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Persistence of reorganized synaptic connectivity in the amygdala of kindled rats
0361-9230/93$6.00 + .OO Copyright0 1993Pergamon Press Ltd.
Brain ResearchBulletin,Vol. 31, pp. 631-635, 1993 Printed in the USA. All rights reserved.
Persistence of Reorganized Synaptic Connectivity in the Amygdala of Kindled Rats REIKO OKADA,* MASAKO NISHIZUKA,f
REIJI IIZUKA* AND YASUMASA ARAIt
*Departments of Psychiatry and fAnatomy, Juntendo University School of Medicine,
Tokyo 113 Japan
Received 27 January 1992; Accepted 7 December 1992 OKADA, R., M. NISHIZUKA, R. IIZUKA AND Y. ARAI. Persistenceof reorganizedsynapticconnectivity in the amygdalaof kindled rats. BRAIN RES BULL X(6) 63 l-635, l993.-Kindling stimulation was applied to the basolateral amygdala of adult rats, and the density of dendritic synapses was examined under the electron microscope in the medial amygdaloid nucleus (MAN) contralateral to the site of stimulation, and an unfolding correction of biasedness was made. When generalized motor seizures had been induced for 5 days consecutively, the kindling was considered to be complete. The number of dendritic synapses, but not the number of somatic synapses, was markedly decreased in the MAN of the kindled rats. Reductions in numb were marked in the case of both dendritic shaft and spine synapses. The reductions in numbers of shaft and spine synapses were similarly evident in the MAN of kindled rats 100 days after stimulation was discontinued. The numbers of dendritic synapses were similarly decreased in the rats that received additional bouts of stimulation subsequent to the completion of kindling. Thus, once the kindling was completed, the newly acquired synaptic connectivity was preserved in the MAN. These findings indicate that the remodeling of synaptic connectivity was a morphological correlate of the kindling in the MAN. Kindling
Amygdala
Synapse
Remodeling
in the MAN, is not transient but is preserved in parallel with the long-lasting nature of the behavioral and neural response. In the present study, synaptic connections in the MAN of kindled rats were examined by electron microscopy after a discontinuation of electrical stimulation and afier additional bouts of stimulation.
KINDLING phenomena involve a progressive increase in behavioral and neural responsiveness as a result of repeated sub-
convulsive electrical stimulation of certain regions of the brain (4,5,2 1,22,24,29). Generalized motor seizures induced by intermittent stimulations elicit epileptiform neural responses at sites of stimulation, and the neural responses are transferred to other regions of the brain which are termed the secondary foci (5,10,24). Several investigators have described the occurrence of neuroplastic changes in the morphology (2,3,7,15,18,23,27) of activated regions of epileptic animals. For example, we have shown that the number of dendritic synapses in the medial amygdaloid nucleus (MAN) was reduced in rats that had received kindling stimulations in the basolateral amygdala (BLA) or other regions of the forebrain (15). Paroxysmal spike discharges, independent of the electrical activity at the sites of stimulation (10,24,29), were seen in electroencephalograms recorded from the MAN ipsi- and contralateral to the stimulation sites (15). Thus, a secondary epileptic focus was likely produced in the MAN (15). The loss of synapses in the MAN seemed, therefore, to be a morphological correlate of the epileptogenesis in this region (15). It is well known that, in animals that acquire epileptic responsiveness, the generalized motor seizures are persistently reproducible when the electrical stimulation is reintroduced after a long interval (5,22,29). These findings imply that any morphological change produced by kindling stimulation, if it occurs
METHOD
Male Sprague-Dawley rats (10-l 1 weeks of age) were used in the present study. An electrode, consisting of a twisted pair of nichrome wires (0.12 mm in diameter) and fully insulated except at the tip, was implanted stereotaxically into the BLA of the right hemisphere of some of the rats while they were deeply anesthetized with sodium pentobarbital(32.5 mg,/kg, b.wt.). The stereotaxic coordinates of the BLA were those given by Pellegrino et al. (19): 5.6 mm anterior to the vertical zero plane; 5.0 mm lateral to the midline; and 0.3 mm ventral to the horizontal zero plane. The electrode was fixed to the skull with a dental resin and screws. An indifferent electrode made of strands of uninsulated stainless steel wire and wrapped around a watch screw was embedded in the skull. One week after the implantation of electrodes, electrical stimulation was applied daily via a sinusoidal current (300 PA, 50 Hz, 1 s) until class 5 generalized motor seizures, as described by Racine (2 l), were elicited for 5 days consecutively. At that point the kindling was considered
’ Requests for reprints should be addressed to Masako Nishizuka, Ph.D., Department of Anatomy, Juntendo University School of Medicine, 2 Hongo, Tokyo, 113, Japan.
