Neuroscience Letters, 133 (1991) 73-76 © 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50
73
NSL 08198
Comparisons between patterns of convulsions induced by two fl-carbolines in 10 inbred strains of mice Benoit Martin, Carole Desforges and Georges Chapouthier Laboratoire G~n~tique, Neurog~n~tique et Comportement, URA 1294, CNRS, UFR Biom~dicale Paris V, Paris (France) (Received 26 May 1990; Revised version received 15 July 1991; Accepted 20 August 1991)
Key words: p-Carboline; Methyl-fl-carboline-3-carboxylate; 6,7-Dimethoxy-4-ethyl-fl-carboline-3-carboxylate; Benzodiazepine inverse agonist; Convulsion; Inbred mouse The fl-carbolines, methyl-fl-carboline-3-carboxylate (fl-CCM) and 6,7-dimethoxy-4-ethyl-fl-carboline-3-carboxylate (DMCM) are known to have pharmacological properties opposite to those of agonistic benzodiazepines. Convulsions induced by these drugs lead to differential patterns, such as clonus, myoclonic or tonic seizures. In 10 different inbred strains of mice we investigated whether the responsiveness to the two drugs was the same, irrespective of the pattern of convulsions. We found the same ranking in the responsiveness of the strains to both drugs in the case of myoclonic seizures. No such correlation could be found for clonus or tonic seizures. Our conclusion is that the same genetic factors determine myoclonic seizures, whereas a plurality of mechanisms underly the other patterns. Thus, myoclonic seizures seem to be the most appropriate index for evaluating the convulsant action of fl-carbolines in genetic experiments.
Classic benzodiazepines are known for their anticonvulsant, anxiolytic, sedative-hypnotic as well as musclerelaxant properties [10]. Ligands of the benzodiazepine receptors have been discovered which produce the opposite effects and are now known as inverse agonists of the benzodiazepine receptor [1, 8, 10]. These include several fl-carbolines such as methyl-fl-carboline-3-carboxylate (fl-CCM) or methyl-6,7-dimethoxy-4-ethyl-fl-carboline3-carboxylate (DMCM), which among other properties, have clear convulsant actions [8]. The first aim of the present study was to investigate possible differences in responsiveness to fl-CCM and DMCM in a systematic way, with a large number of inbred strains of mice (10) and with a substantial number of subjects per strain. The second aim was to evaluate whether for each of these measurable criteria of convulsion (clonus, myoclonic seizures and tonic seizures), a similar ranking in the strain reactions to the two fl-carbolines was found. Such ranking would show that this criterion is a good index for measuring the fl-carboline effects. In the studies on the effects of convulsant agents (fl-carbolines included) already reported by several authors, some have monitored clonus and myoclonic seizures [2, 3, 9], while others measured mortality or tonic seizures [5, 11, 12]. Thus,
Correspondence: B. Martin, Laboratoire Grn&ique, Neurogrnrtique et Comportement, URA 1294, CNRS, U F R Biomrdicale Paris V, 45 Rue des Saints Prres, 75270 Paris Cedex 06, France.
