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Facilitation of amygdala kindling in the rat by transecting ascending noradrenergic pathways
274
Brain Research, 189 (1980) 274 278 ~()/Elsevier/North-Holland Biomedical Press
Facilitation of amygdala kindling in the rat by transecting ascending noradrenergic pathways
CINDY L. EHLERS, DONALD K. CLIFTON and CHARLES H. SAWYER* Department of Anatomy and Brain Research Institute, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted December 27th, 1979) Key words: kindling -- amygdala -- noradrenergic pathway -- epilepsy -- brain norepinephrine --
convulsions
Kindling, a phenomenon described in name by Goddard et al. 6, involves a progressive increase in behavioral and neural responsivity produced by spaced and repeated electrical stimulation of certain brain sites (for review see Racine10). The epileptogenic effect produced by kindling is probably not due to tissue damage, as no degenerative changes have been found in the tissue surrounding the stimulating electrodes using Nissl stains ~ or electron microscope techniques~. This suggests that the kindling effect may be due to more subtle changes in neuroanatomy or possibly neurochemistry. A number of studies have indicated that catecholamines may play a role in the development of kindling. Administration of reserpine, which depletes catecholamines, or 6-hydroxydopamine (6-OHDA), which destroys catecholamine terminals, has been found to facilitate amygdala kindling in rats 1. In addition, various investigators have found depletions of catecholamines in kindled vs non-kindled brains 4,1~. These studies, however, have not resolved whether the effects seen are due to depletion of dopamine (DA), norepinephrine (NE) or both substances. In the present work we have attempted to examine more precisely the role of NE in the development of kindling. In previous studies in our laboratory, Clifton and Sawyer 3 have demonstrated that knife cuts in the dorsal tegmental region of the mesencephalon in the rat deplete hypothalamic NE by 83 ~o. These knife cuts are located caudal to the origin of any dopaminergic pathways and lateral to the serotonin system which originates primarily in the dorsal and median raph6 nuclei. Therefore, the only direct effect these transections could have on monoamines is limited to depletion of NE in the forebrain. We have utilized the same technique in this investi* To whom correspondence should be addressed.
275 gation of the effects of chronic NE depletion on the rate of amygdala kindling in male rats. Thirty-four male Sprague-Dawley rats (Simonsen), weighing 300-500 g, were housed under controlled temperature and lighting conditions. To perform complete transections of the ascending noradrenergic pathway (ANP), animals were anesthetized with sodium methohexital (Brevital), placed in a stereotaxic instrument (TrentWells) and a stainless steel knife blade 2 mm wide was placed bilaterally at A-P 0.0 (deGroot plane) and lowered to the level of 8 mm below dura. Sham transections were made in a similar manner except the knife blade was lowered 6 mm below dura. Animals were then allowed to recover for at least 3 weeks before implantation. Stereotaxic implantation of electrodes was performed on 13 ANP- and 11 sham-transected rats as well as 8 non-transected control rats under sodium pentobarbital (Diabutol)anesthesia. Stainless steel concentric bipolar electrodes were aimed for placement in the amygdala at A 4.7, vertical 2.5 lateral 5.1 (deGroot plane), and screw electrodes were placed in the calvaria over the frontal and parietal cortices. Electrode connections were made through an amphenol connector and the entire assembly attached to the calvaria with dental acrylic. Animals were then allowed at least 2 weeks to recover before amygdala afterdischarge (AD) thresholds were tested and kindling begun. AD thresholds were tested using a Grass $88 stimulator