631
OKADA ET AL. to be complete (21). These experimental paradigms were the same as those described previously ( 15). At the completion of the kindling, the kindled rats were subdivided into three groups. Seven rats were sacrificed when the kindling was completed. Six rats received the same stimulation for 25 days subsequent to the completion of kindling. Another group of six rats received a stimulus every day but the amplitude of the stimulus was decreased by 50 PA steps, until the generalized motor seizures were no longer elicited, to determine the threshold amplitude required to evoke a seizure (29). Then these six rats were housed for 100 days without electrical stimulation. After that time they received the stimulus at the threshold amplitude, and the persistence of the behavioral response was verified. All the rats of these groups were killed on the day of the final stimulation. Other rats had electrodes implanted in the BLA of the right hemisphere and were then housed for 30 days (five rats) or 130 days (five rats) before sacrifice. These rats received no electrical stimulation. At sacrifice, an electrolytic lesion was made at the tip of the electrode in each member of the two sham-operated and three experimental groups, and then the location of each electrode was verified histologically. Five intact rats were used as unoperated controls. Synaptic organization of the left MAN, contralateral to the site of kindling stimulation, were studied under the electron microscope. In brief, all the rats were deeply anesthetized with an overdose of pentobarbital and perfused with a mixture of 4% paraformaldehyde and 2% glutaraldehyde dissolved in 0.1 M phosphate buffer, pH 7.4, through the left cardiac ventricle. The MAN tissue was taken from the left hemisphere and postfixed with OsOI, dehydrated, and embedded in an epoxy resin. Specimens were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined. For semiquantitative evaluation of synapses, ultrathin sections with a silver/gray interference color were used. Electron micrographs of the neuropil of the central part of the left MAN were taken at a magnification of X3,300, and they were enlarged to X33,000. Thirty electron micrographs were prepared for each individual rat, and all electron micrographs were coded so that experimental conditions were unknown during counting. The numbers of synapses formed on the dendritic shafts and on the dendritic spines were counted separately. The number of synapses in the neuropil was computed for a field of 10,000 pm*. These procedures were the same as those described previously ( 15). For correction of biasedness in the counting (I), the length of synaptic discs, that is, the length of paramembranous density in synaptic junctions, on the electron micrographs was measured with a computerized image analyzer (IBAS 2000, Kontron, Munich). The lengths of synaptic discs in dendritic shaft synapses and in dendritic spine synapses were differentially measured. The numerical density of synapses was calculated for a volume of 1,000pm3 using the formula in an unfolding method without a correction for lost caps as indicated previously (1): numerical density =
mean number of synapses in 10,000 pm2 mean length of discs + section thickness
The thickness of ultrathin section with a silver/gray interference color was assumed as 50 pm as measured by Calverley et al. (1). The number of somatic synapses was counted in the left MAN of the rats killed at the completion of kindling and in the left MAN of unoperated control rats. Electron micrographs that covered the circumference of individual neurons on a ultrathin sections were taken and the entire circumference was reconstructed. The number of somatic synapses was counted and the
circumferential length of neurons was measured with a computerized digitizer. Statistical analyses were made using the analysis of variance followed by Duncan’s multiple range test and differences from controls at the level of p < 0.05 were regarded as significant. RESULTS
The behavioral responses described by Racine (2 1) developed after daily electrical stimulation. Generalized motor seizures (21) were eventually produced in all of the kindled rats. The time required to elicit the first generalized motor convulsion was 11.3 + 0.3 (Mean + SEM) days and that to accomplish the kindling was 33.0 +- 3.6 days. All the rats that received the additional stimulations subsequent to the completion of kindling had similar seizures each time when they received the additional stimulations. They experienced seizures on more than 30 occasions but did not show spontaneous convulsion. All the rats that were housed for 100 days after the completion of kindling had seizures in response to the threshold stimulus at 50 or 100 rA when it was applied after 100 days. The behavioral effects of kindling were, thus, reproduced after a long interval between stimulations. Semiquantification of the density of dendritic synapses revealed that the density was reduced in the MAN of the kindled rats (Fig. 1). At the completion of kindling, the number of dendritic synapses in the left MAN in kindled rats was statistically reduced as compared to that in the unoperated and sham-operated rats. Both the dendritic shaft synapses and the dendritic spine synapses were significantly fewer in number at the completion of kindling as compared to control values (Fig. I). By contrast, the number of dendritic synapses in the sham-operated rats with electrodes in place for 30 and 130 days was not statistically different from the values for the unoperated controls (Fig. l), indicating that the presence of an electrode in the right BLA for a long time did not affect the number of synapses in the left MAN. Thus, the reductions in numbers of dendritic synapses in the kindled rats were ascribed to the results of kindling. In contrast to the dendritic synapses, the number of somatic synapses at the completion of kindling was not statistically different from the control values (Table 1) and, hence, additional studies of the somatic synapses were not carried out. In the rats that were housed for 100 days after the completion of kindling, the numbers of dendritic synapses and of their two components were significantly lower than the control values (Fig. 