the second aim of the present study was to investigate whether these criteria could be considered to be equivalent. This equivalence is an important prerequisite for evaluating the conclusions drawn by different teams working on the neuropharmacogenetics of benzodiazepine receptor ligands, because different convulsion criteria have been used [3, 5, 7, 9, 11, 12]. The straightforward method for demonstrating the functional independence between two traits is to test for a decrease in associaton between the observed traits (here the convulsion criteria) from parental strains to segregating generations [6]. However, we used a more economical procedure. The associaton between two traits in two inbred strains does not imply that the traits are genetically linked or that this assocation results from a pleiotropic effect. This association can be the consequence of the development of the inbred strains: the two traits have been selected at random during the development of the strains. In contrast, a large set of inbred strains can be considered as a homozygous segregating population. The probability of observing an association between genetically independent traits across these strains is therefore low. Correlations between traits used to describe the convulsion pattern in inbred strains were thus evaluated; non-significant correlations between the traits provide evidence for a non-genetic association of these traits. Ten inbred strains of mice were selected. The A/J, CBA/H, CBA/J, NZB and SWR/J mice were provided
74 by CSEAL/CNRS, Orlrans, France, BALB/cBY, C3H/ OuJ, C57BL/6J, DBA/2J by Iffa Credo, France, and C3H/HeJ by URA 171 CNRS. Animals were housed and reared under standard conditions of: 24_+0.5°C, a 12:12 h photoperiod with lights on at 08.00 h, water and Souriffarat (IM UAR) food available ad libitum, and dust-free sawdust bedding. Mice were reared in groups except NZB males whose intermale attack behavior does not allow such rearing conditions. These were thus housed singly. fl-CCM was kindly provided by R.H. Dodd (Institut de Chimie des Substances Naturelles, CNRS, 91190 Gifsur-Yvette, France) and DMCM by Schering Co., Berlin, F.R.G. They were dissolved in 1 N HCI (10/A/mg of fl-carboline) and diluted to volume with saline; 0.05 ml solution per 10 g body weight was administered i.p. Preliminary experimental work on BALB/cBy and C57BL/6J mice provided evidence that 5 mg/kg of flCCM and 2 mg/kg of DMCM are the optimal doses to discriminate between the responsiveness of these two inbred strains. These doses are obviously not the ideal dose for each of the criteria but they allow statistical differences between the strains for the indexes to be obtained. Animals were 12 ___3 weeks old at the time of testing. Tests were performed in the housing room from 11.00 h to 15.00 h. Three minutes before injection, individual mice were placed in 20 x 42 x 16 cm plastic cages littered
with dust-free sawdust. The observation period lasted up to 6 min from the time of injection. This length of time was chosen for the observation of seizure occurrence because of the delay of convulsion induced by fl-carbolines in Swiss OF1 mice [9] confirmed in preliminary experiments with the inbred strains used here. Different criteria were noted. Clonus manifests itself as localized jerking movements of the vibrissae, face, ears and forelimbs. A myoclonic seizure was considered present when the mouse fell on its flank with rhythmic jerks. Tonic seizure was reported when the 4 legs were extended caudally. A G2 test of homogeneity was used to evaluate the differences in reactivity between the strains for clonus and myoclonic seizures for the two fl-carbolines. A Spearman rank correlation coefficient was employed to compare the ranking in responsiveness observed (clonus and myoclonic seizures) in the strains for fl-CCM and DMCM. No effect of sex could be observed among the 10 strains for any of the 3 convulsion criteria. Thus the scores of males and females were pooled for each strain. Moreover, for NZB, the absence of differences between males (housed individually) and females (housed in groups) shows that there is no effect of social isolation on these 3 convulsion criteria. The percentage of subjects exhibiting the traits are shown in Fig. 1 for fl-CCM and Fig. 2 for DMCM. Strain differences for the manifestation of individual clo-
8-CCM [22)
100
(30)
(26)
(38) (30)
90 80 70 GO U_,l
(_9
60
[]
CLONUS
[]
MYO-CLONIC S.
•
TONIC S.
z 0 rtI.U
50
(22)
O_
40 30 20 10 0
m
NJ
BALB/cBy C3H/He C3H/OuJ C57BU6J CBNH
m
CBNJ
DBN2J
_
NZB
n
SWR/J
Fig. 1. Percentagesof subjectsfor the 10inbredstrainsexhibitingclonus,myoclonicseizuresand tonicseizuresfor fl-CCM.
75
DMCM
(12)
(lO)
¢./3 LU
¢D
[]
CLONUS
[]
MYO-CLONIC S.
