exhibited different dose response curves for GABA antagonist-stimulated handling-induced convulsions. If so, this could be due to a change in either the density or affinity of the receptor for the drugs. Alternatively, there may be a change in either the GABA or benzodiazepine receptor. These hypotheses were tested by determining the density and affinity of [35S]TBPS and [3H]flunitrazepam binding sites in WSP and WSR mice.
2. Materials and methods
2.1. Materials Picrotoxin, bicuculline and pentylenetetrazole were purchased from Sigma Chemical Co. (St. Louis, MO). [35S]TBPS (60 C i / m m o l ) and [3H] flunitrazepam (90 C i / m m o l ) were obtained from New England Nuclear Co. (Boston, MA). 2.2. Animals Two WSP and two WSR lines of mice, which have been selectively bred for severity of alcohol withdrawal convulsions and maintained in our colonies in Portland, were used. Males and females from selected generations 8-23 were used in the behavioral and binding experiments as indicated in the Results section. Mice ranged in age from 11 to 14 weeks. All mice were naive at the time of testing. 2.3. Handling-induced convulsion scoring Handling-induced convulsion scores were obtained by a procedure described by Goldstein (1972) modified by Crabbe et al. (1985). A mouse was lifted by the tail and observed for convulsions. If a convulsion was not observed the mouse
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was gently spun 180 ° C and examined. A scale of 0-4 was used, as follows; 4, severe tonic-clonic convulsion when lifted by the tail, with a quick onset and long duration, often continuing for several seconds after the mouse is released; 3, tonic-clonic convulsion when lifted by the tail, often with onset delayed by as much as 1-2 s; 2, tonic convulsion when lifted by the tail or tonicclonic convulsion after gentle spin through a 180 o arc; 1, no convulsion when lifted by the tail, but tonic convulsion elicited by spin; 0.5, only facial grimace after spin; and 0, no convulsion. A baseline assessment of handling-induced convulsions was made in each mouse 18 h before drug administration. Handling-induced convulsion scores were measured every 20 rain after picrotoxin administration for 2 h. Handling-induced convulsion measurements were made every 30 rain for 180 min after bicuculline or pentylenetetrazole injection.
2.4. Drug administration Picrotoxin was dissolved in saline and administered by i.p. injection; mice not given drug were injected with saline. Doses were 2, 4, 5, 6 or 8 m g / k g b o d y weight. Pentylenetetrazole was dissolved in saline and administered i.p. 60 m g / k g body weight. Bicuculline was solubilized in several drops of concentrated HC1, brought to p H 7.0 with N a O H , and to a final volume in saline. Mice received 3 m g / k g body weight.
2.5. [35S]TBPS binding assay Naive mice from the WSP and WSR lines were killed by cervical dislocation and decapitated. Brains were quickly removed and dissected into the frontal cortex, remainder of the cortex, hippocampus and cerebelleum according to the procedure of Heffner et al. (1980). Dissected regions from three mice were pooled and placed in 5 ml of 0.32 M sucrose, 3 m M HEPES buffer at p H 7.4 and 4 ° C. The samples were homogenized using 10 complete passes with a motor-driven Thomas ' C ' teflon glass homogenizer, then centrifuged at 1 000 x g for 10 min at 4 ° C . The supernatant was centrifuged at 100000 X g for 30 rain at 4 ° C . The pellet was resuspended in 3 ml of water with a
Polytron, brought to 6 ml and centrifuged at 1 0 0 0 0 0 x g for 30 min at 4 ° C . This step was repeated except with 50 m M citrate-Tris buffer, p H 7.2. Pellets were resuspended in citrate buffer and frozen at a - 8 0 ° C until assayed. Samples were thawed, centrifuged and washed once more with citrate buffer. For the binding assay the pellet was resuspended in 4 ml of assay buffer which contained 200 m M NaCI, 50 m M N a K P O 4 and 10 m M HEPES, p H 7.4. The total assay volume was 1000 #1 and consisted of 0.3 mg protein/ml, increasing concentrations of [35S]TBPS and assay buffer. Non-specific binding was determined by adding 100 ~tM picrotoxin. The mixture was incubated for 2 h at room temperature, then diluted with 5 ml of assay buffer and filtered over GB100R glass fiber filters (Microfiltration Systems, Dublin, CA). The filters were washed twice with 5 ml of assay buffer at room temperature and then counted by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al. (1951).
