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Depletion of noradrenaline fails to affect kindled seizures
EXPERIMENTAL
NEUROLOGY
84,237-240
(1984)
RESEARCH Depletion
of Noradrenaline
NOTE
Fails to Affect Kindled Seizures
VERNERWESTERBERG,JOHNNYE LEWIS,ANDMICHAEL
E. CORCORAN’
Department of Psychology? University of Victoria, P.O. Box 1700, Victoria, B.C. V8 W 2 Y2, Canada Received November 2. 1983 Convulsive seizures were kindled in rats by repeated stimulation of the amygdala, and the subjects then received intracerebral injections of 6-hydroxydopamine into the dorsal noradrenergic bundle. Although this treatment severely depleted noradrenaline, there was no effecton the intensity or duration of seizures, suggesting that noradrenaline does not regulate kindled seizures when they have developed.
Evidence that forebrain noradrenaline (NA) suppresses seizure development (kindling) comes primarily from studies using the specific catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Administration of 6-OHDA either intraventricularly (2, 8) or into the dorsal NA bundle (4, 9) facilitates the rate of amygdaloid, hippocampal(7) or anterior neocortical(1) kindling. Those findings have led to the suggestion that kindling may be based in part on an erosion of the influence of NA (3, 6). Although depletion of NA facilitates the development of kindled seizures, there is indirect evidence that it has no effect on established seizures. A number of investigators (4, 8,9) have found that the duration of clinical and electrographic components of generalized seizures did not differ in control rats and in rats that had received 6-OHDA before kindling. Thus NA may be active against only the mechanisms of seizure development and not against kindled seizures per se. To test this idea more directly, in the present exAbbreviations: AD-afterdischarge, NA-noradrenaline. ’ This work was supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada. We thank Janet Finlay for excellent technical assistance. 237 00144886184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved
238
WESTERBERG,
LEWIS,
AND
CORCORAN
periment we injected 6-OHDA into the dorsal NA bundle u&r rats had been kindled with amygdaloid stimulation. Male hooded rats obtained from Canadian Breeding Farm Laboratories and weighing 250 to 300 g at the time of surgery were used. Eight rats in each group (experimental and control) completed the experiment. The rats were anesthetized with sodium pentobarbital and received implantation of bipolar electrodes into the basolateral amygdala using conventional stereotaxic techniques. The electrodes consisted of twisted Nichrome wire 127 pm in diameter. Beginning 14 days after surgery each animal received 1 s of electrical stimulation daily in the left amygdala. Stimulation was constant-current biphasic square wave with a pulse width of 1.0 ms and a frequency of 60 Hz. Stimulation was administered first at 30 PA (base to peak) and was increased in lo-PA steps every 2 min until afterdischarge (AD) was evoked. The lowest intensity that evoked AD was arbitrarily considered threshold and was used for subsequent stimulation. Kindling continued until three consecutive generalized convulsive seizures [stage 5 in Racine’s (10) terminology] were evoked. Three days after the last stage-5 seizure the rats were matched for threshold and randomly assigned to either the experimental or the vehicle (control) group. They were then anesthetized with sodium pentobarbital, and a 34gauge cannula was lowered into the dorsal NA bundle (4). The experimental group received bilateral injections of 4 pg 6-OHDA dissolved in 2 ~1 0.9% saline with 0.2 mg/ml ascorbic acid as antioxidant. Injections were delivered 1 &min, and the cannula remained in place for an additional minute to permit full diffusion of the drug. Rats in the control group were treated identically except that they received bilateral injections of the saline-ascorbic acid vehicle. Fourteen days after the injections the rats were again stimulated at the previously determined AD threshold once daily, until at least three stage-5 seizures had been produced. After completion of the experiment, concentrations of NA were sampled in the brains of two experimental and two control rats. The rats were killed by cervical fracture, and using the technique of Heffner et al. (5) the brains were quickly dissected on ice into hippocampus, frontal cortex, hypothalamus, and amygdala-pyriform. Concentrations of NA were measured using highperformance liquid chromatography with electrochemical detection. Table 1 indicates that 6-OHDA produced mean depletions of NA that were greater than 93% in all regions sampled except the hypothalamus. This pattern of depletion of NA was comparable to that observed in previous studies (1, 4, 7).