and constant current stimulus isolation units with biphasic square wave pulses of 60 Hz, 1 msec duration in 1-sec trains with a current beginning at 50 #A. If no AD was noted in the amygdala, the current was raised in steps (70 #A, 100/~A, 150 #A, 200 #A) until an AD was recorded. The following day kindling procedures were begun (day 1). The kindling stimulus was the same as that used for AD thresholds except a current of 200/zA was used. Animals were stimulated once a day until 3 fully-kindled convulsions (chewing, rearing, forelimb clonus, falling, class 5 (Racine) a) were noted. Electroencephalograms (EEGs) were taken from all animals (Grass Model 4 Electroencephalograph) prior to, during and after the application of the threshold or kindling stimulus. Animals were sacrificed by decapitation between 09.00-12.00 h and the brains were rapidly removed and frozen. Later the brains were dissected and a section containing the amygdala and periamygdaloid (AMY-periamy) cortex of the nonstimulated side of the brain was removed. Each amygdala section was homogenized in 200/zl 0.4 N perchloric acid and assayed for NE using a modification of the Shellenberger fluorometric techniqueS,IS. The results are summarized in Table I and Fig. 1. Three rats with complete transections of the ANP died within a few days following surgery; the remaining 12 complete- and I 1 sham-transected ANP animals recovered and appeared healthy. After recovery from electrode implant surgery, amygdala AD thresholds in the 3 groups (intact, sham, complete-transected) were tested. No significant differences were found when values for the 3 groups were compared (Student's t-test). Animals were then kindled using a stimulus with a current set at 200 #A. In the sham and intact groups the application of the kindling stimulus on day 1 produced behavioral arrest, closing of the ipsilateral eye and adversive head move-
276
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o
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DAYS TO FULLY K I N O L E D SEIZURE
Fig. 1. Correlation between number of days to fully-kindled seizures in intact controls (circles), rats with brains transected to a depth of 6 mm (squares) and 8 mm (triangles) and concentration of NE O~g/g) in AMY-periamy brain areas. (r = 0.61 ; P < 0.01)
ments in some a n i m a l s ; in addition, one intact a n d two sham-cut animals showed mild m o u t h movements. The complete-cut animals also showed these behaviors on day 1, b u t 5 of the animals also showed chewing, one showed chewing, rearing a n d forelimb clonus, a n d one exhibited a fully-kindled convulsion. A n i m a l s were stimulated once a day until 3 fully-kindled convulsions were noted. One a n i m a l from the sham a n d one from the complete-cut group were eliminated from the study, when following 12 days they still showed n o response to the k i n d l i n g stimulus. The results of the kindling procedure are seen in Table I. N o significant differences were seen when the n u m b e r of stimulations needed to kindle intact control rats were c o m p a r e d with those obtained from sham-transected animals (Student's t-test). However, significant differences were f o u n d (P < 0.001) when values for complete-transected animals were c o m p a r e d with shams or intact controls. More t h a n twice as m a n y stimulations were required to TABLE I Kindling rates and norepinephrine
Effects of dorsal tegmental knife cuts on time, in days, to amygdala kindling in the rat, and concentration of NE in AMY-per±amy of each group. Values are means 4- S.E.
n Days to fully-kindled seizure NE (/~g/g) AMY-per±amy
lntacts
6-mm Transection (sham)
8-mm Transection
8 8.75 ± 0.65 0.45 ± 0.06
10 7.78 ± 0.70 0.24 i 0.04*
11 4.18 ± 0.72** 0.14 ± 0.03***
* P < 0.005 vs intacts (Student's t-test). ** P < 0.001 vs intacts, P < 0.001 vs 6-ram transection. *** P < 0.001 vs intacts, P < 0.05 vs 6-mm transection.