2.6. [ 3H]Flunitrazepam binding assay Animals were killed by cervical dislocation and decapitation. Whole brains were quickly removed, frozen on dry ice and stored at - 8 0 ° C until assayed. Samples were weighed while still frozen and thawed in 50 m M NaKPO4, p H 7.4. The brains were homogenized in 35 ml of 50 m M N a K P O 4 buffer, p H 7.4, using a Brinkman homogenizer at a setting of 6 for 10 s and centrifuged at 40 000 x g for 10 min. This step was repeated four times. The final pellet was resuspended in 240-300 volumes of assay buffer which contained 50 m M N a K P O 4, 200 m M NaC1 at p H 7.4. The assay volume was 1 000 ~1 and contained approximately 0.4 mg p r o t e i n / m l . The K D and Bma× for [3H]flunitrazepam binding was determined by adding a fixed concentration of the labeled drug, 0.58 nM, and an increasing concentration of unlabeled flunitrazepam, 1-1000 nM. The mixture was incubated for 30 min at 3 7 ° C and the reaction terminated by filtration over W h a t m a n G F / B filters. The filters were washed twice with 4 ml of assay buffer at 4 ° C , then counted by liquid scintillation spectroscopy. The K D and Bmax val-
150 and pentylenetetrazole; fig. 3 shows the handlinginduced convulsion scores at 30 and 90 min. All three G A B A antagonists caused an initial increase in handling-induced convulsion scores for WSP and W S R mice. At 30 min WSP mice were more sensitive than W S R mice to the enhancement of handling-induced convulsions by bicuculiine (P < 0.01) and pentylenetetrazole (P < 0.05), but their response to picrotoxin did not differ. By 90 min after injection, handling-induced convulsion severity in W S R mice had declined to the level of the saline-treated group for all drugs, while scores for WSP mice treated with picrotoxin or pentylenetetrazole remained significantly elevated (P < 0.01). The results for picrotoxin and bicuculline have been replicated using mice from different selected generations. One potential explanation for the differential sensitivity to picrotoxin and pentylenetetrazole between WSP and W S R mice could be a difference either in density or affinity of the receptor for picrotoxin. In preliminary experiments we found no consistent differences between the lines in TBPS binding to the whole brain from WSP versus W S R mice (densities of 0.9-1.3 p m o l / m g protein and
ues were determined using the computer program of MacPherson (1985) based on the method of Munson and Rodbard (1980). The density of sites was expressed as p m o l / m g protein. In experiments testing the potency of G A B A to increase [3H]flunitrazepam binding the concentration of labeled ligand was 1 nM.
3. Results Picrotoxin, bicuculline and pentylenetetrazole exacerbated handling-induced convulsions in both lines of mice. The WSP and W S R lines exhibited a different time course for the development of handling-induced convulsions, which is shown for 6 m g / k g picrotoxin in fig. 1. Both groups peaked at around 20 min (P < 0.01), but the decay in response was slower in WSP mice (P < 0.05 at 120 min). The area under the time course curve, which represents the total handling-induced convulsion score, was significantly greater (P < 0.01-0.001) in the WSP compared to the W S R line at all drug doses tested (fig. 2). Similar results were observed for WSP and W S R mice treated with bicuculline
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values are the mean + S.E. handling-induced convulsion scores for 5-8 mice/group.
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Fig. 3. Effect of bicuculline (BIC, 3 m g / k g ) , pentylenetetrazole (PTZ, 60 m g / k g ) and picrotoxin (PIC, 4 m g / k g ) on handlinginduced convulsions in WSP and WSR mice from selected generation 8. The values are the mean + S.E. handling-induced convulsion scores for 8-11 m i c e / g r o u p .
K D was 39-44 nM, respectively). However, the selection pressure for WSP versus W S R mice may only have altered receptor properties in a discrete region of the brain which may not have been detected in an assay of the whole brain. Therefore, the binding of TBPS was characterized in the frontal cortex, remainder of the cortex, hippocampus and cerebellum. The results are presented in table 1. In agreement with the results from the whole brain there was no significant difference between WSP and W S R mice in TBPS binding to any area. An alternate site of action may involve the receptors for flunitrazepam and G A B A on the GABA-chloride channel complex. The density of [3H]flunitrazepam receptors in whole brain from W S R mice (selected generation 23) was 1.95 _+ 0.06
152 TABLE 1 [35S]TBPS binding to different regions of the brain from WSP and W S R mice a. Region
N
Frontal cortex Remainder cortex Hippocampus Cerebellum
K D (nM)
3 3 3 2
Bmax ( p m o l / m g protein)
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WSP
WSR
WSP
42.6 ± 15.7 b 38.8 +- 10.0 48.1 + 8.5 47.6 ± 20.9
42.1+ 6.9 37.1 ± 6.6 42.5 ± 10,5 53.3 +__2%8
0.37+0.13 1.00±0.28 0.21 ±0.04 0.40 ± 0.15
0.34 ± 0.05 0.95+_0.21 0.25 ±0,08 0.30 +_0.06
a Selected generations 20-21. b Mean + S.D.