Z
LLI
rr" LLI a-
(10)
TONIC S.
m
:L NJ
BALB/cBy C3H/He C3H/OuJ C57BU6J
CBNH
CBNJ
DBN2J
NZB
SWR/J
Fig. 2. Percentages of subjects fo the 10 inbred strains exhibiting clonus, myoclonic seizures and tonic seizures for DMCM.
nus and myoclonic seizures are significant (respectively for fl-CCM: G2=23.61, P=0.0050; and DMCM: G2=17.23, P
73
NSL 08198
Comparisons between patterns of convulsions induced by two fl-carbolines in 10 inbred strains of mice Benoit Martin, Carole Desforges and Georges Chapouthier Laboratoire G~n~tique, Neurog~n~tique et Comportement, URA 1294, CNRS, UFR Biom~dicale Paris V, Paris (France) (Received 26 May 1990; Revised version received 15 July 1991; Accepted 20 August 1991)
Key words: p-Carboline; Methyl-fl-carboline-3-carboxylate; 6,7-Dimethoxy-4-ethyl-fl-carboline-3-carboxylate; Benzodiazepine inverse agonist; Convulsion; Inbred mouse The fl-carbolines, methyl-fl-carboline-3-carboxylate (fl-CCM) and 6,7-dimethoxy-4-ethyl-fl-carboline-3-carboxylate (DMCM) are known to have pharmacological properties opposite to those of agonistic benzodiazepines. Convulsions induced by these drugs lead to differential patterns, such as clonus, myoclonic or tonic seizures. In 10 different inbred strains of mice we investigated whether the responsiveness to the two drugs was the same, irrespective of the pattern of convulsions. We found the same ranking in the responsiveness of the strains to both drugs in the case of myoclonic seizures. No such correlation could be found for clonus or tonic seizures. Our conclusion is that the same genetic factors determine myoclonic seizures, whereas a plurality of mechanisms underly the other patterns. Thus, myoclonic seizures seem to be the most appropriate index for evaluating the convulsant action of fl-carbolines in genetic experiments.
Classic benzodiazepines are known for their anticonvulsant, anxiolytic, sedative-hypnotic as well as musclerelaxant properties [10]. Ligands of the benzodiazepine receptors have been discovered which produce the opposite effects and are now known as inverse agonists of the benzodiazepine receptor [1, 8, 10]. These include several fl-carbolines such as methyl-fl-carboline-3-carboxylate (fl-CCM) or methyl-6,7-dimethoxy-4-ethyl-fl-carboline3-carboxylate (DMCM), which among other properties, have clear convulsant actions [8]. The first aim of the present study was to investigate possible differences in responsiveness to fl-CCM and DMCM in a systematic way, with a large number of inbred strains of mice (10) and with a substantial number of subjects per strain. The second aim was to evaluate whether for each of these measurable criteria of convulsion (clonus, myoclonic seizures and tonic seizures), a similar ranking in the strain reactions to the two fl-carbolines was found. Such ranking would show that this criterion is a good index for measuring the fl-carboline effects. In the studies on the effects of convulsant agents (fl-carbolines included) already reported by several authors, some have monitored clonus and myoclonic seizures [2, 3, 9], while others measured mortality or tonic seizures [5, 11, 12]. Thus,
Correspondence: B. Martin, Laboratoire Grn&ique, Neurogrnrtique et Comportement, URA 1294, CNRS, U F R Biomrdicale Paris V, 45 Rue des Saints Prres, 75270 Paris Cedex 06, France.