277 kindle shams or intact control rats than were needed to kindle ANP-transected animals. Since these results suggest that rats are more susceptible to the kindling procedure following removal of the noradrenergic input to the forebrain, it was necessary to verify that the transections depleted NE. The concentration of NE in the AMY-periamy regions was reduced to about 30 % of uncut control levels, i.e. 70 reduction, whereas NE in sham-cut rats was reduced to 50 ~o o f control, as seen in Table I. Since previous studies in our laboratory3 have shown that hypothalamic NE in complete-cut animals is reduced to 17 % whereas sham-cut animals show essentially no depletion of hypothalamic NE, it appears that sham cuts probably partially disrupt the dorsal bundles but not the ventral bundles of the ANP. However, as seen in Fig. 1, there was a significant correlation between the values for NE obtained from amygdala and periamygdaloid cortex and the number of days to fully-kindled seizure in all 3 groups (r ---- 0.61 ; P < 0.01). The results reported here indicate that NE may play an important inhibitory role in the development of convulsions by the kindling method. These results confirm and extend the pharmacological studies of Arnold et al. 1, who found that reserpine and 6OHDA facilitate amygdala kindling in rats. Since no differences in the threshold to AD in the amygdala were found in these NE-depleted animals, the results seen may represent a general effect on the proclivity of the brain to epileptogenic stimuli and not necessarily local changes in amygdala neuronal thresholds. This idea is also supported by Mason and Corcoran 7, who found a greater susceptibility to metrazol-induced seizures and electroshock-induced seizures in animals depleted of NE (but not DA) by injections of 6-OHDA into sites in the brain stem. The fact that the AMY-periamy regions in our sham-cut animals were depleted of NE by 50 % and showed no facilitation of kindling indicates that the amount of depletion or the destination of the ANP fibers may be of importance. Racine and Paxinos 11, utilizing knife cut techniques, have suggested that the ventral amygdalofugal pathway (VAF) rather than other pathways tested may be more strongly involved in the propagation of amygdala discharges. Since the VAF sends projections to lateral preoptic and hypothalamic areas 2, it may be of significance that the complete ANP-transected animals, which have been found to be depleted of hypothalamic NE by 84 ~oa, kindled twice as fast as sham or intact controls in which hypothalamic NE is known to be unchanged. In conclusion, depletion of forebrain NE by transection of the ANP facilitates amygdala kindling in rats. This evidence lends support to the idea that NE may play a role in the development of human temporal lobe epilepsy. This study was supported by grants from NIH (NS01162) and the Ford Foundation. C.L.E. was a postdoctoral scholar supported by the Giannini Foundation and D.K.C. was a predoctoral trainee in Neuroscience supported by USPHS Grant 5 T01 MH06415. The authors wish to thank Ms. Peggy Chappus for technical assistance, Ms. Lois Fels for secretarial and editorial aid and Mr. Bob McAlister for the drawing.
278 1 Arnold, P. S., Racine, R. J. and Wise, P. A., Effects of atropine, reserpine, 6-hydroxydopamine and handling on seizure development in the rat, Exp. Neurol., 40 (1973) 457~,70. 2 Carpenter, M. B., Human Neuroanatomy, Williams and Wilkins, Baltimore, 1977, 541 pp. 3 Clifton, D. K. and Sawyer, C. H., LH release and ovulation in the rat following depletion of hypothalamic norepinephrine: chronic vs. acute effects, Neuroendocrinolagy, 28 (I 979) 442-449. 4 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. 5 Goddard, G. V. and Douglas, R. M., Does the engram of kindling model the engram of normal long term memory? Canad. J. neurol. Sci., 2 (1975) 385-394. 6 Goddard, G. V., McIntyre, D. C. and Leech, C. K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 7 Mason, S. T. and Corcoran, M. E., Catecholamines and convulsions, Brain Research, 170 (1979) 497-507. 8 Nance, D. M., Shryne, J. E., Gordon, J. H. and Gorski, R. A., Examination of some factors that control the effects of septal lesions on lordosis behavior, Pharmacol. Biochem. Behav., 6 (1977) 227-234. 9 Racine, R. J., Modification of seizure activity by electrical stimulation, II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281-299. 10 Racine, R., Kindling, the first decade, Neurosurgery, 3 (1978) 234-252. 11 Racine, R. J. and Paxinos, G., cited in Racine, R., Kindling: the first decade, Neurosurgery, 3 (1978) 240. 12 Sato, M. and Nakashima, T., Kindling: secondary epileptogenesis, sleep and catecholamines, Canad. J. neurol. Sci., 2 (1975) 439~146. 13 SheUenberger, M. L. and Gordon, S. H., A rapid simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxy-tryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372.