1). However, there was no statistical difference in the numbers of synapses between these rats and the rats killed when the kindling has been completed (Fig. l), indicating that recovery of synaptic connections did not occur after stimulation was terminated. Furthermore, the numbers of dendritic synapses and of their components in the rats that received the additional stimulations were significantly lower than those in the controls (Fig. I). Thus, the numbers of dendritic shaft synapses and of spine synapses in the rats that acquired the kindling were significantly decreased as compared to those of control rats. The subsequent electrical stimulations did not, however, enhance the decrease in numbers of dendritic synapses in the MAN. The numbers of synapses after subsequent additional stimulations were comparable to the numbers at the completion of kindling (Fig. 1). There were no statistically significant differences in the patterns of distribution of the dendritic synapses in the MAN between the rats that received additional stimulations and the rats that were housed without stimulation. Table 2 summarizes the numerical densities (mean + SEM) ofdendritic synapses that were calculated using the mean length
SYNAPTIC
REMODELING
IN AMYGDALOID
633
KINDLING
i?
:ztooo45
E
0
; 500n 5 Z
L
per&d
km-*perated Sham-operated and
and housed
(30 days)
housed (130days)
Kind~d
Kindled
Kindled
and
and
housed
stimulated
FIG. 1. Numbers of dendritic synapses (mean + SEM) in a field of 10,000 pm* in the neuropil of the left MAN of unoperated rats, of rats housed for 30 and I30 days, of rats killed at the completion ofthe kindling (kindled), of rats that were housed for 100 days after the completion of kindling (kindled and housed), and of rats that received additional stimulation for 25 days subsequent to the completion of kindling {kindled and stimulated). The stimulation was applied to the right BLA. The numbers of dendritic shaft synapses (dotted columns), dendritic spine synapses (densely dotted columns), and total dendritic synapses (open columns) are shown. The numbers at the bottom of the open columns show the numbers of rats studied. All the differences between the control groups and the kindled groups, as examined by the analysis of variance followed by Duncan’s multiple range test, are significant (p < 0.05), while differences among three kinds of control groups and among three kinds of the kindled rats are not significant. sham-operated
(mean + SEM) of synaptic discs. At the completion of kindling, both the dendritic shaft synapses and the dendritic spine synapses were significantly lower in density as compared to the control groups. In the rats that were housed for 100days after the completion of kindling and in the rats that received subsequent stimulation, the densities of synapses were significantly lower than the control values. However, there was no statistical difference in the densities of synapses between these rats and the rats killed when the kindling had been completed. These results obtained by the unfolding correction (1) indicated that the reductions in numbers of dendritic synapses and the persistence of the reduced densities in the MAN of the kindled rats were likely ascribed to the results of kindling but not of severe biasedness ofthe counting methods. DISCUSSION
The present results clearly indicate that the density of dendritic synapses was decreased in the MAN of the kindled rats. This decrease was not due to tissue damage because the reduction was seen in the MAN contralateral to the sites of stimulation. The occurrence of~ductions in numbers of dendritic shaft and spine synapses in the MAN coincides with our previous observations of decreased numbers of synapses in the same region of rats after kindling stimulation of the BLA, the septum, or the corpus callosum (15). Because the appearance of epileptiform neural activity, independent of the spike discharges occurring in the stimulation site, suggested that transfer of the kindling effects from the sites of stimulation to the ipsi- and contralateral sides of the MAN had occurred ( 1S), the decrease in number of synapses was considered to be correlated with the formation of the secondary focus in the MAN. The most important among the present findings is the observation that, once the kindling had been completed, the re-
duced densities of dendritic shaft and of dendritic spine synapses were preserved in the MAN. Elimination of a certain population of synapses may result in reorganization of the remaining neuronal circuits in the MAN. Once the reduction in numbers of dendritic synapses had occurred, enhancement ofthe remodeling and recovery of the connectivity was not encountered in the MAN, the newly acquired connectivity of the MAN being ap parently stable. Such preservation of the remodeled circuits obviously has implications with respect to the classical observation that cessation of kindling stimulation does not result in disap pearance of the acquired electrical and behavioral responses (5,22,29). Recent advance in stereology has revealed that the disector method that is based on making counts in two parallel sections offers the possibility of unbiased estimates of the numerical density of biological particles including synapses (26). Another correction technique, the unfolding method, can give essentially similar values for the numerical density of synapses to those estimated by the disector method, even though the unfolding method is still biased (1). Because serial ultrathin sections a known distance apart were not obtained, the unfolding correction (1) was applied in the present study. Because the somatic synapses in the MAN were not influenced by the kindling stimulation, it appears that, in the MAN, the kindling remodeled the synaptic connectivity primarily on the dendrites. The possibility remains that the loss of dendritic synapses is attributable to a decrease in or remodeling of axonal ~mi~cations in the MAN. The BLA in the present study included a part of the basomedial and the basal nuclei and these regions send axons to the MAN ipsi- and contralateral directions (8,16,20). These int~yg~oid inputs that formed synaptic connections in the MAN presumably played a role in the kindling and the reduction of synapses in the MAN.
