Brain Research, 153 1"1978)423426 :(~) Elsevier/North-Holland Biomedical Press
423
Reduction in tyrosine hydroxylase activity in the rat amygdala induced by kindling stimulation,
1. B. FARJO* and D. H. R. BLACKWOOD MRC Brain Metabolism Unit, Department of Pharmacology, Edinburgh EH8 9JZ (Great Britain)
(Accepted April 20th, 1978)
Kindling, as described by Goddard et al.V involves electrical stimulation of certain brain structures, in particular the amygdala in the limbic system, until a previously ineffective stimulus comes to elicit a full motor seizure. Kindling has become an important experimental model for the study of epileptogenesis 14,17. However, the mechanisms underlying kindling are still largely unknown. Some studies have suggested that catecholamine in the brain are inhibitory to kindling 1, and to seizures in general a3. It has been shown that cortical noradrenaline and dopamine levels are reduced in the brain of kindled cats 15. Furthermore, it has been suggested that dopamine is an inhibitory transmitter in the amygdala 2,1e. More recently, it has been shown that unilateral lesion of the stria terminalis which contains the catecholamine-inhibitory afferents to the amygdala facilitates the early stages of development of kindling induced by ipsilateral amygdaloid stimulation in rats 5. Moreover, the dopamine content was found to be locally reduced at the site of the stimulated amygdala in kindled rats 6. However, other workers have reported a reduction in noradrenaline content in the hypothalamus but found no change in brain dopamine levels in kindled rats 4. In the present study, the activity of the enzyme tyrosine hydroxylase, the ratelimiting step of catecholamine biosynthesis11,16, was measured in the amygdala and other brain regions in rats kindled by amygdaloid stimulation. Twenty-one male Wistar rats, 180-200 g in weight, were used for this study; 7 of these rats served as unoperated controls. Under halothane anaesthesia, the left basolateral amygdalae of 14 rats were stereotaxically implanted with bipolar stimulating electrodes of nichrome wire. The coordinates were : 4.5 mm anterior to the interaural line, 4.4 mm lateral to the midline, and 7.9 mm deep to the cortical surfacO °. Rats were allowed one week for recovery and daily stimulation was begun on the eighth day. The rats were divided into two groups. Seven were stimulated and 7 were non-stimulated sham controls. To the stimulated rats, a suprathreshold constant current, ranging between 100-300/~A, and consisting of trains of 1 msec biphasic square wave pulses at a frequency of 60 Hz, was delivered for one second daily via the stimulating electrode * 1. B. Farjo is on a Ph. D grant from the College of Medicine, University of Baghdad, Iraq.
424 using a c o n s t a n t c u r r e n t physiological s t i m u l a t o r (Farnell). Electrical activity was r e c o r d e d f r o m the a m y g d a l a on a G r a s s p o l y g r a p h ( m o d e l 7) before and after stimulus delivery a n d the d u r a t i o n o f the ensuing afterdischarge was measured. D a i l y st imulation was c o n t i n u e d until a generalised convulsion d e v e l o p e d 14 o n 3 consecutive days. The 7 e x p e r i m e n t a l rats d e m o n s t r a t e d afterdischarges on the first d a y o f s t i m u l a t i o n , a n d d e v e l o p e d generalised convulsions between 9 and 13 days. The p a t t e r n o f dev e l o p m e n t o f kindling did n o t differ from t h a t r e p o r t e d by o t h e r w o r k e r s 7,l L F o r weeks after the last stimulation, rats were killed by d e c a p i t a t i o n a n d their brains dissected o u t quickly on a cold stage at 4 °C. The following dissection was done with the aid o f the dissecting microscope. The entire h i p p o c a m p u s was carefully dissected o u t on b o t h sides o f the brain. T w o vertical cuts were then made from the inferior surface o f the brain; the first at the level o f the optic c h i a s m a (A 5500 # m ) 10 a n d the second at the level o f j u n c t i o n o f the cerebral peduncles with the base o f the cerebral hemispheres (A 3000 ~ m ) 1°, thus dividing,She brain into 3 parts, F r o m the a n t e r i o r part, tissue p o r t i o n s were t a k e n f r o m the frontal cortex and s t r i a t u m on both TABLE I Tyrosine hydroxylase activity nmole /3H/L-DOPA ]brmed/h/g wet wt.) in different brain regions hi amygdaloid kindled rats Each value represents the mean activity :k S.E. (n). Right