p m o l / m g protein (N = 5) versus 1.88 + 0.15 (N = 5) in WSP mice. Ligand affinities ranged from 11 to 14 nM. There were no significant differences in flunitrazepam receptor density or affinity among the four lines of mice. The binding of GABA to its receptor can decrease the affinity of TBPS and increase the affinity of flunitrazepam to their respective binding sites. A change in the coupling between these receptors might account for the differential enhancement of handling-induced convulsions by picrotoxin and pentylenetetrazole in the selected lines. As expected, the addition of increasing concentrations of GABA to the [3H]flunitrazepam binding assay elevated flunitrazepam binding to its receptor. The EDs0 for G A B A stimulation was 2 /zM; a value in close agreement with others reported in the literature (Marley and Wehner,
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1986). The WSP and WSR mice exhibited similar EDs0 values for this action of GABA (fig. 4).
4. Discussion
It had previously been shown that WSP and WSR mice from selected generations 8-10 did not differ in their sensitivity to tonic hindlimb extensor seizures induced by either electroconvulsive shock, generalized seizure-producing drugs or the GABA antagonists picrotoxin and bicuculline (McSwigan et al., 1984). However, in the present study it was found that WSP mice were more sensitive than WSR mice to subconvulsant doses of picrotoxin, bicuculline and pentylenetetrazole when they were subsequently tested for handlinginduced convulsions. Methods which produce tonic hindlimb extensor seizures are probably causing maximal CNS activity, and handling-induced seizures are more likely to reflect subtle changes in CNS excitability. The difference between W S P and WSR mice at any given dose of picrotoxin (see fig. 2) predominantly reflected a larger area under the time course curve due to a slower rate of decline in convulsions to baseline in WSP mice (see fig. 1). The response of WSP mice to pentylenetetrazole also decayed more slowly compared to WSR mice. The peak effect of bicuculline was lower in WSR compared to WSP mice. One possible explanation is that the mice may differ in their rate of metabolism or elimination of picrotoxin, bicuculline and pentylenetetrazole. However, an examination of the structures of these compounds would suggest
153 that their mode of elimination is probably not the same. Prone and resistant mice of the l l t h selected generation metabolized and eliminated ethanol at approximately the same rate, after either an acute dose or chronic treatment (Kosobud and Crabbe, 1986). In addition, acute doses of five alcohols, diazepam, and three barbiturates produced similar brain concentrations in both lines (Crabbe et al., 1986). These results suggest that WSP and WSR mice have not been selected for differences in drug metabolism. However, studies are being initiated to address this question in more detail. We considered the possibility that the prolongation of the handling-induced convulsion response in the WSP mice compared to the WSR mice may simply reflect a sensitization of the WSP mice to repeated testing. However, the group of WSP mice administered saline showed seizure scores which remained stable over repeated tests (data not shown). Furthermore, we have previously reported that repeated testing for handlinginduced convulsions leads to a slight, but significant reduction in scores over time (Crabbe et al., 1981). Thus, we discount this possibility as an explanation of the difference between WSP and WSR mice. Alternatively, the behavioral results could be explained by an alteration in the GABA-chloride channel between the WSP and WSR lines. A difference in receptor-mediated activity could involve three possible mechanisms: (1) receptor density, (2) receptor affinity or (3) the coupling process between the picrotoxin receptor and other components of the GABA-benzodiazepine-chloride channel complex. The affinity of [35]TBPS for the picrotoxin binding site in whole brain was the same for both the WSP and WSR lines. While regional differences could be more important for understanding the mechanisms involved in the development of neuroexcitability during withdrawal, the results from the present binding studies did not support the hypothesis that changes in TBPS receptors in specific brain regions mediates the alteration in picrotoxin sensitivity between WSP and WSR mice. These results do not rule out that there may be a change in more discrete regions such as the inferior colliculus in the midbrain or nuclei in the brainstem. We plan to apply
