Pharmacology Biochemistry & Behavior, Vol. 7, pp. 221--226. Copyright © 1977 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Pharmacogenetic Differences in Audiogenic Seizure Priming of C57BL/6Bg and DBA/1Bg-asr Mice S T E P H E N C. M A X S O N , 2 J O H N S. COWEN, s A N D P A U L Y. SZE
Department o f Biobehavioral Sciences, The University o f Connecticut, Storrs, CT 06268 (Received 14 May 1977)
MAXSON, S. C., J. S. COWEN AND P. Y. SZE. Pharmacogeneticdifferences in audiogenic seizure priming of C57BL/6Bg and DBA/1Bg-asr mice. PHARMAC. BIOCHEM. BEHAV. 7(3) 221-226, 1977. - Susceptibility to audiogenic seizures can be induced in some strains of resistant mice by exposure to an initial auditory stimulus (acoustic priming). Aminooxyacetic acid, hydrazine, glutamic acid, gamma-aminobutyric acid (GABA), cycloheximide, and metyrapone antagonize the acoustic priming of audiogenic seizure susceptibility in C57BL/6Bg mice, whereas only metyrapone attenuates that of DBA/1Bg-asr mice. The strain difference in the effect of AOAA and cycloheximide is correlated with a small, transient fall in level of brain GABA in C57BL/6Bg but not DBA/1Bg-asr mice. These findings support our hypothesis that there are at least two neural mechanisms of acoustic priming, each with its own genetic basis and that corticosteroids are required by both mechanisms for the development of primed seizures. Gamma-aminobutyric acid
Pharmacogenetics
Acoustic priming
Audiogenic seizures
Inbred mice
thesis is that the genetic variation may be due to a difference in susceptibility of the acoustic receptors to damage by intense auditory stimulation [2,3]. Under this hypothesis, mice with a cochlea susceptible to damage by stimuli would suffer hearing loss with c o n s e q u e n t hyperreactivity to later a u d i t o r y input, whereas mice with a cochlea resistant to damage would be nonprimable. However, o t h e r findings indicate that genetic variation may result in qualitatively different mechanisms of acoustic priming. In one of the first genetic analyses of acoustic priming, Henry and B o w m a n [ 11 ] exposed D B A / 2 J , C 5 7 B L / 6 J , and their F~ hybrids to the IAS at one of 15 ages from Day 0 to 28 and tested t h e m at 28 days of age. The F1 hydrids, unlike the t w o parental strains, showed a bimodal sensitivity period, suggesting that there are different mechanisms of acoustic priming in the two parental strains. Also, a qualitative difference was found b e t w e e n the priming prone and m o d e r a t e l y priming prone lines developed by selective breeding [3]. Exposure to the IAS, as well as t y m p a n i c m e m b r a n e perforation, induced audiogenic seizure susceptibility in the priming prone mice; whereas exposure to the IAS, but not t y m p a n i c m e m b r a n e perforation, induced susceptibility in m o d e r a t e l y priming prone mice.
A U D I O G E N I C seizure susceptibility has been p r o d u c e d in several strains of "seizure resistant m i c e " by exposing t h e m to an initial a u d i t o r y stimulus (IAS) during a sensitive period in postnatal d e v e l o p m e n t [2, 6, 9, 12, 2 3 ] . Several studies have indicated that this acoustic priming effect can be influenced by genetic variation. Starting with a heterogeneous stock of mice, Chen and Fuller [3] selectively bred for seizure prone, priming prone, m o d e r a t e l y priming prone, and seizure resistant lines. The rapid response to selection suggested to them that only a few genes might be involved in the genetic variation of each p h e n o t y p e . F r o m a n o t h e r h e t e r o g e n e o u s stock o f mice, Deckard et al. [4] also selectively bred for high and low lines of acoustic priming. The realized heritability at the eighth generation of selective breeding was 0.22 ± 0.029 in the high line and 0.29 -+ 0.037 in the low line. Genetic variation in acoustic priming also exists between closely related inbred strains of mice. The C57BL/6 R u b b o appears to be n o n p r i m a b l e under conditions of a u d i t o r y stimulation which are effective for acoustic priming in C 5 7 B L / J and C 5 7 B L / K Bradley [ 2 ] , as well as in C 5 7 B L / 6 J [ 9 ] , and C 5 7 B L / 1 0 B g (Maxson, unpublished data). Despite the genetic variation, it has been suggested that acoustic priming m a y involve a c o m m o n d e v e l o p m e n t a l and neural mechanism. One h y p o -