LeJt
1.96 i 0.13 2.11 ± 0.17 1.95 ~ 0.10
1.31 ± 0.09*,** 2.10 -~ 0.15" t.98 -3 0.07
0.36 ± 0.02 0.38 ± 0.03 0.38 _-k0.02
0.34 A_0.04 0.42 :k 0.02 0.40 ~ 0.03
4.17 i: 0.28 4.31 ± 0.19 5.01 -% 0.18
3.58 -~ 0.28 4.45 t: 0.25 4.89 :k 0.27
Thalamus Kindled (6) Sham-operated (6) Unoperated (6)
1.13 ~ 0.11 0.89 ± 0.10 1.10 ± 0.18
0.82 i 0.16 0.88 i 0.10 0.92 ~ 0.16
Striatum Kindled (6) Sham-operated (6) Unoperated (6)
23.99 i: 1.05 24.02 i 1.63 23.99 t: 1.66
21.79 ± 0.68 24.62 i: 1.24 25.14 :£ 1.85
Cortex Kindled (7) Sham-operated (7) Unoperated (7)
0.28 _-k0.01 0.27 :k 0.02 0.27 _~ 0.02
0.26 ± 0.02 0.27 ± 0.02 0.28 :k 0.02
Amygdala Kindled (7) Sham-operated (7) Unoperated (7) Hippocampus Kindled (6) Sham-operated (6) Unoperated (6) Hypothalamus Kindled (6) Sham-operated (6) Unoperated (6)
* Site of electrode implantation into the left amygdala. ** P < 0.002 for comparison of right and left sides (Mann-Whitney U-test)
425 sides. From the middle part, both amygdalae were dissected out under the dissecting microscope. Tissue portions from the hypothalamus and thalamus were then taken. Kindled, implanted and unoperated control rats were treated together in all phases of sacrifice and biochemical analysis. Tissue samples were stored in liquid nitrogen before the subsequent biochemical estimation of tyrosine hydroxylase activity. Tissues were homogenised with 20 mM potassium phosphate buffer, pH 7.4, so that 10 #1 buffer was used for each 1 mg tissue. Homogenates of tissue samples were assayed, in duplicates, for tyrosine hydroxylase activity according to the method of Hendry and lversen (1971) 8. Tissue and reagent blanks, in duplicate, were assayed simultaneously with tissue samples. Recovery was estimated by taking 10 #1 of [3H]LDOPA (specific activity 2.5 Ci/mmole, Radiochemical Centre, Amersham) through the assay, in duplicate, and compared to 10 ,ul [aH]L-DOPA counted directly after the addition of scintillant. The estimated percentage recovery of the method ranged between 49 and 52 °/~I. The counting efficiency of tritium was estimated to be 34 %. The results of assaying the enzyme activity are shown in Table I. Tyrosine hydroxylase activity in brain areas of unoperated control rats were not significantly different from the normal levels previously reported 9. An analysis of variance showed no significant difference in the enzyme activity in the amygdala of both the sham-operated and unoperated control rats. However, the data showed a consistent and a significant decrease in tyrosine hydroxylase activity in the left stimulated, but not the contralateral, amygdala of all kindled rats. This reduction in enzyme activity was significant at the level of P < 0.002 (Mann-Whitney U-test, U - 3) when compared to that of the contralateral amygdala. However, no correlation was found between the reduced enzyme activity and the rate of seizure development or the duration of the evoked afterdischarge. No significant difference was found in tyrosine hydroxylase activity in the hippocampus, thalamus, hypothalamus, frontal cortex and striatum on the right and left sides of both implanted and unoperated control rats. Similarly, no significant difference in the enzyne activity was found in the hippocampus, thalamus, frontal cortex on both sides of the brain in kindled rats. However, the enzyme activity tended to be reduced in the hypothalamus and the striatum in kindled rats, but this reduction was not statistically significant. The fact that tyrosine hydroxylase activity is significantly reduced at the site of stimulation might suggest that a persistent reduction in catecholamine production is implicated in reducing inhibition with consequent focal hyperexcitability changes in the stimulated amygdala in kindled rats. It has already been shown that kindlinginduced hypersensitivity may result, in part, from a persistent decrease in postsynaptic inhibitory influences3. The absence of any significant change in catecholamine production in other brain regions studied might suggest that the mechanisms underlying the spread of the afterdischarge are different from those initiating the focal hyperexcitability in kindling convulsions. Consequently, it is concluded that kindling produced a definite persistent decrement in tyrosine hydroxylase activity at the site of stimulation in the amygdala.