the method of receptor autoradiography in brain slices to provide this information. A change in the coupling between the picrotoxin site and either the GABA or benzodiazepine binding sites or the chloride channel is another possible explanation for the difference between WSP and WSR mice in neurosensitivity to picrotoxin and ethanol withdrawal. The proposal that behavioral selection for mice with different sensitivities to ethanol can produce lines of mice with altered GABA-chloride channel activity is supported by work on the long sleep (LS) and short sleep (SS) mice. These mouse lines were genetically selected for high and low sensitivity, respectively, to ethanol's hypnotic effect (McClearn and Kakihana, 1981). The density and affinity of [3 H]flunitrazepam binding sites in whole brain was found to be the same in both the LS and SS lines (Marley and Wehner, 1986). Comparable results were found in the current study with the WSP and WSR mice. On the other hand, the action of GABA to increase [3H]flunitrazepam binding was greater in the cortex and cerebellum of SS than in LS mice (Marley and Wehner, 1986), while differential enhancement of [3H]flunitrazepam binding to whole brain by GABA was not observed in the WSP and WSR mice. It has been shown that the GABA receptorchloride channel in the cerebellum of LS mice compared to SS mice was more sensitive to muscimol stimulated 36C1- uptake into microsacs, and to the inhibition of [35S]TBPS binding to membranes. Ethanol at revevant concentrations potentiated muscimol-stimulated chloride uptake in LS, but not SS mice (Allan and Harris, 1986). It is possible that the latter differences may be responsible for the difference between these selectively bred lines in sensitivity to the hypnotic effect of ethanol. Similar 36C1- uptake studies should be initiated on the cortex, cerebellum and hippocampus of WSP and WSR mice. In summary, we found that WSP mice were more sensitive than WSR mice to the ability of picrotoxin, bicuculline and pentylenetetrazole to potentiate handling-induced seizures. This finding cannot currently be explained by a difference in the binding characteristics of [35S]TBPS or [3H]flunitrazepam. More detailed studies of the
154
GABA-chloride channel complex in these lines will be required to elucidate the pharmacological mechanism of this behavioral difference.
Acknowledgements We thank Cathy Deutsch, Ann Kosobud, Brenda Tam, Emmett Young and Virginia Bleck for assistance in performing these experiments. These studies were supported by grants from the Veterans Administration and by PHS-ADAMHA Grants AA 03527, AA 05828, AA 06243, AA 06399 and AA 06498.
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Elsevier EJP 50533
Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice D a n i e l J. F e l l e r
1,2,.,
R. A d r o n H a r r i s 3 a n d J o h n C. C r a b b e 1,2
1 Research Service, VA Medical Center, Portland, OR 97201, : Departments of Medical Psychology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201, and 3 Research Service, VA Medical Center and Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, U.S.A.
Received 25 April 1988, revised MS received 3 August 1988, accepted 30 August 1988
Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice~ have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [3Ss]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [ 3H]flunitrazepam and the potency of "t-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities. Picrotoxin; Bicuculline; Pentylenetetrazole; Convulsions (handling-induced); [35S]TBPS; [3H]Flunitrazepam; (Withdrawal seizure-prone and withdrawal seizure-resistant lines, Selected lines, Pharmacogenetics)
1. Introduction Seizures are a c o m m o n sign of withdrawal behavior after chronic ethanol exposure (Friedman, 1980). Mice chronically intoxicated with ethanol and then withdrawn have increased susceptibility to handling-induced convulsions (Goldstein, 1972). F o u r lines of mice have been genetically selected based on the severity of handling-induced convulsions observed during withdrawal from chronic ethanol exposure (Crabbe et al., 1985). T w o lines of withdrawal seizure prone (WSP-1 and WSP-2)
* To whom all correspondence should be addressed: Research Service (151-W), VA Medical Center, Portland, OR 97201, U.S.A.
