Life Sciences, Vol. 47, pp. 1519-1525 Printed in the U.S.A.
Pergamon Press
DIAZEPAM SENSITIVE MICE: DIFFERENTIAL SENSITIVITY TO THE DEPRESSANT AND ANTICONVULSANT EFFECTS OF DIAZEPAM P. T.-H. Wong and W. L. Teo
Department of Phaxmacology, Faculty of Medicine National University of Singapore, Singapore 0511 (Received in final form August 13, 1990)
SUMMARY A mouse line was developed by selecting for increased sensitivityto the hypnotic effect of diazepam. These "diazepam sensitive" mice showed a mean duration of loss of righting reflex (LORR) of approximately 150 rain at a dose of 20 mg/kg diazepam, this dose failed to induce L O R R in the control outbred mice. Rotarod treading times of the diazepam sensitive mice wore. significantlyshorter than that of the control mice over the same dose
range indicating that these mice arc also more sensitive to the sedative/muscle relaxant effects of diazcpam. On the contrary, the ability of diazcpam to protect against pcntylcnctctrazolc-induced convulsion was found ~>to be the same in the sensitive and control mice. These observations strongly ;,,suggest that the heightened sensitivity to the sedative-hypnotic effects of dlazcpam in the sensitive mice is unlikely to be due entirely to changes in drug disposition. Swiss albino mice are known to exhibit marked variation in their sensitivity to the hypnotic effect of diaz~pam (1). By selecting according to the duration of LORR induced by an hypnotic dose of diazepam, we have developed a line of mice with significantly higher sensitivity to diazepam (2). After six generations of littcrmate inbreexling, the realized hcritability for these sensitive mice was found to be 0.7. Selection for diazepam insensitivity, however, showed a realized hcrimbility of only 0.3. This shows that upward selection for increased sensitivity to diaz~pam is more favored and effective. We report here further findings and characterization of these diazcpam sensitive mice. In particular, the heightened sensitivity to the hypnotic effect may or may not extend to the other effects of diazcpam. The sedative/muscle relaxant and anticonvulsant effects of diazepam in these sensitive mice were therefore compared to those in control mice. METHODS Chen~als Diazepam in the injection form (Valium 10) was purchased from Roche while diazepam in the powder form was a gift. Oxazepam was a gift from Wyeth. Pentylenetetrazole was obtained from Sigma. All other chemicals used were of analytical grade.
0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
1520
Diazepam Sensitive Mice
Vol. 47, No. 17, 1990
Selective breeding Selective breeding was carried out as described previously (2). At first, only siblings from the same litter were allowed to mate in order to maintain a high selection pressure. As it appeared that the diazepam sensitive (DS) line was reasonably well established by the sixth generation, selection was relaxed from the seventh generation onward whereby only mating of non-littermates (cousins) were allowed. The testing dose of diazepam was also reduced from 35 to 20 mg/kg for the DS line while that for the insensitive line remained at 35 mg/kg. v
Duration of loss of rightin~ reflex 0ADRR) Having been injected intraperitoneally with diazepam (Valium 10) or oxazepam dissolved in dimethyl sulfoxide (DMSO), the mice were observed for the loss of righting reflex when being placed on their backs. The righting reflex was considered lost if they could not right themselves within 1 min and regained when they managed to fight themselves three times within 1 min. The duration of LORR was recorded for each v
mouse.
Rotaro;1 and anticonvulsant tests Control and DS mice were tested for their rotarod performance exactly as described previously (3) 15 rain after administration of diazepam (0.25-4 mg/kg, i.p.). The length of time each mouse managed to stay on the accelerating rod was recorded. At 30 rain, these animals were injected with pentylenetetrazole (100 mg/kg, s.c, in saline). The number of mice that showed any seizure activity with or without full tonic extension of the hindlimbs was recorded. Diazepam was dissolved in DMSO which by itself has no detectable anticonvulsant effect (4) or causes any significant reduction in treading time on the rotarod
(3). Benzodiazevine recevtor bindine assav [rl-I]i~lunitrazeparn bindings in seven brain regions were determined as described before (4) in the presence or absence of 10 ~tM GABA. The final concentration of [~l-I]flunitrazepam was 1.2 nM. All protein determinations were performed according to Lowry et al. (5).