634
OKADA ET AL. TABLE I
NUMBERS
OF SOMATIC
SYNAPSES
Gr0lJps
No. of Neurons
Unoperated Kindled
296 300
ON NEURONS
IN THE MEDIAL
AMYGDALOID
Circumferential Length (pm)/Neuron
NUCLEUS
IN KINDLED
No. of Synapses
36.9 + 1.2* 38.3 -+ 1.3
AND UNOPERATED
No. of Synapses/40 pm Circumference
No. of Synapses/Neuron
418 415
RATS
1.5 t 0.2* 1.7 + 0.3
1.5 c 0.2* I .7 + 0.2
* Mean f SEM of results from six rats in a given group. The differences, as examined by the anaylsis of variance, are not significant between the two groups.
With regard to hippocampal kindling that was produced by stimulation of the perforant path in the rat, the active zones of the presynaptic terminals of the perforant path axons were enlarged in the dentate gyrus (2). A similar type of kindling induced a marked decrease in number of spine synapses that were made by the perforant path axons on the granule cells of the dentate gyms (3), and it also caused a long-lasting regrowth of the axon of the granule cells (27). In the hippocampal neurons of Mongolian gerbils that exhibited spontaneous seizures, the density of dendritic spines on which the axons of the granule cells synapsed was reduced as compared to that in seizure-resistant animals (18). These findings in the hippocampus suggest the involvement of multiple neural substrates in the selective synaptic remodeling that occurs in the MAN. Involvement of gamma-aminobutyric acid (GABA) in epileptogenesis had been discussed repeatedly (6,7,9,17,23-25). A decrease in GABA-specific immunoreactivity was particularly obvious in the interneurons and terminals in the hippocampus of kindled rats (27). Although no information about interneurons
was provided by our earlier morphological classification of the MAN neurons (14) previous observations that numerous synapses of the MAN survived complete deatferentation (13) indicate that either the interneurons or the axon collaterals of some neurons make intrinsic synaptic connections in the MAN. Neurons that contain GABA are constituents of the MAN in the rat (12). Levels of GABA-synthesizing enzymes (6,9) and of receptors for GABA (9) were apparently decreased in the amygdala of BLA-kindled rats. Taken all together, these results indicate that some GABAergic neurons, in addition to the peptidergic (24,25,28) and aminergic (11,24) elements suggested by others (11,24,25,28), participate in the kindling-induced remodeling of the neurocircuits in the MAN. ACKNOWLEDGEMENTS
We thank to M. Sato in Tohoku University who generously provided the nichrome wire that was used for the electrodes, Y. Kojima for her valuable suggestions in operation of the computerized image analyzer, and Y. Tajima and M. Ishida for their excellent technical assistance.
TABLE 2 LENGTH OF SYNAPTIC DISCS AND NUMERICAL DENSITY OF DENDRITIC SHAFT SYNAPSES AND OF DENDRITIC SPINE SYNAPSES IN THE MEDIAL AMYGDALOID NUCLEUS IN KINDLED RATS Dendritic Spine Synapses
Dendritic Shaft Synapses
Length of Discs (nm)
Unoperated [5] Sham-operated 30 days (51 130 days [5] Kindled [7] Kindled and housed [6] Kindled and stimulated [6]
419 f 389 * 403 + 361 f 395 + 390 f
6.2* 6.8 10.9 5.5 7.0 6.9
Numerical
Numerical Density/ 1,000 pm’
Length of Discs (nm)
Density/ I ,ooO pm3
4.6 + 0.09
339 rt 5.0
4.8 t 0.04
4.4 * 3.8 + 2.8 ? 3.2 + 3.0 f
347 + 309 + 314 f 338 + 341 +
4.5 * 5.1 f 3.2 + 3.1 f 3.1 +
0.07 0.11 0.04 0.05 0.06
6.1 4.3 4.8 5.0 5.9
0.09 0.11 0.06 0.04 0.06
Numbers in brackets = number of rats. Unoperated rats, sham-operated rats that were housed for 30 and 130 days, the rats killed at the completion of the kindling (kindled), the rats that were housed for 100 days after the completion of kindling (kindled and housed), and the rats that received additional stimulation for 25 days subsequent to the completion of kindling (kindled and stimulated) were studied. The stimulation was applied to the right BLA and the left MAN was observed. As for the numerical density, the differences between the control groups and the kindled groups, as examined by the analysis of variance followed by Duncan’s multiple range test, are significant (P i 0.05). * Mean +- SEM.