and two lines of withdrawal seizure resistant (WSR-1 and WSR-2) mice have been developed which differ by at least 10-fold in withdrawal handling-induced convulsion severity. Both W S P lines also exhibit more severe tremors than both W S R lines while undergoing withdrawal (Kosobud and Crabbe, 1986). These lines should provide a useful experimental model for studying the mechanism by which convulsant activity develops during withdrawal, and the relationship between withdrawal behavior and alcohol dependence. Since neither the two W S P lines nor the two W S R lines differ from each other on any relevant variables, these lines will henceforth be referred to as W S P and WSR, respectively. We considered the possibility that the difference in the handling-induced convulsion scores
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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of WSP compared to WSR mice simply reflects a non-specific difference in CNS excitability. However, the EDs0 of strychnine, flurothyl, GABA antagonists, and the CAs0 of electroconvulsive shock to produce a maximum seizure response did not differ significantly between the lines (McSwigan et al., 1984). It is also possible that eliciting a maximum seizure response may not be a sensitive enough approach for comparing the general seizure susceptibility of WSP and WSR mice to treatments other than ethanol withdrawal. The handling-induced convulsion behavior is likely a more subtle measure of CNS excitability. The initial experiments described in this paper were designed to test the hypothesis that WSP mice were more sensitive than WSR mice to the effect of subconvulsant doses of the GABA antagonists picrotoxin, bicuculline and pentylenetetrazole to exacerbate handling-induced convulsions. Acute and chronic ethanol treatment have been reported to alter brain GABA concentrations and to affect the GABA-benzodiazepine-picrotoxin receptor ionophore system at multiple sites (for review see Hunt, 1983; 1985; Allan and Harris, 1987). GABA agonists and inhibitors of GABA degradation can attenuate some signs of ethanol withdrawal behavior, while GABA antagonists potentiate withdrawal signs (Goldstein, 1973; Hillbom, 1975; Cooper et al., 1979; Frye et al., 1983a,b). [35S]t_Butylbicyclophosphorothionate (TBPS), a cage convulsant, binds to the GABAchloride channel complex at the same site as picrotoxin and its binding is modulated by GABA agonists, depressant and convulsant drugs (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984). It has been proposed that pentylenetetrazole's mechanism of action to cause convulsions involves the picrotoxin-TBPS binding site (Ramanjaneyulu and Ticku, 1984; Maksay and Ticku, 1984). Bicuculline specifically acts to block the binding of GABA to its receptor site. The GABA-chloride channel complex also contains a site which binds benzodiazepines and is allosterically coupled to the G A B A site. Benzodiazepines enhance GABA mediated chloride flux; conversely, GABA increases the affinity of [3H]flunitrazepam. Other sedative-hypnotic drugs which interact with this system can also cause
withdrawal seizure behavior in WSP and WSR mice: significantly higher handling-induced convulsion scores measured during withdrawal were observed in WSP compared to WSR mice after they were made physically dependent on either phenobarbital or diazepam (Belknap et al., 1988; in press). The reported experiments were designed to see whether WSP and WSR mice exhibited different dose response curves for GABA antagonist-stimulated handling-induced convulsions. If so, this could be due to a change in either the density or affinity of the receptor for the drugs. Alternatively, there may be a change in either the GABA or benzodiazepine receptor. These hypotheses were tested by determining the density and affinity of [35S]TBPS and [3H]flunitrazepam binding sites in WSP and WSR mice.
2. Materials and methods
2.1. Materials Picrotoxin, bicuculline and pentylenetetrazole were purchased from Sigma Chemical Co. (St. Louis, MO). [35S]TBPS (60 C i / m m o l ) and [3H] flunitrazepam (90 C i / m m o l ) were obtained from New England Nuclear Co. (Boston, MA). 2.2. Animals Two WSP and two WSR lines of mice, which have been selectively bred for severity of alcohol withdrawal convulsions and maintained in our colonies in Portland, were used. Males and females from selected generations 8-23 were used in the behavioral and binding experiments as indicated in the Results section. Mice ranged in age from 11 to 14 weeks. All mice were naive at the time of testing. 2.3. Handling-induced convulsion scoring Handling-induced convulsion scores were obtained by a procedure described by Goldstein (1972) modified by Crabbe et al. (1985). A mouse was lifted by the tail and observed for convulsions. If a convulsion was not observed the mouse
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was gently spun 180 ° C and examined. A scale of 0-4 was used, as follows; 4, severe tonic-clonic convulsion when lifted by the tail, with a quick onset and long duration, often continuing for several seconds after the mouse is released; 3, tonic-clonic convulsion when lifted by the tail, often with onset delayed by as much as 1-2 s; 2, tonic convulsion when lifted by the tail or tonicclonic convulsion after gentle spin through a 180 o arc; 1, no convulsion when lifted by the tail, but tonic convulsion elicited by spin; 0.5, only facial grimace after spin; and 0, no convulsion. A baseline assessment of handling-induced convulsions was made in each mouse 18 h before drug administration. Handling-induced convulsion scores were measured every 20 rain after picrotoxin administration for 2 h. Handling-induced convulsion measurements were made every 30 rain for 180 min after bicuculline or pentylenetetrazole injection.