Brain Research, 189 (1980) 274 278 ~()/Elsevier/North-Holland Biomedical Press
Facilitation of amygdala kindling in the rat by transecting ascending noradrenergic pathways
CINDY L. EHLERS, DONALD K. CLIFTON and CHARLES H. SAWYER* Department of Anatomy and Brain Research Institute, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted December 27th, 1979) Key words: kindling -- amygdala -- noradrenergic pathway -- epilepsy -- brain norepinephrine --
convulsions
Kindling, a phenomenon described in name by Goddard et al. 6, involves a progressive increase in behavioral and neural responsivity produced by spaced and repeated electrical stimulation of certain brain sites (for review see Racine10). The epileptogenic effect produced by kindling is probably not due to tissue damage, as no degenerative changes have been found in the tissue surrounding the stimulating electrodes using Nissl stains ~ or electron microscope techniques~. This suggests that the kindling effect may be due to more subtle changes in neuroanatomy or possibly neurochemistry. A number of studies have indicated that catecholamines may play a role in the development of kindling. Administration of reserpine, which depletes catecholamines, or 6-hydroxydopamine (6-OHDA), which destroys catecholamine terminals, has been found to facilitate amygdala kindling in rats 1. In addition, various investigators have found depletions of catecholamines in kindled vs non-kindled brains 4,1~. These studies, however, have not resolved whether the effects seen are due to depletion of dopamine (DA), norepinephrine (NE) or both substances. In the present work we have attempted to examine more precisely the role of NE in the development of kindling. In previous studies in our laboratory, Clifton and Sawyer 3 have demonstrated that knife cuts in the dorsal tegmental region of the mesencephalon in the rat deplete hypothalamic NE by 83 ~o. These knife cuts are located caudal to the origin of any dopaminergic pathways and lateral to the serotonin system which originates primarily in the dorsal and median raph6 nuclei. Therefore, the only direct effect these transections could have on monoamines is limited to depletion of NE in the forebrain. We have utilized the same technique in this investi* To whom correspondence should be addressed.
275 gation of the effects of chronic NE depletion on the rate of amygdala kindling in male rats. Thirty-four male Sprague-Dawley rats (Simonsen), weighing 300-500 g, were housed under controlled temperature and lighting conditions. To perform complete transections of the ascending noradrenergic pathway (ANP), animals were anesthetized with sodium methohexital (Brevital), placed in a stereotaxic instrument (TrentWells) and a stainless steel knife blade 2 mm wide was placed bilaterally at A-P 0.0 (deGroot plane) and lowered to the level of 8 mm below dura. Sham transections were made in a similar manner except the knife blade was lowered 6 mm below dura. Animals were then allowed to recover for at least 3 weeks before implantation. Stereotaxic implantation of electrodes was performed on 13 ANP- and 11 sham-transected rats as well as 8 non-transected control rats under sodium pentobarbital (Diabutol)anesthesia. Stainless steel concentric bipolar electrodes were aimed for placement in the amygdala at A 4.7, vertical 2.5 lateral 5.1 (deGroot plane), and screw electrodes were placed in the calvaria over the frontal and parietal cortices. Electrode connections were made through an amphenol connector and the entire assembly attached to the calvaria with dental acrylic. Animals were then allowed at least 2 weeks to recover before amygdala afterdischarge (AD) thresholds were tested and kindling begun. AD thresholds were tested using a Grass $88 stimulator and constant current stimulus isolation units with biphasic square wave pulses of 60 Hz, 1 msec