RESULTS Table I shows that the duration of LORR of the F 7 generation of the DS mice was 139-~6 rain, only 74% of that of the previous generation (189-'x.25 rain) (2). The mean duration of LORR drifted further downward to 86 rain for Fg. The diazepam sensitivity then did not change significantly until it increased sharply from the F1, to F13 generations as seen by the increase in the mean duration of LORR from 88 to 146 rain. From there on, the mean duration of LORR of the next seven generations appeared consistent and only these mice were used in further experiments. There were no significant difference between the two sexes in their duration of LORR for all generations. In contrast to the success in the DS mouse line, selection for diazepam insensitivity was only effective initially with the mean duration of LORR reduced to 28+17 rain in the F~ generation (2). Although the duration of LORR of the next two generations were maintained at similar levels, there were marked increases in the diazepam-induced duration of LORR from the F 9 to Fx2 generations (Table I). In the meantime, the mice were losing their vigor very rapidly and were afflicted with infertility and general ill-health. Selection for diazepam insensitive mice was thus abandoned at this point.
Vol. 47, No. 17, 1990
Diazepam Sensitive Mice
1521
TABLE I Duration of diazepam-induced LORR of mice selected for differential diazeparn sensitivity. Diazepam sensitive mice F7 Fs F9 F~o Fll F12 Fl3 F14 F15 F16 F17 Fls F19 F~o
139 110 86 93 88 118 146 145 150 156 160 156 155 164
+ 6 + 3 + 4 _+ 3 + 4 + 7 + 3 + 2 + 2 + 2 + 2 + 2 + 3 + 3
Diazepam insensitive mice 23 _+ 2 (101) 30 + 4 (86) 71 + 6 (87) 77 + 6 (34) 76 + 11 (15) 122 5:10 (18) abandoned
(74) (74) (150) (194) (185) (170) (196) (184) (113) (155) (228) (198) (93) (79)
Mice were housed in standard plastic cages with feed and water available ad lib and a 12 h light dark cycle. One adult male was kept with one or two females for mating. Pregnant females were housed singly. The young were weaned at 3-4 weeks old and littermates of the same sex were caged together. They were tested for diazepam sensitivity at 5-6 weeks old with mice selected for sensitivity receiving 20 mg/kg diazepam except for F7 which received 25 mg/kg. Mice selected for insensitivity also received 35 mg/kg. Duration of LORR were expressed as mean + SEM (N) in rain. Analysis of variance (ANOVA): F~3.1~>=66.06 for sensitive mice, F~5,33~=36.92 for insensitive mice, P
Pergamon Press
DIAZEPAM SENSITIVE MICE: DIFFERENTIAL SENSITIVITY TO THE DEPRESSANT AND ANTICONVULSANT EFFECTS OF DIAZEPAM P. T.-H. Wong and W. L. Teo
Department of Phaxmacology, Faculty of Medicine National University of Singapore, Singapore 0511 (Received in final form August 13, 1990)
SUMMARY A mouse line was developed by selecting for increased sensitivityto the hypnotic effect of diazepam. These "diazepam sensitive" mice showed a mean duration of loss of righting reflex (LORR) of approximately 150 rain at a dose of 20 mg/kg diazepam, this dose failed to induce L O R R in the control outbred mice. Rotarod treading times of the diazepam sensitive mice wore. significantlyshorter than that of the control mice over the same dose
range indicating that these mice arc also more sensitive to the sedative/muscle relaxant effects of diazcpam. On the contrary, the ability of diazcpam to protect against pcntylcnctctrazolc-induced convulsion was found ~>to be the same in the sensitive and control mice. These observations strongly ;,,suggest that the heightened sensitivity to the sedative-hypnotic effects of dlazcpam in the sensitive mice is unlikely to be due entirely to changes in drug disposition. Swiss albino mice are known to exhibit marked variation in their sensitivity to the hypnotic effect of diaz~pam (1). By selecting according to the duration of LORR induced by an hypnotic dose of diazepam, we have developed a line of mice with significantly higher sensitivity to diazepam (2). After six generations of littcrmate inbreexling, the realized hcritability for these sensitive mice was found to be 0.7. Selection for diazepam insensitivity, however, showed a realized hcrimbility of only 0.3. This shows that upward selection for increased sensitivity to diaz~pam is more favored and effective. We report here further findings and characterization of these diazcpam sensitive mice. In particular, the heightened sensitivity to the hypnotic effect may or may not extend to the other effects of diazcpam. The sedative/muscle relaxant and anticonvulsant effects of diazepam in these sensitive mice were therefore compared to those in control mice. METHODS Chen~als Diazepam in the injection form (Valium 10) was purchased from Roche while diazepam in the powder form was a gift. Oxazepam was a gift from Wyeth. Pentylenetetrazole was obtained from Sigma. All other chemicals used were of analytical grade.