SYNAPTIC REMODELING IN AMYGDALOID
KINDLING
REFERENCES 1. Calverley, R. K. S.; Bendi, K. S.; Jones, D. G. Estimation of the
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numerical density of synapses in rat neocortex. Comparison of the ‘disector’ with an ‘unfolding’ method. J. Neurosci. Methods 23: 195205; 1988. Geinisman, Y.; de Toledo-Morrell, L.; Morrell, F. The brain’s record of experience: Kindling induced enlargement of the active zone in hippocampal perforanted synapses. Brain Res. 5 13:175- 179; 1990. Geinisman, Y.; Morrell, F.; de Toledo-Morrell, L. Remodeling of synaptic architecture during hippocampal “kindling.” Proc. Natl. Acad. Sci. USA 85:3260-3264; 1988. Goddard, G. V.; Douglass, R. M. Does the engram of kindling model engram of normal long term memory? Can. J. Neurosci. 2:385-394; 1975. Goddard, G. V.; McIntyre, D. C.; Leech, C. K. A permanent changes in brain function resulting from daily electrical stimulation. Exp. Neurol. 25:295-330; 1969. Itagaki, S.; Kimura, H. Retardation of resynthesis of GABA-transaminase in some brain regions of amygdala-kindled rats. Brain Res. 381:77-84; 1986. Kamphuis, W.; Wadman, W. J.; Buijs, R. M.; Lopes da Silva, F. H. The development of changes in hippocampal GABA immunoreactivity in the rat kindling model of epilepsy: A light microscopic study with GABA antibodies. Neuroscience 23:433-446; 1987. Krettek, J. E.; Price, J. L. A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid axonal connections. J. Comp. Neurol. 178:255-280; 1978. L&cher, W.; Schwark, W. S. Further evidence for abnormal GABAeraic circuits in amvadala-kindled rats. Brain Res. 420~385-390: 1987: McIntyre, D. C.; Goddard, G. V. Transfer, interference and spontaneous recovery of convulsions kindled from the rat amygdala. Electroencenhaloar. Clin. Neuronhvsiol. 35:533-543: 1973. McIntyre, D. C.; Saari, M.; Papp-&,-B. A. Potentiatioh of amygdala kindling in adult or infant rats by injection of 6-hydroxydopamine. Exp. Neurol. 63:527-544; 1979. Mugnaini, E.; Oertel, W. H. An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In: Bjorklund, A.; Htikfelt, T., eds. Handbook of chemical neuroanatomy. vol. 4. GABA and neuropeptides in the CNS. part 1. Amsterdam: Elsevier; 1985:436-608. Nishizuka, M.; Arai, Y. Intrinsic connections in the medial amygdala as revealed by complete deafferentation. Neurosci. Lett. 35:247251; 1983. Nishizuka, M.; Murakami, S.; Arai, Y. Neuronal structure and malefemale difference in the medial amygdala of rats. Biomed. Res. lO(S3):323-327; 1990. -1
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15. Nishizuka, M.; Okada, R.; Seki, K.; Arai, Y.; Iizuka, R. Loss of dendritic synapses in the medial amygdala associated with kindling. Brain Res. 552:351-355; 1991. 16. Nitecka, L.; Amerski, L.; Narkiewicz, 0. Interamygdaloid connections in the rat studied by the horseradish peroxidase method. Neurosci. Lett. 26: l-4; 198 1. 17. Okada, R.; Negishi, N.; Nagaya, H. The role of the nigro-tegmental GABAetgic pathway in the propagation of pentylenetetrazol-induced seizures. Brain Res. 480~383-387; 1989. 18. Paul, L. A.; Fried, I.; Watanabe, K.; Forsythe, A. B.; Scheibel, A. B. Structural correlates of seizure behavior in the Mongolian gerbil. Science 2 13:924-926; 198 1. 19. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain, 2nd ed. New York: Plenum Press; 1979. 20. Price, J. L.; Russchen, F. T.; Amaral, D. G. The limbic region. II. The amygdaloid complex. In: Bjorklund, A.; Hbkfelt, T.; Swanson, L. W., eds. Handbook of chemical neuroanatomy. vol. 5. Integrated systems of the CNS, part 1. Hypothalamus, hippocampus, amygdala, retina. Amsterdam: Elsevier; 1987:279-388. 