426 Special a c k n o w l e d g e m e n t is e x t e n d e d to Dr. J. M c Q u e e n o f the M R C Brain M e t a b o l i s m Unit, D e p a r t m e n t o f P h a r m a c o l o g y , E d i n b u r g h , for her supervision t h r o u g h o u t the course o f this piece o f work. This w o r k is included in p a r t i a l fulfillment for the degree o f Ph.D. for 1. B. F a r j o f r o m the D e p a r t m e n t o f P h a r m a c o l o g y , F a c u l t y o f Medicine, E d i n b u r g h University.
1 Arnold, P., Racine, R. and Wise, R., Effect of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat, Exp. Neurol., 40 (1973) 457--460. 2 Ben-Ari, Y., and Kelly, J. S., Dopamine evoked inhibition of single cells of the feline putamen and basolateral amygdala, J. Physiol. (Lond.), 256 (1976) 1-22. 3 Bliss, T. V. P. and Gardner-Medwin, A. R., Long lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 357-374. 4 Callaghan, D. A. and Schwark, W. S., Neurochemical change and drug effects in a model of epilepsy in the rat, Neurosci. Abstr., 2 (1976) 257. 5 Engel, J., Jr. and Katzman, R., Facilitation of amygdaloid kindling by lesions of the stria terminalis, Brain Research, 122 (1977) 137-142. 6 Engel, J., Jr. and Sharpless, N. S., Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 7 Goddard, G. V., Mclntyre, D. C. and Leech, C. K., A permanent change in brain function resulting from daily electrical stimulation, Exp. NeuroL, 25 (1969) 295-330. 8 Hendry, I. A. and Iversen, L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in the mouse superior cervical sympathetic ganglion, Brain Research, 29 (1971) 159-162. 9 Iversen, L. L. and Uretsky, N. J., Regional effects of 6-hydroxydopamine on catecholamine containing neurones in rat brain and spinal cord, Brain Research, 24 (1970) 364-367. 10 K6nig, J. F. R. and Klippel, R~ A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and lower Parts of Brain Stem, Williams and Wilkins, Baltimore, 1963. 11 Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmae. exp. Ther., 148 (1965) 1-8. 12 McCrea, D. A., Jordan, L. M. and Lake, N., Microiontophoresis in the amygdala of rat. Canad. Physiol., 4 (1973) 189. 13 Maynert, E. W., The role of biochemical and neurochemical factors in the laboratory evaluation of antiepileptic drugs, Epilepsia (Amst.), 10 (1969) 145-162. 14 Racine, R. J., Modification of seizure activity by electrical stimulation, I1. Motor seizure, Electroenceph, clin. Neurophysiol., 32 (1972) 281-294. 15 Sato, M. and Nakashima, J., Kindling: secondary epileptogenesis, sleep and catecholamines, Canad. J. neurol. Sci., 2 (1975) 439-446. 16 Spector, S., Inhibition of endogenous catecholamine biosynthesis, Pharma¢vl. Rev., 18 (1966), 599-609. 17 Wada, J. A. (Ed.), Kindling, Raven Press, New York, 1976, p. 230.