For statistical analysis, data on durations of AD and clinical seizures were collapsed and averaged over the three stage-5 seizures before drug or vehicle injections and over the three stage-5 seizures after the injections. As shown in Table 2, there were no differences between groups or conditions on any
NORADRENALINE
239
AND KINDLING
TABLE 1 Effects of 6-Hydroxydopamine on Mean (SE) Regional Concentrations of Not-adrenaline“ Groupb
Frontal cortex
Control Experimental
417 + 4 0 (100%)
Hippocampus 36Ok27 17 rt 17 (95.5%)
Hypothalamus 2281 f 249 568 + 50 (75.1%)
Amy&la-pyriform 642 2 31 42 + 25 (93.3%)
’ Results are expressed as nanograms of amine per gram of wet weight of tissue, with samples from both hemispheres averaged. Value in parentheses is percentage depletion. b Eiit rats in each group.
of the measures: NA-depleted rats did not differ from controls in either duration of AD (F(1, 14) = 0.002, P = 0.88) or duration of clinical seizures (F( 1, 14) = 1.138, P = 0.30). Similarly no overall within-group differences were found between the kindling and rekindling sessions for either duration of AD (F( 1, 14) = 1.59, P = 0.23) or duration of clinical seizures (fll, 14) = 4.49, P = 0.52). There also were no significant interaction effects either for duration of AD (F( 1, 14) = 0.20, P = 0.66) or duration of clinical seizures (F( 1, 14) = 0.07, P = 0.80). A further similarity between the groups was that TABLE 2 Effects of Depletion of Noradrenaline on Previously Established Amygdaloid Seizures Group Control (N = 8) Kindle R iE Rekindle d r SE Experimental (N = 8) Kindle d :E Rekindle R iE
Duration of afterdischarge on first three stage-5 seizures (s)
Duration of first three stage-5 clinical seizures (s)
48.3 15-96 11.2
36.9 14-64 6.7
61.5 17-122 14.3
45.6 22-75 7.3
53.7 12-116 12.1
42.9 22-60 5.1
59.9 19-86 7.6
54.1 27-70 5.9
240
WESTERBERG,
LEWIS,
AND
CORCORAN
all rats showed a stage-5 seizure on the first stimulation after injection of 6OHDA or vehicle, and seizures of this intensity were reliably elicited in all subjects for the duration of subsequent testing (data not shown). These results indicate that depletion of forebrain NA with 6-OHDA has no significant effect on the strength or duration of kindled amygdaloid seizures that were established before injection of the drug. This is consistent with previous findings in rats that had received 6-OHDA before kindling (4, 8, 9), and it suggests that NA acts to suppress the development of kindled epileptogenicity but fails to affect already established seizures. Whether this differential involvement of NA is based on a kindling-induced progressive decline in postsynaptic sensitivity to NA (6) or some other mechanism (3) remains to be elucidated. REFERENCES 1. ALTMAN, I. M., AND M. E. CORCORAN.1983. Facilitation of neocortical kindling by depletion of forebrain noradrenaline. Bruin Rex 270: 174- 177. 2. ARNOLD, P. S., R. J. RACINE, AND R. A. WISE. 1973. Effects of atropine, reserpine, 6hydroxydopamine, and handling on seizure development in the rat. Exp. Neural. 40: 457-470. 3.
4. 5. 6. 7. 8. 9. 10.
CORCORAN, M. E. 1981. Catecholamines and kindling. Pages 87-104 in J. A. WADA, Ed., Kindling Two. Raven Press, New York. CORCORAN,M. E., AND S. T. MASON. 1980. Role of forebrain catecholamines in amygdaloid kindling. Brain Rex 190: 473-484. HEFFNER,T. G., J. A. HARTMANN, AND L. S. SEIDEN. 1980. A rapid method for the regional dissection of the rat brain. Pharmacol. Biochem. Behav. 13: 453-456. MCINTYRE, D. C. 198 1. Catecholamine involvement in amygdala kindling of the rat. Pages 67-85 in J. A. WADA, Ed., Kindling Two. Raven Press, New York. MCINTYRE, D. C., AND N. EDSON. 1982. Effect of norepinephrine depletion on dorsal hippocampal kindling in rats. Exp. Neural. 77: 700-704. MCINTYRE, D. C., M. SAARI, AND B. A. PAPPAS. 1979. Potentiation of amygdala kindling in adult or infant rats by injection of 6-hydroxydopamine. Exp. Neural. 63: 527-544. MOHR, E., AND M. E. CORCORAN,198 1. Depletion of noradrenaline and amygdaloid kindling. Exp. Neural. 72: 507-5 11. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 28 l-294.