2.4. Drug administration Picrotoxin was dissolved in saline and administered by i.p. injection; mice not given drug were injected with saline. Doses were 2, 4, 5, 6 or 8 m g / k g b o d y weight. Pentylenetetrazole was dissolved in saline and administered i.p. 60 m g / k g body weight. Bicuculline was solubilized in several drops of concentrated HC1, brought to p H 7.0 with N a O H , and to a final volume in saline. Mice received 3 m g / k g body weight.
2.5. [35S]TBPS binding assay Naive mice from the WSP and WSR lines were killed by cervical dislocation and decapitated. Brains were quickly removed and dissected into the frontal cortex, remainder of the cortex, hippocampus and cerebelleum according to the procedure of Heffner et al. (1980). Dissected regions from three mice were pooled and placed in 5 ml of 0.32 M sucrose, 3 m M HEPES buffer at p H 7.4 and 4 ° C. The samples were homogenized using 10 complete passes with a motor-driven Thomas ' C ' teflon glass homogenizer, then centrifuged at 1 000 x g for 10 min at 4 ° C . The supernatant was centrifuged at 100000 X g for 30 rain at 4 ° C . The pellet was resuspended in 3 ml of water with a
Polytron, brought to 6 ml and centrifuged at 1 0 0 0 0 0 x g for 30 min at 4 ° C . This step was repeated except with 50 m M citrate-Tris buffer, p H 7.2. Pellets were resuspended in citrate buffer and frozen at a - 8 0 ° C until assayed. Samples were thawed, centrifuged and washed once more with citrate buffer. For the binding assay the pellet was resuspended in 4 ml of assay buffer which contained 200 m M NaCI, 50 m M N a K P O 4 and 10 m M HEPES, p H 7.4. The total assay volume was 1000 #1 and consisted of 0.3 mg protein/ml, increasing concentrations of [35S]TBPS and assay buffer. Non-specific binding was determined by adding 100 ~tM picrotoxin. The mixture was incubated for 2 h at room temperature, then diluted with 5 ml of assay buffer and filtered over GB100R glass fiber filters (Microfiltration Systems, Dublin, CA). The filters were washed twice with 5 ml of assay buffer at room temperature and then counted by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al. (1951).
2.6. [ 3H]Flunitrazepam binding assay Animals were killed by cervical dislocation and decapitation. Whole brains were quickly removed, frozen on dry ice and stored at - 8 0 ° C until assayed. Samples were weighed while still frozen and thawed in 50 m M NaKPO4, p H 7.4. The brains were homogenized in 35 ml of 50 m M N a K P O 4 buffer, p H 7.4, using a Brinkman homogenizer at a setting of 6 for 10 s and centrifuged at 40 000 x g for 10 min. This step was repeated four times. The final pellet was resuspended in 240-300 volumes of assay buffer which contained 50 m M N a K P O 4, 200 m M NaC1 at p H 7.4. The assay volume was 1 000 ~1 and contained approximately 0.4 mg p r o t e i n / m l . The K D and Bma× for [3H]flunitrazepam binding was determined by adding a fixed concentration of the labeled drug, 0.58 nM, and an increasing concentration of unlabeled flunitrazepam, 1-1000 nM. The mixture was incubated for 30 min at 3 7 ° C and the reaction terminated by filtration over W h a t m a n G F / B filters. The filters were washed twice with 4 ml of assay buffer at 4 ° C , then counted by liquid scintillation spectroscopy. The K D and Bmax val-
150 and pentylenetetrazole; fig. 3 shows the handlinginduced convulsion scores at 30 and 90 min. All three G A B A antagonists caused an initial increase in handling-induced convulsion scores for WSP and W S R mice. At 30 min WSP mice were more sensitive than W S R mice to the enhancement of handling-induced convulsions by bicuculiine (P < 0.01) and pentylenetetrazole (P < 0.05), but their response to picrotoxin did not differ. By 90 min after injection, handling-induced convulsion severity in W S R mice had declined to the level of the saline-treated group for all drugs, while scores for WSP mice treated with picrotoxin or pentylenetetrazole remained significantly elevated (P < 0.01). The results for picrotoxin and bicuculline have been replicated using mice from different selected generations. One potential explanation for the differential sensitivity to picrotoxin and pentylenetetrazole between WSP and W S R mice could be a difference either in density or affinity of the receptor for picrotoxin. In preliminary experiments we found no consistent differences between the lines in TBPS binding to the whole brain from WSP versus W S R mice (densities of 0.9-1.3 p m o l / m g protein and
ues were determined using the computer program of MacPherson (1985) based on the method of Munson and Rodbard (1980). The density of sites was expressed as p m o l / m g protein. In experiments testing the potency of G A B A to increase [3H]flunitrazepam binding the concentration of labeled ligand was 1 nM.