1This research was supported in part by NIH grants MH 17088 and RR 00602 and by a grant from the University of Connecticut Research Foundation. 2 Reprints may be obtained from Dr. Stephen C. Maxson, Department of Biobehavioral Sciences, Box U-154, The University of Connecticut, Storrs,CT 06268. s Present address: Department of Neurosciences, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, MA 02115. 221
222
MAXSON, COWEN AND SZE
Thus, acoustic priming of mice of the m o d e r a t e l y priming prone line cannot be due to hearing loss p r o d u c e d by auditory stimulus with c o n s e q u e n t hyperreactivity of central structures to later auditory input. Strain differences have also been r e p o r t e d for effects of a m i n o o x y a c e t i c acid ( A O A A ) on acoustic priming. Siporin and Fuller [22] showed that A O A A a t t e n u a t e d acoustic priming in C 5 7 B L / 6 J , but not in S J L / J mice. We have also r e p o r t e d in a preliminary c o m m u n i c a t i o n [17] that A O A A a t t e n u a t e d the acoustic priming in C57BL/6Bg, but not in DBA/1Bgasr. The latter appears to be a single gene m u t a t i o n from D B A / 1 B g which blocks genetic but not primed susceptibility to audiogenic seizures [ 1 4 , 1 7 ] . It is therefore possible to study acoustic priming in the DBA/1 g e n o t y p e w i t h o u t the c o n f o u n d i n g presence of genetic susceptibility. In the experiments described in this study, we have further c o m p a r e d in C 5 7 B L / 6 B g and DBA/1Bg-asr the effects o f the following pharmacological agents on acoustic priming: drugs acting on the G A B A system, inhibitors of protein synthesis, and inhibitors of adrenocortical steroidogenesis. Previous studies [17, 19, 24] have shown that each of these t r e a t m e n t s can attenuate acoustic priming in C 5 7 B L / 6 B g and suggested that there may be at least two neural mechanisms involved in acoustic priming of these mice. The present study investigates further qualitative variation for the neural mechanisms of acoustic priming in genetically different mice. EXPERIMENT 1
Method An a p p r o x i m a t e l y equal n u m b e r of male and female C 5 7 B L / 6 B g and DBA/1Bg-asr mice were used since no sex differences were observed in acoustic priming. The mice were obtained from our specific-pathogen-free mouse colony. Conditions of rearing and m a i n t e n a n c e have been previously described [ 24 ]. F o u r t r e a t m e n t groups were used for each strain: a n o n p r i m e d control group injected with 0.1 m l / 1 0 g of saline (C57BL/6Bg, N = 74; DBA/1Bg-asr, N = 79); a primed control group injected with saline prior to the IAS (C57BL/6Bg, N = 120; DBA/1Bg-asr, N = 35); an A O A A t r e a t m e n t group injected with drug (20 mg/kg, SC) 5 hr prior to the IAS (C57BL/6Bg, N = 44; DBA/1Bg-asr, N = 41); and a hydrazine hydrate t r e a t m e n t group injected with drug (50 mg/kg, SC) at 3 hr prior to the IAS (C57BL/6Bg, N = 1 15; DBA/1Bg-asr, N = 108). F o r acoustic priming at 19 days of age, the IAS was applied in a c h a m b e r 38 x 43 × 38 cm. An Edwards Lungen 7.6 electric bell was used to provide a sound stimulus of 9 5 - 1 0 5 dB (re: 2 x 10 -4 d y n e / c m 2 ) . The mouse was placed on the chamber floor and 60 sec later the bell was activated for 90 sec. All mice were tested for susceptibility to audiogenic seizures on Day 28 of age using the same a u d i t o r y stimulus. Both incidence of wild circling activity (WCA) and that of clonic-tonic seizures ( C - T ) were recorded. All priming and testing occurred b e t w e e n 1 p.m. and 5 p.m. The test of significance b e t w e e n p r o p o r t i o n s was used to analyze the data here and in later experiments [11.