423
Reduction in tyrosine hydroxylase activity in the rat amygdala induced by kindling stimulation,
1. B. FARJO* and D. H. R. BLACKWOOD MRC Brain Metabolism Unit, Department of Pharmacology, Edinburgh EH8 9JZ (Great Britain)
(Accepted April 20th, 1978)
Kindling, as described by Goddard et al.V involves electrical stimulation of certain brain structures, in particular the amygdala in the limbic system, until a previously ineffective stimulus comes to elicit a full motor seizure. Kindling has become an important experimental model for the study of epileptogenesis 14,17. However, the mechanisms underlying kindling are still largely unknown. Some studies have suggested that catecholamine in the brain are inhibitory to kindling 1, and to seizures in general a3. It has been shown that cortical noradrenaline and dopamine levels are reduced in the brain of kindled cats 15. Furthermore, it has been suggested that dopamine is an inhibitory transmitter in the amygdala 2,1e. More recently, it has been shown that unilateral lesion of the stria terminalis which contains the catecholamine-inhibitory afferents to the amygdala facilitates the early stages of development of kindling induced by ipsilateral amygdaloid stimulation in rats 5. Moreover, the dopamine content was found to be locally reduced at the site of the stimulated amygdala in kindled rats 6. However, other workers have reported a reduction in noradrenaline content in the hypothalamus but found no change in brain dopamine levels in kindled rats 4. In the present study, the activity of the enzyme tyrosine hydroxylase, the ratelimiting step of catecholamine biosynthesis11,16, was measured in the amygdala and other brain regions in rats kindled by amygdaloid stimulation. Twenty-one male Wistar rats, 180-200 g in weight, were used for this study; 7 of these rats served as unoperated controls. Under halothane anaesthesia, the left basolateral amygdalae of 14 rats were stereotaxically implanted with bipolar stimulating electrodes of nichrome wire. The coordinates were : 4.5 mm anterior to the interaural line, 4.4 mm lateral to the midline, and 7.9 mm deep to the cortical surfacO °. Rats were allowed one week for recovery and daily stimulation was begun on the eighth day. The rats were divided into two groups. Seven were stimulated and 7 were non-stimulated sham controls. To the stimulated rats, a suprathreshold constant current, ranging between 100-300/~A, and consisting of trains of 1 msec biphasic square wave pulses at a frequency of 60 Hz, was delivered for one second daily via the stimulating electrode * 1. B. Farjo is on a Ph. D grant from the College of Medicine, University of Baghdad, Iraq.
424 using a c o n s t a n t c u r r e n t physiological s t i m u l a t o r (Farnell). Electrical activity was r e c o r d e d f r o m the a m y g d a l a on a G r a s s p o l y g r a p h ( m o d e l 7) before and after stimulus delivery a n d the d u r a t i o n o f the ensuing afterdischarge was measured. D a i l y st imulation was c o n t i n u e d until a generalised convulsion d e v e l o p e d 14 o n 3 consecutive days. The 7 e x p e r i m e n t a l rats d e m o n s t r a t e d afterdischarges on the first d a y o f s t i m u l a t i o n , a n d d e v e l o p e d generalised convulsions between 9 and 13 days. The p a t t e r n o f dev e l o p m e n t o f kindling did n o t differ from t h a t r e p o r t e d by o t h e r w o r k e r s 7,l L F o r weeks after the last stimulation, rats were killed by d e c a p i t a t i o n a n d their brains dissected o u t quickly on a cold stage at 4 °C. The following dissection was done with the aid o f the dissecting microscope. The entire h i p p o c a m p u s was carefully dissected o u t on b o t h sides o f the brain. T w o vertical cuts were then made from the inferior surface o f the brain; the first at the level o f the optic c h i a s m a (A 5500 # m ) 10 a n d the second at the level o f j u n c t i o n o f the cerebral peduncles with the base o f the cerebral hemispheres (A 3000 ~ m ) 1°, thus dividing,She brain into 3 parts, F r o m the a n t e r i o r part, tissue p o r t i o n s were t a k e n f r o m the frontal cortex and s t r i a t u m on both TABLE I Tyrosine hydroxylase activity nmole /3H/L-DOPA ]brmed/h/g wet wt.) in different brain regions hi amygdaloid kindled rats Each value represents the mean activity :k S.E. (n). Right