duration in 1-sec trains with a current beginning at 50 #A. If no AD was noted in the amygdala, the current was raised in steps (70 #A, 100/~A, 150 #A, 200 #A) until an AD was recorded. The following day kindling procedures were begun (day 1). The kindling stimulus was the same as that used for AD thresholds except a current of 200/zA was used. Animals were stimulated once a day until 3 fully-kindled convulsions (chewing, rearing, forelimb clonus, falling, class 5 (Racine) a) were noted. Electroencephalograms (EEGs) were taken from all animals (Grass Model 4 Electroencephalograph) prior to, during and after the application of the threshold or kindling stimulus. Animals were sacrificed by decapitation between 09.00-12.00 h and the brains were rapidly removed and frozen. Later the brains were dissected and a section containing the amygdala and periamygdaloid (AMY-periamy) cortex of the nonstimulated side of the brain was removed. Each amygdala section was homogenized in 200/zl 0.4 N perchloric acid and assayed for NE using a modification of the Shellenberger fluorometric techniqueS,IS. The results are summarized in Table I and Fig. 1. Three rats with complete transections of the ANP died within a few days following surgery; the remaining 12 complete- and I 1 sham-transected ANP animals recovered and appeared healthy. After recovery from electrode implant surgery, amygdala AD thresholds in the 3 groups (intact, sham, complete-transected) were tested. No significant differences were found when values for the 3 groups were compared (Student's t-test). Animals were then kindled using a stimulus with a current set at 200 #A. In the sham and intact groups the application of the kindling stimulus on day 1 produced behavioral arrest, closing of the ipsilateral eye and adversive head move-
276
0.7'- • INTACT CONTROL
•
0 6-ram TRAN~CTION Z~8-ram TRANSECTION 0.6• "~
•
0
0.5-
0.4 4
~
•
0.3 O O.2 ~
o
~
~
o
o
/
0.1 i
OI
~
z~
~
~
,
2
4
6
13
I0
12
14
DAYS TO FULLY K I N O L E D SEIZURE
Fig. 1. Correlation between number of days to fully-kindled seizures in intact controls (circles), rats with brains transected to a depth of 6 mm (squares) and 8 mm (triangles) and concentration of NE O~g/g) in AMY-periamy brain areas. (r = 0.61 ; P < 0.01)
ments in some a n i m a l s ; in addition, one intact a n d two sham-cut animals showed mild m o u t h movements. The complete-cut animals also showed these behaviors on day 1, b u t 5 of the animals also showed chewing, one showed chewing, rearing a n d forelimb clonus, a n d one exhibited a fully-kindled convulsion. A n i m a l s were stimulated once a day until 3 fully-kindled convulsions were noted. One a n i m a l from the sham a n d one from the complete-cut group were eliminated from the study, when following 12 days they still showed n o response to the k i n d l i n g stimulus. The results of the kindling procedure are seen in Table I. N o significant differences were seen when the n u m b e r of stimulations needed to kindle intact control rats were c o m p a r e d with those obtained from sham-transected animals (Student's t-test). However, significant differences were f o u n d (P < 0.001) when values for complete-transected animals were c o m p a r e d with shams or intact controls. More t h a n twice as m a n y stimulations were required to TABLE I Kindling rates and norepinephrine
Effects of dorsal tegmental knife cuts on time, in days, to amygdala kindling in the rat, and concentration of NE in AMY-per±amy of each group. Values are means 4- S.E.
n Days to fully-kindled seizure NE (/~g/g) AMY-per±amy
lntacts
6-mm Transection (sham)
8-mm Transection
8 8.75 ± 0.65 0.45 ± 0.06
10 7.78 ± 0.70 0.24 i 0.04*
11 4.18 ± 0.72** 0.14 ± 0.03***
* P < 0.005 vs intacts (Student's t-test). ** P < 0.001 vs intacts, P < 0.001 vs 6-ram transection. *** P < 0.001 vs intacts, P < 0.05 vs 6-mm transection.