Results and Discussion Table 1 shows that administration of A O A A or hydrazine reduced acoustic priming, as measured by either the
TABLE 1 PYRADOXAL ANTAGONISTS AND ACOUSTIC PRIMING Number Strain
Treatment
% WCA
% C-T
Tested
C57BL/6Bg
Non-primed control Primed control AOAA Hydrazine
16" 87 30* 70t
9* 85 25* 68t
74 120 44 115
DBA/1Bg-asr Non-primed control
18" 88 76 80
16" 80 71 74
79 35 41 108
Primed control AOAA Hydrazine
All mice except non-primed control were exposed to the IAS at 19 days of age and all w e r e tested at 28 days of age for a 9-day primingtest interval. The controls were injected (SC) with saline. AOAA (20 mg/kg, SC) was injected 5 hr prior to priming. Hydrazine hydrate (50 mg/kg, SC) was injected 3 hr prior to priming. *Significant difference from primed control, p
Pharmacogenetic Differences in Audiogenic Seizure Priming of C57BL/6Bg and DBA/1Bg-asr Mice S T E P H E N C. M A X S O N , 2 J O H N S. COWEN, s A N D P A U L Y. SZE
Department o f Biobehavioral Sciences, The University o f Connecticut, Storrs, CT 06268 (Received 14 May 1977)
MAXSON, S. C., J. S. COWEN AND P. Y. SZE. Pharmacogeneticdifferences in audiogenic seizure priming of C57BL/6Bg and DBA/1Bg-asr mice. PHARMAC. BIOCHEM. BEHAV. 7(3) 221-226, 1977. - Susceptibility to audiogenic seizures can be induced in some strains of resistant mice by exposure to an initial auditory stimulus (acoustic priming). Aminooxyacetic acid, hydrazine, glutamic acid, gamma-aminobutyric acid (GABA), cycloheximide, and metyrapone antagonize the acoustic priming of audiogenic seizure susceptibility in C57BL/6Bg mice, whereas only metyrapone attenuates that of DBA/1Bg-asr mice. The strain difference in the effect of AOAA and cycloheximide is correlated with a small, transient fall in level of brain GABA in C57BL/6Bg but not DBA/1Bg-asr mice. These findings support our hypothesis that there are at least two neural mechanisms of acoustic priming, each with its own genetic basis and that corticosteroids are required by both mechanisms for the development of primed seizures. Gamma-aminobutyric acid
Pharmacogenetics
Acoustic priming
Audiogenic seizures
Inbred mice
thesis is that the genetic variation may be due to a difference in susceptibility of the acoustic receptors to damage by intense auditory stimulation [2,3]. Under this hypothesis, mice with a cochlea susceptible to damage by stimuli would suffer hearing loss with c o n s e q u e n t hyperreactivity to later a u d i t o r y input, whereas mice with a cochlea resistant to damage would be nonprimable. However, o t h e r findings indicate that genetic variation may result in qualitatively different mechanisms of acoustic priming. In one of the first genetic analyses of acoustic priming, Henry and B o w m a n [ 11 ] exposed D B A / 2 J , C 5 7 B L / 6 J , and their F~ hybrids to the IAS at one of 15 ages from Day 0 to 28 and tested t h e m at 28 days of age. The F1 hydrids, unlike the t w o parental strains, showed a bimodal sensitivity period, suggesting that there are different mechanisms of acoustic priming in the two parental strains. Also, a qualitative difference was found b e t w e e n the priming prone and m o d e r a t e l y priming prone lines developed by selective breeding [3]. Exposure to the IAS, as well as t y m p a n i c m e m b r a n e perforation, induced audiogenic seizure susceptibility in the priming prone mice; whereas exposure to the IAS, but not t y m p a n i c m e m b r a n e perforation, induced susceptibility in m o d e r a t e l y priming prone mice.
A U D I O G E N I C seizure susceptibility has been p r o d u c e d in several strains of "seizure resistant m i c e " by exposing t h e m to an initial a u d i t o r y stimulus (IAS) during a sensitive period in postnatal d e v e l o p m e n t [2, 6, 9, 12, 2 3 ] . Several studies have indicated that this acoustic priming effect can be influenced by genetic variation. Starting with a heterogeneous stock of mice, Chen and Fuller [3] selectively bred for seizure prone, priming prone, m o d e r a t e l y priming prone, and seizure resistant lines. The rapid response to selection suggested to them that only a few genes might be involved in the genetic variation of each p h e n o t y p e . F r o m a n o t h e r h e t e r o g e n e o u s stock o f mice, Deckard et al. [4] also selectively bred for high and low lines of acoustic priming. The realized heritability at the eighth generation of selective breeding was 0.22 ± 0.029 in the high line and 0.29 -+ 0.037 in the low line. Genetic variation in acoustic priming also exists between closely related inbred strains of mice. The C57BL/6 R u b b o appears to be n o n p r i m a b l e under conditions of a u d i t o r y stimulation which are effective for acoustic priming in C 5 7 B L / J and C 5 7 B L / K Bradley [ 2 ] , as well as in C 5 7 B L / 6 J [ 9 ] , and C 5 7 B L / 1 0 B g (Maxson, unpublished data). Despite the genetic variation, it has been suggested that acoustic priming m a y involve a c o m m o n d e v e l o p m e n t a l and neural mechanism. One h y p o -