LeJt
1.96 i 0.13 2.11 ± 0.17 1.95 ~ 0.10
1.31 ± 0.09*,** 2.10 -~ 0.15" t.98 -3 0.07
0.36 ± 0.02 0.38 ± 0.03 0.38 _-k0.02
0.34 A_0.04 0.42 :k 0.02 0.40 ~ 0.03
4.17 i: 0.28 4.31 ± 0.19 5.01 -% 0.18
3.58 -~ 0.28 4.45 t: 0.25 4.89 :k 0.27
Thalamus Kindled (6) Sham-operated (6) Unoperated (6)
1.13 ~ 0.11 0.89 ± 0.10 1.10 ± 0.18
0.82 i 0.16 0.88 i 0.10 0.92 ~ 0.16
Striatum Kindled (6) Sham-operated (6) Unoperated (6)
23.99 i: 1.05 24.02 i 1.63 23.99 t: 1.66
21.79 ± 0.68 24.62 i: 1.24 25.14 :£ 1.85
Cortex Kindled (7) Sham-operated (7) Unoperated (7)
0.28 _-k0.01 0.27 :k 0.02 0.27 _~ 0.02
0.26 ± 0.02 0.27 ± 0.02 0.28 :k 0.02
Amygdala Kindled (7) Sham-operated (7) Unoperated (7) Hippocampus Kindled (6) Sham-operated (6) Unoperated (6) Hypothalamus Kindled (6) Sham-operated (6) Unoperated (6)
* Site of electrode implantation into the left amygdala. ** P < 0.002 for comparison of right and left sides (Mann-Whitney U-test)
425 sides. From the middle part, both amygdalae were dissected out under the dissecting microscope. Tissue portions from the hypothalamus and thalamus were then taken. Kindled, implanted and unoperated control rats were treated together in all phases of sacrifice and biochemical analysis. Tissue samples were stored in liquid nitrogen before the subsequent biochemical estimation of tyrosine hydroxylase activity. Tissues were homogenised with 20 mM potassium phosphate buffer, pH 7.4, so that 10 #1 buffer was used for each 1 mg tissue. Homogenates of tissue samples were assayed, in duplicates, for tyrosine hydroxylase activity according to the method of Hendry and lversen (1971) 8. Tissue and reagent blanks, in duplicate, were assayed simultaneously with tissue samples. Recovery was estimated by taking 10 #1 of [3H]LDOPA (specific activity 2.5 Ci/mmole, Radiochemical Centre, Amersham) through the assay, in duplicate, and compared to 10 ,ul [aH]L-DOPA counted directly after the addition of scintillant. The estimated percentage recovery of the method ranged between 49 and 52 °/~I. The counting efficiency of tritium was estimated to be 34 %. The results of assaying the enzyme activity are shown in Table I. Tyrosine hydroxylase activity in brain areas of unoperated control rats were not significantly different from the normal levels previously reported 9. An analysis of variance showed no significant difference in the enzyme activity in the amygdala of both the sham-operated and unoperated control rats. However, the data showed a consistent and a significant decrease in tyrosine hydroxylase activity in the left stimulated, but not the contralateral, amygdala of all kindled rats. This reduction in enzyme activity was significant at the level of P < 0.002 (Mann-Whitney U-test, U - 3) when compared to that of the contralateral amygdala. However, no correlation was found between the reduced enzyme activity and the rate of seizure development or the duration of the evoked afterdischarge. No significant difference was found in tyrosine hydroxylase activity in the hippocampus, thalamus, hypothalamus, frontal cortex and striatum on the right and left sides of both implanted and unoperated control rats. Similarly, no significant difference in the enzyne activity was found in the hippocampus, thalamus, frontal cortex on both sides of the brain in kindled rats. However, the enzyme activity tended to be reduced in the hypothalamus and the striatum in kindled rats, but this reduction was not statistically significant. The fact that tyrosine hydroxylase activity is significantly reduced at the site of stimulation might suggest that a persistent reduction in catecholamine production is implicated in reducing inhibition with consequent focal hyperexcitability changes in the stimulated amygdala in kindled rats. It has already been shown that kindlinginduced hypersensitivity may result, in part, from a persistent decrease in postsynaptic inhibitory influences3. The absence of any significant change in catecholamine production in other brain regions studied might suggest that the mechanisms underlying the spread of the afterdischarge are different from those initiating the focal hyperexcitability in kindling convulsions. Consequently, it is concluded that kindling produced a definite persistent decrement in tyrosine hydroxylase activity at the site of stimulation in the amygdala.