2 1. Racine, R. J. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroncenhaloar. 32:28 l. _ Clin. Neuroohvsiol. . . 294; 1972. 22. Racine, R. J. Kindling: The first decade. Neurosurgery 3:234-252; 1918. 23. Ribak, C. E.; Bradbume, R. M.; Harris, A. B. A preferential loss of GABAergic, symmetric synapses in epileptic foci: A quantitative ultrastructural analysis of monkey neocortex. J. Neurosci. 2: 17251735; 1982. 24. Sato, M.; Racine, R. J.; McIntyre, D. C. Kindling: Basic mechanisms and clinical validity. Electroencephalogr. Clin. Neurophysiol. 76~459-472; 1990. 25. Shinoda, H.; Schwartz, J. P.; Nadi, N. S. Amygdaloid kindling of rats increases preprosomatostatin mRNA and somatostatin without affectingglutamic acid decarboxylase (GAD) mRNA or GAD. Mol. Brain Res. 5:243-246; 1989. 26. Sterio, D. C. The unbiased estimation of number and sizes ofarbitrary particles using the disector. J. Microsconv 134: 127-136: 1984. 27. Sutula, T.; Xiao-Xian, H.; Cavazos, J.; Scott, G. Synaptic reorganization in the hippocampus induced by abnormal functional activity. Science 239:1147-l 150; 1988. 28. Talavera, E.; Omana-Zapata, I.; Asai, M.; Comes-Lam, M. Regional brain ir-Met-, ir-Leu-enkephalin concentrations during progress and full electrical amygdaloid kindling. Brain Res. 485:141-148; 1989. 29. Wada, J. A.; Sato, M. Generalized convulsive seizures induced by electrical stimulation of the amygdala in cat: Correlative electrographic and behavioral features. Neurology 24:565-574; 1974.
Brain ResearchBulletin,Vol. 31, pp. 631-635, 1993 Printed in the USA. All rights reserved.
Persistence of Reorganized Synaptic Connectivity in the Amygdala of Kindled Rats REIKO OKADA,* MASAKO NISHIZUKA,f
REIJI IIZUKA* AND YASUMASA ARAIt
*Departments of Psychiatry and fAnatomy, Juntendo University School of Medicine,
Tokyo 113 Japan
Received 27 January 1992; Accepted 7 December 1992 OKADA, R., M. NISHIZUKA, R. IIZUKA AND Y. ARAI. Persistenceof reorganizedsynapticconnectivity in the amygdalaof kindled rats. BRAIN RES BULL X(6) 63 l-635, l993.-Kindling stimulation was applied to the basolateral amygdala of adult rats, and the density of dendritic synapses was examined under the electron microscope in the medial amygdaloid nucleus (MAN) contralateral to the site of stimulation, and an unfolding correction of biasedness was made. When generalized motor seizures had been induced for 5 days consecutively, the kindling was considered to be complete. The number of dendritic synapses, but not the number of somatic synapses, was markedly decreased in the MAN of the kindled rats. Reductions in numb were marked in the case of both dendritic shaft and spine synapses. The reductions in numbers of shaft and spine synapses were similarly evident in the MAN of kindled rats 100 days after stimulation was discontinued. The numbers of dendritic synapses were similarly decreased in the rats that received additional bouts of stimulation subsequent to the completion of kindling. Thus, once the kindling was completed, the newly acquired synaptic connectivity was preserved in the MAN. These findings indicate that the remodeling of synaptic connectivity was a morphological correlate of the kindling in the MAN. Kindling
Amygdala
Synapse
Remodeling
in the MAN, is not transient but is preserved in parallel with the long-lasting nature of the behavioral and neural response. In the present study, synaptic connections in the MAN of kindled rats were examined by electron microscopy after a discontinuation of electrical stimulation and afier additional bouts of stimulation.