1This research was supported in part by NIH grants MH 17088 and RR 00602 and by a grant from the University of Connecticut Research Foundation. 2 Reprints may be obtained from Dr. Stephen C. Maxson, Department of Biobehavioral Sciences, Box U-154, The University of Connecticut, Storrs,CT 06268. s Present address: Department of Neurosciences, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, MA 02115. 221
222
MAXSON, COWEN AND SZE
Thus, acoustic priming of mice of the m o d e r a t e l y priming prone line cannot be due to hearing loss p r o d u c e d by auditory stimulus with c o n s e q u e n t hyperreactivity of central structures to later auditory input. Strain differences have also been r e p o r t e d for effects of a m i n o o x y a c e t i c acid ( A O A A ) on acoustic priming. Siporin and Fuller [22] showed that A O A A a t t e n u a t e d acoustic priming in C 5 7 B L / 6 J , but not in S J L / J mice. We have also r e p o r t e d in a preliminary c o m m u n i c a t i o n [17] that A O A A a t t e n u a t e d the acoustic priming in C57BL/6Bg, but not in DBA/1Bgasr. The latter appears to be a single gene m u t a t i o n from D B A / 1 B g which blocks genetic but not primed susceptibility to audiogenic seizures [ 1 4 , 1 7 ] . It is therefore possible to study acoustic priming in the DBA/1 g e n o t y p e w i t h o u t the c o n f o u n d i n g presence of genetic susceptibility. In the experiments described in this study, we have further c o m p a r e d in C 5 7 B L / 6 B g and DBA/1Bg-asr the effects o f the following pharmacological agents on acoustic priming: drugs acting on the G A B A system, inhibitors of protein synthesis, and inhibitors of adrenocortical steroidogenesis. Previous studies [17, 19, 24] have shown that each of these t r e a t m e n t s can attenuate acoustic priming in C 5 7 B L / 6 B g and suggested that there may be at least two neural mechanisms involved in acoustic priming of these mice. The present study investigates further qualitative variation for the neural mechanisms of acoustic priming in genetically different mice. EXPERIMENT 1
Method An a p p r o x i m a t e l y equal n u m b e r of male and female C 5 7 B L / 6 B g and DBA/1Bg-asr mice were used since no sex differences were observed in acoustic priming. The mice were obtained from our specific-pathogen-free mouse colony. Conditions of rearing and m a i n t e n a n c e have been previously described [ 24 ]. F o u r t r e a t m e n t groups were used for each strain: a n o n p r i m e d control group injected with 0.1 m l / 1 0 g of saline (C57BL/6Bg, N = 74; DBA/1Bg-asr, N = 79); a primed control group injected with saline prior to the IAS (C57BL/6Bg, N = 120; DBA/1Bg-asr, N = 35); an A O A A t r e a t m e n t group injected with drug (20 mg/kg, SC) 5 hr prior to the IAS (C57BL/6Bg, N = 44; DBA/1Bg-asr, N = 41); and a hydrazine hydrate t r e a t m e n t group injected with drug (50 mg/kg, SC) at 3 hr prior to the IAS (C57BL/6Bg, N = 1 15; DBA/1Bg-asr, N = 108). F o r acoustic priming at 19 days of age, the IAS was applied in a c h a m b e r 38 x 43 × 38 cm. An Edwards Lungen 7.6 electric bell was used to provide a sound stimulus of 9 5 - 1 0 5 dB (re: 2 x 10 -4 d y n e / c m 2 ) . The mouse was placed on the chamber floor and 60 sec later the bell was activated for 90 sec. All mice were tested for susceptibility to audiogenic seizures on Day 28 of age using the same a u d i t o r y stimulus. Both incidence of wild circling activity (WCA) and that of clonic-tonic seizures ( C - T ) were recorded. All priming and testing occurred b e t w e e n 1 p.m. and 5 p.m. The test of significance b e t w e e n p r o p o r t i o n s was used to analyze the data here and in later experiments [11.
Results and Discussion Table 1 shows that administration of A O A A or hydrazine reduced acoustic priming, as measured by either the
TABLE 1 PYRADOXAL ANTAGONISTS AND ACOUSTIC PRIMING Number Strain
Treatment
% WCA
% C-T
Tested
C57BL/6Bg
Non-primed control Primed control AOAA Hydrazine
16" 87 30* 70t
9* 85 25* 68t
74 120 44 115
DBA/1Bg-asr Non-primed control
18" 88 76 80
16" 80 71 74
79 35 41 108
Primed control AOAA Hydrazine
All mice except non-primed control were exposed to the IAS at 19 days of age and all w e r e tested at 28 days of age for a 9-day primingtest interval. The controls were injected (SC) with saline. AOAA (20 mg/kg, SC) was injected 5 hr prior to priming. Hydrazine hydrate (50 mg/kg, SC) was injected 3 hr prior to priming. *Significant difference from primed control, p