3. Results Picrotoxin, bicuculline and pentylenetetrazole exacerbated handling-induced convulsions in both lines of mice. The WSP and W S R lines exhibited a different time course for the development of handling-induced convulsions, which is shown for 6 m g / k g picrotoxin in fig. 1. Both groups peaked at around 20 min (P < 0.01), but the decay in response was slower in WSP mice (P < 0.05 at 120 min). The area under the time course curve, which represents the total handling-induced convulsion score, was significantly greater (P < 0.01-0.001) in the WSP compared to the W S R line at all drug doses tested (fig. 2). Similar results were observed for WSP and W S R mice treated with bicuculline
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Fig. 3. Effect of bicuculline (BIC, 3 m g / k g ) , pentylenetetrazole (PTZ, 60 m g / k g ) and picrotoxin (PIC, 4 m g / k g ) on handlinginduced convulsions in WSP and WSR mice from selected generation 8. The values are the mean + S.E. handling-induced convulsion scores for 8-11 m i c e / g r o u p .
K D was 39-44 nM, respectively). However, the selection pressure for WSP versus W S R mice may only have altered receptor properties in a discrete region of the brain which may not have been detected in an assay of the whole brain. Therefore, the binding of TBPS was characterized in the frontal cortex, remainder of the cortex, hippocampus and cerebellum. The results are presented in table 1. In agreement with the results from the whole brain there was no significant difference between WSP and W S R mice in TBPS binding to any area. An alternate site of action may involve the receptors for flunitrazepam and G A B A on the GABA-chloride channel complex. The density of [3H]flunitrazepam receptors in whole brain from W S R mice (selected generation 23) was 1.95 _+ 0.06
152 TABLE 1 [35S]TBPS binding to different regions of the brain from WSP and W S R mice a. Region
N
Frontal cortex Remainder cortex Hippocampus Cerebellum
K D (nM)
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Bmax ( p m o l / m g protein)
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WSP
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WSP
42.6 ± 15.7 b 38.8 +- 10.0 48.1 + 8.5 47.6 ± 20.9
42.1+ 6.9 37.1 ± 6.6 42.5 ± 10,5 53.3 +__2%8
0.37+0.13 1.00±0.28 0.21 ±0.04 0.40 ± 0.15
0.34 ± 0.05 0.95+_0.21 0.25 ±0,08 0.30 +_0.06
a Selected generations 20-21. b Mean + S.D.
p m o l / m g protein (N = 5) versus 1.88 + 0.15 (N = 5) in WSP mice. Ligand affinities ranged from 11 to 14 nM. There were no significant differences in flunitrazepam receptor density or affinity among the four lines of mice. The binding of GABA to its receptor can decrease the affinity of TBPS and increase the affinity of flunitrazepam to their respective binding sites. A change in the coupling between these receptors might account for the differential enhancement of handling-induced convulsions by picrotoxin and pentylenetetrazole in the selected lines. As expected, the addition of increasing concentrations of GABA to the [3H]flunitrazepam binding assay elevated flunitrazepam binding to its receptor. The EDs0 for G A B A stimulation was 2 /zM; a value in close agreement with others reported in the literature (Marley and Wehner,
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1986). The WSP and WSR mice exhibited similar EDs0 values for this action of GABA (fig. 4).