the second aim of the present study was to investigate whether these criteria could be considered to be equivalent. This equivalence is an important prerequisite for evaluating the conclusions drawn by different teams working on the neuropharmacogenetics of benzodiazepine receptor ligands, because different convulsion criteria have been used [3, 5, 7, 9, 11, 12]. The straightforward method for demonstrating the functional independence between two traits is to test for a decrease in associaton between the observed traits (here the convulsion criteria) from parental strains to segregating generations [6]. However, we used a more economical procedure. The associaton between two traits in two inbred strains does not imply that the traits are genetically linked or that this assocation results from a pleiotropic effect. This association can be the consequence of the development of the inbred strains: the two traits have been selected at random during the development of the strains. In contrast, a large set of inbred strains can be considered as a homozygous segregating population. The probability of observing an association between genetically independent traits across these strains is therefore low. Correlations between traits used to describe the convulsion pattern in inbred strains were thus evaluated; non-significant correlations between the traits provide evidence for a non-genetic association of these traits. Ten inbred strains of mice were selected. The A/J, CBA/H, CBA/J, NZB and SWR/J mice were provided
74 by CSEAL/CNRS, Orlrans, France, BALB/cBY, C3H/ OuJ, C57BL/6J, DBA/2J by Iffa Credo, France, and C3H/HeJ by URA 171 CNRS. Animals were housed and reared under standard conditions of: 24_+0.5°C, a 12:12 h photoperiod with lights on at 08.00 h, water and Souriffarat (IM UAR) food available ad libitum, and dust-free sawdust bedding. Mice were reared in groups except NZB males whose intermale attack behavior does not allow such rearing conditions. These were thus housed singly. fl-CCM was kindly provided by R.H. Dodd (Institut de Chimie des Substances Naturelles, CNRS, 91190 Gifsur-Yvette, France) and DMCM by Schering Co., Berlin, F.R.G. They were dissolved in 1 N HCI (10/A/mg of fl-carboline) and diluted to volume with saline; 0.05 ml solution per 10 g body weight was administered i.p. Preliminary experimental work on BALB/cBy and C57BL/6J mice provided evidence that 5 mg/kg of flCCM and 2 mg/kg of DMCM are the optimal doses to discriminate between the responsiveness of these two inbred strains. These doses are obviously not the ideal dose for each of the criteria but they allow statistical differences between the strains for the indexes to be obtained. Animals were 12 ___3 weeks old at the time of testing. Tests were performed in the housing room from 11.00 h to 15.00 h. Three minutes before injection, individual mice were placed in 20 x 42 x 16 cm plastic cages littered
with dust-free sawdust. The observation period lasted up to 6 min from the time of injection. This length of time was chosen for the observation of seizure occurrence because of the delay of convulsion induced by fl-carbolines in Swiss OF1 mice [9] confirmed in preliminary experiments with the inbred strains used here. Different criteria were noted. Clonus manifests itself as localized jerking movements of the vibrissae, face, ears and forelimbs. A myoclonic seizure was considered present when the mouse fell on its flank with rhythmic jerks. Tonic seizure was reported when the 4 legs were extended caudally. A G2 test of homogeneity was used to evaluate the differences in reactivity between the strains for clonus and myoclonic seizures for the two fl-carbolines. A Spearman rank correlation coefficient was employed to compare the ranking in responsiveness observed (clonus and myoclonic seizures) in the strains for fl-CCM and DMCM. No effect of sex could be observed among the 10 strains for any of the 3 convulsion criteria. Thus the scores of males and females were pooled for each strain. Moreover, for NZB, the absence of differences between males (housed individually) and females (housed in groups) shows that there is no effect of social isolation on these 3 convulsion criteria. The percentage of subjects exhibiting the traits are shown in Fig. 1 for fl-CCM and Fig. 2 for DMCM. Strain differences for the manifestation of individual clo-
8-CCM [22)
100
(30)
(26)
(38) (30)
90 80 70 GO U_,l
(_9
60
[]
CLONUS
[]
MYO-CLONIC S.
•
TONIC S.
z 0 rtI.U
50
(22)
O_
40 30 20 10 0
m
NJ
BALB/cBy C3H/He C3H/OuJ C57BU6J CBNH
m
CBNJ
DBN2J
_
NZB
n
SWR/J
Fig. 1. Percentagesof subjectsfor the 10inbredstrainsexhibitingclonus,myoclonicseizuresand tonicseizuresfor fl-CCM.
75
DMCM
(12)
(lO)
¢./3 LU
¢D
[]
CLONUS
[]
MYO-CLONIC S.
Z
LLI
rr" LLI a-
(10)
TONIC S.
m
:L NJ
BALB/cBy C3H/He C3H/OuJ C57BU6J
CBNH
CBNJ
DBN2J
NZB
SWR/J
Fig. 2. Percentages of subjects fo the 10 inbred strains exhibiting clonus, myoclonic seizures and tonic seizures for DMCM.
nus and myoclonic seizures are significant (respectively for fl-CCM: G2=23.61, P=0.0050; and DMCM: G2=17.23, P