NEUROLOGY
84,237-240
(1984)
RESEARCH Depletion
of Noradrenaline
NOTE
Fails to Affect Kindled Seizures
VERNERWESTERBERG,JOHNNYE LEWIS,ANDMICHAEL
E. CORCORAN’
Department of Psychology? University of Victoria, P.O. Box 1700, Victoria, B.C. V8 W 2 Y2, Canada Received November 2. 1983 Convulsive seizures were kindled in rats by repeated stimulation of the amygdala, and the subjects then received intracerebral injections of 6-hydroxydopamine into the dorsal noradrenergic bundle. Although this treatment severely depleted noradrenaline, there was no effecton the intensity or duration of seizures, suggesting that noradrenaline does not regulate kindled seizures when they have developed.
Evidence that forebrain noradrenaline (NA) suppresses seizure development (kindling) comes primarily from studies using the specific catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Administration of 6-OHDA either intraventricularly (2, 8) or into the dorsal NA bundle (4, 9) facilitates the rate of amygdaloid, hippocampal(7) or anterior neocortical(1) kindling. Those findings have led to the suggestion that kindling may be based in part on an erosion of the influence of NA (3, 6). Although depletion of NA facilitates the development of kindled seizures, there is indirect evidence that it has no effect on established seizures. A number of investigators (4, 8,9) have found that the duration of clinical and electrographic components of generalized seizures did not differ in control rats and in rats that had received 6-OHDA before kindling. Thus NA may be active against only the mechanisms of seizure development and not against kindled seizures per se. To test this idea more directly, in the present exAbbreviations: AD-afterdischarge, NA-noradrenaline. ’ This work was supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada. We thank Janet Finlay for excellent technical assistance. 237 00144886184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved
238
WESTERBERG,
LEWIS,
AND
CORCORAN
periment we injected 6-OHDA into the dorsal NA bundle u&r rats had been kindled with amygdaloid stimulation. Male hooded rats obtained from Canadian Breeding Farm Laboratories and weighing 250 to 300 g at the time of surgery were used. Eight rats in each group (experimental and control) completed the experiment. The rats were anesthetized with sodium pentobarbital and received implantation of bipolar electrodes into the basolateral amygdala using conventional stereotaxic techniques. The electrodes consisted of twisted Nichrome wire 127 pm in diameter. Beginning 14 days after surgery each animal received 1 s of electrical stimulation daily in the left amygdala. Stimulation was constant-current biphasic square wave with a pulse width of 1.0 ms and a frequency of 60 Hz. Stimulation was administered first at 30 PA (base to peak) and was increased in lo-PA steps every 2 min until afterdischarge (AD) was evoked. The lowest intensity that evoked AD was arbitrarily considered threshold and was used for subsequent stimulation. Kindling continued until three consecutive generalized convulsive seizures [stage 5 in Racine’s (10) terminology] were evoked. Three days after the last stage-5 seizure the rats were matched for threshold and randomly assigned to either the experimental or the vehicle (control) group. They were then anesthetized with sodium pentobarbital, and a 34gauge cannula was lowered into the dorsal NA bundle (4). The experimental group received bilateral injections of 4 pg 6-OHDA dissolved in 2 ~1 0.9% saline with 0.2 mg/ml ascorbic acid as antioxidant. Injections were delivered 1 &min, and the cannula remained in place for an additional minute to permit full diffusion of the drug. Rats in the control group were treated identically except that they received bilateral injections of the saline-ascorbic acid vehicle. Fourteen days after the injections the rats were again stimulated at the previously determined AD threshold once daily, until at least three stage-5 seizures had been produced. After completion of the experiment, concentrations of NA were sampled in the brains of two experimental and two control rats. The rats were killed by cervical fracture, and using the technique of Heffner et al. (5) the brains were quickly dissected on ice into hippocampus, frontal cortex, hypothalamus, and amygdala-pyriform. Concentrations of NA were measured using highperformance liquid chromatography with electrochemical detection. Table 1 indicates that 6-OHDA produced mean depletions of NA that were greater than 93% in all regions sampled except the hypothalamus. This pattern of depletion of NA was comparable to that observed in previous studies (1, 4, 7).