KINDLING phenomena involve a progressive increase in behavioral and neural responsiveness as a result of repeated sub-
convulsive electrical stimulation of certain regions of the brain (4,5,2 1,22,24,29). Generalized motor seizures induced by intermittent stimulations elicit epileptiform neural responses at sites of stimulation, and the neural responses are transferred to other regions of the brain which are termed the secondary foci (5,10,24). Several investigators have described the occurrence of neuroplastic changes in the morphology (2,3,7,15,18,23,27) of activated regions of epileptic animals. For example, we have shown that the number of dendritic synapses in the medial amygdaloid nucleus (MAN) was reduced in rats that had received kindling stimulations in the basolateral amygdala (BLA) or other regions of the forebrain (15). Paroxysmal spike discharges, independent of the electrical activity at the sites of stimulation (10,24,29), were seen in electroencephalograms recorded from the MAN ipsi- and contralateral to the stimulation sites (15). Thus, a secondary epileptic focus was likely produced in the MAN (15). The loss of synapses in the MAN seemed, therefore, to be a morphological correlate of the epileptogenesis in this region (15). It is well known that, in animals that acquire epileptic responsiveness, the generalized motor seizures are persistently reproducible when the electrical stimulation is reintroduced after a long interval (5,22,29). These findings imply that any morphological change produced by kindling stimulation, if it occurs
METHOD
Male Sprague-Dawley rats (10-l 1 weeks of age) were used in the present study. An electrode, consisting of a twisted pair of nichrome wires (0.12 mm in diameter) and fully insulated except at the tip, was implanted stereotaxically into the BLA of the right hemisphere of some of the rats while they were deeply anesthetized with sodium pentobarbital(32.5 mg,/kg, b.wt.). The stereotaxic coordinates of the BLA were those given by Pellegrino et al. (19): 5.6 mm anterior to the vertical zero plane; 5.0 mm lateral to the midline; and 0.3 mm ventral to the horizontal zero plane. The electrode was fixed to the skull with a dental resin and screws. An indifferent electrode made of strands of uninsulated stainless steel wire and wrapped around a watch screw was embedded in the skull. One week after the implantation of electrodes, electrical stimulation was applied daily via a sinusoidal current (300 PA, 50 Hz, 1 s) until class 5 generalized motor seizures, as described by Racine (2 l), were elicited for 5 days consecutively. At that point the kindling was considered
’ Requests for reprints should be addressed to Masako Nishizuka, Ph.D., Department of Anatomy, Juntendo University School of Medicine, 2 Hongo, Tokyo, 113, Japan.
631
OKADA ET AL. to be complete (21). These experimental paradigms were the same as those described previously ( 15). At the completion of the kindling, the kindled rats were subdivided into three groups. Seven rats were sacrificed when the kindling was completed. Six rats received the same stimulation for 25 days subsequent to the completion of kindling. Another group of six rats received a stimulus every day but the amplitude of the stimulus was decreased by 50 PA steps, until the generalized motor seizures were no longer elicited, to determine the threshold amplitude required to evoke a seizure (29). Then these six rats were housed for 100 days without electrical stimulation. After that time they received the stimulus at the threshold amplitude, and the persistence of the behavioral response was verified. All the rats of these groups were killed on the day of the final stimulation. Other rats had electrodes implanted in the BLA of the right hemisphere and were then housed for 30 days (five rats) or 130 days (five rats) before sacrifice. These rats received no electrical stimulation. At sacrifice, an electrolytic lesion was made at the tip of the electrode in each member of the two sham-operated and three experimental groups, and then the location of each electrode was verified histologically. Five intact rats were used as unoperated controls. Synaptic organization of the left MAN, contralateral to the site of kindling stimulation, were studied under the electron microscope. In brief, all the rats were deeply anesthetized with an overdose of pentobarbital and perfused with a mixture of 4% paraformaldehyde and 2% glutaraldehyde dissolved in 0.1 M phosphate buffer, pH 7.4, through the left cardiac ventricle. The MAN tissue was taken from the left hemisphere and postfixed with OsOI, dehydrated, and embedded in an epoxy resin. Specimens were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined. For semiquantitative evaluation of synapses, ultrathin sections with a silver/gray interference color were used. Electron micrographs of the neuropil of the central part of the left MAN were taken at a magnification of X3,300, and they were enlarged to X33,000. Thirty electron micrographs were prepared for each individual rat, and all electron micrographs were coded so that experimental conditions were unknown during counting. The numbers of synapses formed on the dendritic shafts and on the dendritic spines were counted separately. The number of synapses in the neuropil was computed for a field of 10,000 pm*. These procedures were the same as those described previously ( 15). For correction of biasedness in the counting (I), the length of synaptic discs, that is, the length of paramembranous density in synaptic junctions, on the electron micrographs was measured with a computerized image analyzer (IBAS 2000, Kontron, Munich). The lengths of synaptic discs in dendritic shaft synapses and in dendritic spine synapses were differentially measured. The numerical density of synapses was calculated for a volume of 1,000pm3 using the formula in an unfolding method without a correction for lost caps as indicated previously (1): numerical density =