426 Special a c k n o w l e d g e m e n t is e x t e n d e d to Dr. J. M c Q u e e n o f the M R C Brain M e t a b o l i s m Unit, D e p a r t m e n t o f P h a r m a c o l o g y , E d i n b u r g h , for her supervision t h r o u g h o u t the course o f this piece o f work. This w o r k is included in p a r t i a l fulfillment for the degree o f Ph.D. for 1. B. F a r j o f r o m the D e p a r t m e n t o f P h a r m a c o l o g y , F a c u l t y o f Medicine, E d i n b u r g h University.
1 Arnold, P., Racine, R. and Wise, R., Effect of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat, Exp. Neurol., 40 (1973) 457--460. 2 Ben-Ari, Y., and Kelly, J. S., Dopamine evoked inhibition of single cells of the feline putamen and basolateral amygdala, J. Physiol. (Lond.), 256 (1976) 1-22. 3 Bliss, T. V. P. and Gardner-Medwin, A. R., Long lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 357-374. 4 Callaghan, D. A. and Schwark, W. S., Neurochemical change and drug effects in a model of epilepsy in the rat, Neurosci. Abstr., 2 (1976) 257. 5 Engel, J., Jr. and Katzman, R., Facilitation of amygdaloid kindling by lesions of the stria terminalis, Brain Research, 122 (1977) 137-142. 6 Engel, J., Jr. and Sharpless, N. S., Long-lasting depletion of dopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 7 Goddard, G. V., Mclntyre, D. C. and Leech, C. K., A permanent change in brain function resulting from daily electrical stimulation, Exp. NeuroL, 25 (1969) 295-330. 8 Hendry, I. A. and Iversen, L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in the mouse superior cervical sympathetic ganglion, Brain Research, 29 (1971) 159-162. 9 Iversen, L. L. and Uretsky, N. J., Regional effects of 6-hydroxydopamine on catecholamine containing neurones in rat brain and spinal cord, Brain Research, 24 (1970) 364-367. 10 K6nig, J. F. R. and Klippel, R~ A., The Rat Brain: a Stereotaxic Atlas of the Forebrain and lower Parts of Brain Stem, Williams and Wilkins, Baltimore, 1963. 11 Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmae. exp. Ther., 148 (1965) 1-8. 12 McCrea, D. A., Jordan, L. M. and Lake, N., Microiontophoresis in the amygdala of rat. Canad. Physiol., 4 (1973) 189. 13 Maynert, E. W., The role of biochemical and neurochemical factors in the laboratory evaluation of antiepileptic drugs, Epilepsia (Amst.), 10 (1969) 145-162. 14 Racine, R. J., Modification of seizure activity by electrical stimulation, I1. Motor seizure, Electroenceph, clin. Neurophysiol., 32 (1972) 281-294. 15 Sato, M. and Nakashima, J., Kindling: secondary epileptogenesis, sleep and catecholamines, Canad. J. neurol. Sci., 2 (1975) 439-446. 16 Spector, S., Inhibition of endogenous catecholamine biosynthesis, Pharma¢vl. Rev., 18 (1966), 599-609. 17 Wada, J. A. (Ed.), Kindling, Raven Press, New York, 1976, p. 230.