0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
1520
Diazepam Sensitive Mice
Vol. 47, No. 17, 1990
Selective breeding Selective breeding was carried out as described previously (2). At first, only siblings from the same litter were allowed to mate in order to maintain a high selection pressure. As it appeared that the diazepam sensitive (DS) line was reasonably well established by the sixth generation, selection was relaxed from the seventh generation onward whereby only mating of non-littermates (cousins) were allowed. The testing dose of diazepam was also reduced from 35 to 20 mg/kg for the DS line while that for the insensitive line remained at 35 mg/kg. v
Duration of loss of rightin~ reflex 0ADRR) Having been injected intraperitoneally with diazepam (Valium 10) or oxazepam dissolved in dimethyl sulfoxide (DMSO), the mice were observed for the loss of righting reflex when being placed on their backs. The righting reflex was considered lost if they could not right themselves within 1 min and regained when they managed to fight themselves three times within 1 min. The duration of LORR was recorded for each v
mouse.
Rotaro;1 and anticonvulsant tests Control and DS mice were tested for their rotarod performance exactly as described previously (3) 15 rain after administration of diazepam (0.25-4 mg/kg, i.p.). The length of time each mouse managed to stay on the accelerating rod was recorded. At 30 rain, these animals were injected with pentylenetetrazole (100 mg/kg, s.c, in saline). The number of mice that showed any seizure activity with or without full tonic extension of the hindlimbs was recorded. Diazepam was dissolved in DMSO which by itself has no detectable anticonvulsant effect (4) or causes any significant reduction in treading time on the rotarod
(3). Benzodiazevine recevtor bindine assav [rl-I]i~lunitrazeparn bindings in seven brain regions were determined as described before (4) in the presence or absence of 10 ~tM GABA. The final concentration of [~l-I]flunitrazepam was 1.2 nM. All protein determinations were performed according to Lowry et al. (5).
RESULTS Table I shows that the duration of LORR of the F 7 generation of the DS mice was 139-~6 rain, only 74% of that of the previous generation (189-'x.25 rain) (2). The mean duration of LORR drifted further downward to 86 rain for Fg. The diazepam sensitivity then did not change significantly until it increased sharply from the F1, to F13 generations as seen by the increase in the mean duration of LORR from 88 to 146 rain. From there on, the mean duration of LORR of the next seven generations appeared consistent and only these mice were used in further experiments. There were no significant difference between the two sexes in their duration of LORR for all generations. In contrast to the success in the DS mouse line, selection for diazepam insensitivity was only effective initially with the mean duration of LORR reduced to 28+17 rain in the F~ generation (2). Although the duration of LORR of the next two generations were maintained at similar levels, there were marked increases in the diazepam-induced duration of LORR from the F 9 to Fx2 generations (Table I). In the meantime, the mice were losing their vigor very rapidly and were afflicted with infertility and general ill-health. Selection for diazepam insensitive mice was thus abandoned at this point.
Vol. 47, No. 17, 1990
Diazepam Sensitive Mice
1521
TABLE I Duration of diazepam-induced LORR of mice selected for differential diazeparn sensitivity. Diazepam sensitive mice F7 Fs F9 F~o Fll F12 Fl3 F14 F15 F16 F17 Fls F19 F~o
139 110 86 93 88 118 146 145 150 156 160 156 155 164
+ 6 + 3 + 4 _+ 3 + 4 + 7 + 3 + 2 + 2 + 2 + 2 + 2 + 3 + 3
Diazepam insensitive mice 23 _+ 2 (101) 30 + 4 (86) 71 + 6 (87) 77 + 6 (34) 76 + 11 (15) 122 5:10 (18) abandoned
(74) (74) (150) (194) (185) (170) (196) (184) (113) (155) (228) (198) (93) (79)
Mice were housed in standard plastic cages with feed and water available ad lib and a 12 h light dark cycle. One adult male was kept with one or two females for mating. Pregnant females were housed singly. The young were weaned at 3-4 weeks old and littermates of the same sex were caged together. They were tested for diazepam sensitivity at 5-6 weeks old with mice selected for sensitivity receiving 20 mg/kg diazepam except for F7 which received 25 mg/kg. Mice selected for insensitivity also received 35 mg/kg. Duration of LORR were expressed as mean + SEM (N) in rain. Analysis of variance (ANOVA): F~3.1~>=66.06 for sensitive mice, F~5,33~=36.92 for insensitive mice, P