For statistical analysis, data on durations of AD and clinical seizures were collapsed and averaged over the three stage-5 seizures before drug or vehicle injections and over the three stage-5 seizures after the injections. As shown in Table 2, there were no differences between groups or conditions on any
NORADRENALINE
239
AND KINDLING
TABLE 1 Effects of 6-Hydroxydopamine on Mean (SE) Regional Concentrations of Not-adrenaline“ Groupb
Frontal cortex
Control Experimental
417 + 4 0 (100%)
Hippocampus 36Ok27 17 rt 17 (95.5%)
Hypothalamus 2281 f 249 568 + 50 (75.1%)
Amy&la-pyriform 642 2 31 42 + 25 (93.3%)
’ Results are expressed as nanograms of amine per gram of wet weight of tissue, with samples from both hemispheres averaged. Value in parentheses is percentage depletion. b Eiit rats in each group.
of the measures: NA-depleted rats did not differ from controls in either duration of AD (F(1, 14) = 0.002, P = 0.88) or duration of clinical seizures (F( 1, 14) = 1.138, P = 0.30). Similarly no overall within-group differences were found between the kindling and rekindling sessions for either duration of AD (F( 1, 14) = 1.59, P = 0.23) or duration of clinical seizures (fll, 14) = 4.49, P = 0.52). There also were no significant interaction effects either for duration of AD (F( 1, 14) = 0.20, P = 0.66) or duration of clinical seizures (F( 1, 14) = 0.07, P = 0.80). A further similarity between the groups was that TABLE 2 Effects of Depletion of Noradrenaline on Previously Established Amygdaloid Seizures Group Control (N = 8) Kindle R iE Rekindle d r SE Experimental (N = 8) Kindle d :E Rekindle R iE
Duration of afterdischarge on first three stage-5 seizures (s)
Duration of first three stage-5 clinical seizures (s)
48.3 15-96 11.2
36.9 14-64 6.7
61.5 17-122 14.3
45.6 22-75 7.3
53.7 12-116 12.1
42.9 22-60 5.1
59.9 19-86 7.6
54.1 27-70 5.9
240
WESTERBERG,
LEWIS,
AND
CORCORAN
all rats showed a stage-5 seizure on the first stimulation after injection of 6OHDA or vehicle, and seizures of this intensity were reliably elicited in all subjects for the duration of subsequent testing (data not shown). These results indicate that depletion of forebrain NA with 6-OHDA has no significant effect on the strength or duration of kindled amygdaloid seizures that were established before injection of the drug. This is consistent with previous findings in rats that had received 6-OHDA before kindling (4, 8, 9), and it suggests that NA acts to suppress the development of kindled epileptogenicity but fails to affect already established seizures. Whether this differential involvement of NA is based on a kindling-induced progressive decline in postsynaptic sensitivity to NA (6) or some other mechanism (3) remains to be elucidated. REFERENCES 1. ALTMAN, I. M., AND M. E. CORCORAN.1983. Facilitation of neocortical kindling by depletion of forebrain noradrenaline. Bruin Rex 270: 174- 177. 2. ARNOLD, P. S., R. J. RACINE, AND R. A. WISE. 1973. Effects of atropine, reserpine, 6hydroxydopamine, and handling on seizure development in the rat. Exp. Neural. 40: 457-470. 3.
4. 5. 6. 7. 8. 9. 10.
CORCORAN, M. E. 1981. Catecholamines and kindling. Pages 87-104 in J. A. WADA, Ed., Kindling Two. Raven Press, New York. CORCORAN,M. E., AND S. T. MASON. 1980. Role of forebrain catecholamines in amygdaloid kindling. Brain Rex 190: 473-484. HEFFNER,T. G., J. A. HARTMANN, AND L. S. SEIDEN. 1980. A rapid method for the regional dissection of the rat brain. Pharmacol. Biochem. Behav. 13: 453-456. MCINTYRE, D. C. 198 1. Catecholamine involvement in amygdala kindling of the rat. Pages 67-85 in J. A. WADA, Ed., Kindling Two. Raven Press, New York. MCINTYRE, D. C., AND N. EDSON. 1982. Effect of norepinephrine depletion on dorsal hippocampal kindling in rats. Exp. Neural. 77: 700-704. MCINTYRE, D. C., M. SAARI, AND B. A. PAPPAS. 1979. Potentiation of amygdala kindling in adult or infant rats by injection of 6-hydroxydopamine. Exp. Neural. 63: 527-544. MOHR, E., AND M. E. CORCORAN,198 1. Depletion of noradrenaline and amygdaloid kindling. Exp. Neural. 72: 507-5 11. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32: 28 l-294.