4. Discussion
It had previously been shown that WSP and WSR mice from selected generations 8-10 did not differ in their sensitivity to tonic hindlimb extensor seizures induced by either electroconvulsive shock, generalized seizure-producing drugs or the GABA antagonists picrotoxin and bicuculline (McSwigan et al., 1984). However, in the present study it was found that WSP mice were more sensitive than WSR mice to subconvulsant doses of picrotoxin, bicuculline and pentylenetetrazole when they were subsequently tested for handlinginduced convulsions. Methods which produce tonic hindlimb extensor seizures are probably causing maximal CNS activity, and handling-induced seizures are more likely to reflect subtle changes in CNS excitability. The difference between W S P and WSR mice at any given dose of picrotoxin (see fig. 2) predominantly reflected a larger area under the time course curve due to a slower rate of decline in convulsions to baseline in WSP mice (see fig. 1). The response of WSP mice to pentylenetetrazole also decayed more slowly compared to WSR mice. The peak effect of bicuculline was lower in WSR compared to WSP mice. One possible explanation is that the mice may differ in their rate of metabolism or elimination of picrotoxin, bicuculline and pentylenetetrazole. However, an examination of the structures of these compounds would suggest
153 that their mode of elimination is probably not the same. Prone and resistant mice of the l l t h selected generation metabolized and eliminated ethanol at approximately the same rate, after either an acute dose or chronic treatment (Kosobud and Crabbe, 1986). In addition, acute doses of five alcohols, diazepam, and three barbiturates produced similar brain concentrations in both lines (Crabbe et al., 1986). These results suggest that WSP and WSR mice have not been selected for differences in drug metabolism. However, studies are being initiated to address this question in more detail. We considered the possibility that the prolongation of the handling-induced convulsion response in the WSP mice compared to the WSR mice may simply reflect a sensitization of the WSP mice to repeated testing. However, the group of WSP mice administered saline showed seizure scores which remained stable over repeated tests (data not shown). Furthermore, we have previously reported that repeated testing for handlinginduced convulsions leads to a slight, but significant reduction in scores over time (Crabbe et al., 1981). Thus, we discount this possibility as an explanation of the difference between WSP and WSR mice. Alternatively, the behavioral results could be explained by an alteration in the GABA-chloride channel between the WSP and WSR lines. A difference in receptor-mediated activity could involve three possible mechanisms: (1) receptor density, (2) receptor affinity or (3) the coupling process between the picrotoxin receptor and other components of the GABA-benzodiazepine-chloride channel complex. The affinity of [35]TBPS for the picrotoxin binding site in whole brain was the same for both the WSP and WSR lines. While regional differences could be more important for understanding the mechanisms involved in the development of neuroexcitability during withdrawal, the results from the present binding studies did not support the hypothesis that changes in TBPS receptors in specific brain regions mediates the alteration in picrotoxin sensitivity between WSP and WSR mice. These results do not rule out that there may be a change in more discrete regions such as the inferior colliculus in the midbrain or nuclei in the brainstem. We plan to apply
the method of receptor autoradiography in brain slices to provide this information. A change in the coupling between the picrotoxin site and either the GABA or benzodiazepine binding sites or the chloride channel is another possible explanation for the difference between WSP and WSR mice in neurosensitivity to picrotoxin and ethanol withdrawal. The proposal that behavioral selection for mice with different sensitivities to ethanol can produce lines of mice with altered GABA-chloride channel activity is supported by work on the long sleep (LS) and short sleep (SS) mice. These mouse lines were genetically selected for high and low sensitivity, respectively, to ethanol's hypnotic effect (McClearn and Kakihana, 1981). The density and affinity of [3 H]flunitrazepam binding sites in whole brain was found to be the same in both the LS and SS lines (Marley and Wehner, 1986). Comparable results were found in the current study with the WSP and WSR mice. On the other hand, the action of GABA to increase [3H]flunitrazepam binding was greater in the cortex and cerebellum of SS than in LS mice (Marley and Wehner, 1986), while differential enhancement of [3H]flunitrazepam binding to whole brain by GABA was not observed in the WSP and WSR mice. It has been shown that the GABA receptorchloride channel in the cerebellum of LS mice compared to SS mice was more sensitive to muscimol stimulated 36C1- uptake into microsacs, and to the inhibition of [35S]TBPS binding to membranes. Ethanol at revevant concentrations potentiated muscimol-stimulated chloride uptake in LS, but not SS mice (Allan and Harris, 1986). It is possible that the latter differences may be responsible for the difference between these selectively bred lines in sensitivity to the hypnotic effect of ethanol. Similar 36C1- uptake studies should be initiated on the cortex, cerebellum and hippocampus of WSP and WSR mice. In summary, we found that WSP mice were more sensitive than WSR mice to the ability of picrotoxin, bicuculline and pentylenetetrazole to potentiate handling-induced seizures. This finding cannot currently be explained by a difference in the binding characteristics of [35S]TBPS or [3H]flunitrazepam. More detailed studies of the
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GABA-chloride channel complex in these lines will be required to elucidate the pharmacological mechanism of this behavioral difference.
Acknowledgements We thank Cathy Deutsch, Ann Kosobud, Brenda Tam, Emmett Young and Virginia Bleck for assistance in performing these experiments. These studies were supported by grants from the Veterans Administration and by PHS-ADAMHA Grants AA 03527, AA 05828, AA 06243, AA 06399 and AA 06498.
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