277 kindle shams or intact control rats than were needed to kindle ANP-transected animals. Since these results suggest that rats are more susceptible to the kindling procedure following removal of the noradrenergic input to the forebrain, it was necessary to verify that the transections depleted NE. The concentration of NE in the AMY-periamy regions was reduced to about 30 % of uncut control levels, i.e. 70 reduction, whereas NE in sham-cut rats was reduced to 50 ~o o f control, as seen in Table I. Since previous studies in our laboratory3 have shown that hypothalamic NE in complete-cut animals is reduced to 17 % whereas sham-cut animals show essentially no depletion of hypothalamic NE, it appears that sham cuts probably partially disrupt the dorsal bundles but not the ventral bundles of the ANP. However, as seen in Fig. 1, there was a significant correlation between the values for NE obtained from amygdala and periamygdaloid cortex and the number of days to fully-kindled seizure in all 3 groups (r ---- 0.61 ; P < 0.01). The results reported here indicate that NE may play an important inhibitory role in the development of convulsions by the kindling method. These results confirm and extend the pharmacological studies of Arnold et al. 1, who found that reserpine and 6OHDA facilitate amygdala kindling in rats. Since no differences in the threshold to AD in the amygdala were found in these NE-depleted animals, the results seen may represent a general effect on the proclivity of the brain to epileptogenic stimuli and not necessarily local changes in amygdala neuronal thresholds. This idea is also supported by Mason and Corcoran 7, who found a greater susceptibility to metrazol-induced seizures and electroshock-induced seizures in animals depleted of NE (but not DA) by injections of 6-OHDA into sites in the brain stem. The fact that the AMY-periamy regions in our sham-cut animals were depleted of NE by 50 % and showed no facilitation of kindling indicates that the amount of depletion or the destination of the ANP fibers may be of importance. Racine and Paxinos 11, utilizing knife cut techniques, have suggested that the ventral amygdalofugal pathway (VAF) rather than other pathways tested may be more strongly involved in the propagation of amygdala discharges. Since the VAF sends projections to lateral preoptic and hypothalamic areas 2, it may be of significance that the complete ANP-transected animals, which have been found to be depleted of hypothalamic NE by 84 ~oa, kindled twice as fast as sham or intact controls in which hypothalamic NE is known to be unchanged. In conclusion, depletion of forebrain NE by transection of the ANP facilitates amygdala kindling in rats. This evidence lends support to the idea that NE may play a role in the development of human temporal lobe epilepsy. This study was supported by grants from NIH (NS01162) and the Ford Foundation. C.L.E. was a postdoctoral scholar supported by the Giannini Foundation and D.K.C. was a predoctoral trainee in Neuroscience supported by USPHS Grant 5 T01 MH06415. The authors wish to thank Ms. Peggy Chappus for technical assistance, Ms. Lois Fels for secretarial and editorial aid and Mr. Bob McAlister for the drawing.
278 1 Arnold, P. S., Racine, R. J. and Wise, P. A., Effects of atropine, reserpine, 6-hydroxydopamine and handling on seizure development in the rat, Exp. Neurol., 40 (1973) 457~,70. 2 Carpenter, M. B., Human Neuroanatomy, Williams and Wilkins, Baltimore, 1977, 541 pp. 3 Clifton, D. K. and Sawyer, C. H., LH release and ovulation in the rat following depletion of hypothalamic norepinephrine: chronic vs. acute effects, Neuroendocrinolagy, 28 (I 979) 442-449. 4 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. 5 Goddard, G. V. and Douglas, R. M., Does the engram of kindling model the engram of normal long term memory? Canad. J. neurol. Sci., 2 (1975) 385-394. 6 Goddard, G. V., McIntyre, D. C. and Leech, C. K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 7 Mason, S. T. and Corcoran, M. E., Catecholamines and convulsions, Brain Research, 170 (1979) 497-507. 8 Nance, D. M., Shryne, J. E., Gordon, J. H. and Gorski, R. A., Examination of some factors that control the effects of septal lesions on lordosis behavior, Pharmacol. Biochem. Behav., 6 (1977) 227-234. 9 Racine, R. J., Modification of seizure activity by electrical stimulation, II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281-299. 10 Racine, R., Kindling, the first decade, Neurosurgery, 3 (1978) 234-252. 11 Racine, R. J. and Paxinos, G., cited in Racine, R., Kindling: the first decade, Neurosurgery, 3 (1978) 240. 12 Sato, M. and Nakashima, T., Kindling: secondary epileptogenesis, sleep and catecholamines, Canad. J. neurol. Sci., 2 (1975) 439~146. 13 SheUenberger, M. L. and Gordon, S. H., A rapid simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxy-tryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372.