mean number of synapses in 10,000 pm2 mean length of discs + section thickness
The thickness of ultrathin section with a silver/gray interference color was assumed as 50 pm as measured by Calverley et al. (1). The number of somatic synapses was counted in the left MAN of the rats killed at the completion of kindling and in the left MAN of unoperated control rats. Electron micrographs that covered the circumference of individual neurons on a ultrathin sections were taken and the entire circumference was reconstructed. The number of somatic synapses was counted and the
circumferential length of neurons was measured with a computerized digitizer. Statistical analyses were made using the analysis of variance followed by Duncan’s multiple range test and differences from controls at the level of p < 0.05 were regarded as significant. RESULTS
The behavioral responses described by Racine (2 1) developed after daily electrical stimulation. Generalized motor seizures (21) were eventually produced in all of the kindled rats. The time required to elicit the first generalized motor convulsion was 11.3 + 0.3 (Mean + SEM) days and that to accomplish the kindling was 33.0 +- 3.6 days. All the rats that received the additional stimulations subsequent to the completion of kindling had similar seizures each time when they received the additional stimulations. They experienced seizures on more than 30 occasions but did not show spontaneous convulsion. All the rats that were housed for 100 days after the completion of kindling had seizures in response to the threshold stimulus at 50 or 100 rA when it was applied after 100 days. The behavioral effects of kindling were, thus, reproduced after a long interval between stimulations. Semiquantification of the density of dendritic synapses revealed that the density was reduced in the MAN of the kindled rats (Fig. 1). At the completion of kindling, the number of dendritic synapses in the left MAN in kindled rats was statistically reduced as compared to that in the unoperated and sham-operated rats. Both the dendritic shaft synapses and the dendritic spine synapses were significantly fewer in number at the completion of kindling as compared to control values (Fig. I). By contrast, the number of dendritic synapses in the sham-operated rats with electrodes in place for 30 and 130 days was not statistically different from the values for the unoperated controls (Fig. l), indicating that the presence of an electrode in the right BLA for a long time did not affect the number of synapses in the left MAN. Thus, the reductions in numbers of dendritic synapses in the kindled rats were ascribed to the results of kindling. In contrast to the dendritic synapses, the number of somatic synapses at the completion of kindling was not statistically different from the control values (Table 1) and, hence, additional studies of the somatic synapses were not carried out. In the rats that were housed for 100 days after the completion of kindling, the numbers of dendritic synapses and of their two components were significantly lower than the control values (Fig. 1). However, there was no statistical difference in the numbers of synapses between these rats and the rats killed when the kindling has been completed (Fig. l), indicating that recovery of synaptic connections did not occur after stimulation was terminated. Furthermore, the numbers of dendritic synapses and of their components in the rats that received the additional stimulations were significantly lower than those in the controls (Fig. I). Thus, the numbers of dendritic shaft synapses and of spine synapses in the rats that acquired the kindling were significantly decreased as compared to those of control rats. The subsequent electrical stimulations did not, however, enhance the decrease in numbers of dendritic synapses in the MAN. The numbers of synapses after subsequent additional stimulations were comparable to the numbers at the completion of kindling (Fig. 1). There were no statistically significant differences in the patterns of distribution of the dendritic synapses in the MAN between the rats that received additional stimulations and the rats that were housed without stimulation. Table 2 summarizes the numerical densities (mean + SEM) ofdendritic synapses that were calculated using the mean length
SYNAPTIC
REMODELING
IN AMYGDALOID
633
KINDLING
i?
:ztooo45
E
0
; 500n 5 Z
L
per&d
km-*perated Sham-operated and
and housed
(30 days)
housed (130days)
Kind~d
Kindled
Kindled
and
and
housed
stimulated
FIG. 1. Numbers of dendritic synapses (mean + SEM) in a field of 10,000 pm* in the neuropil of the left MAN of unoperated rats, of rats housed for 30 and I30 days, of rats killed at the completion ofthe kindling (kindled), of rats that were housed for 100 days after the completion of kindling (kindled and housed), and of rats that received additional stimulation for 25 days subsequent to the completion of kindling {kindled and stimulated). The stimulation was applied to the right BLA. The numbers of dendritic shaft synapses (dotted columns), dendritic spine synapses (densely dotted columns), and total dendritic synapses (open columns) are shown. The numbers at the bottom of the open columns show the numbers of rats studied. All the differences between the control groups and the kindled groups, as examined by the analysis of variance followed by Duncan’s multiple range test, are significant (p < 0.05), while differences among three kinds of control groups and among three kinds of the kindled rats are not significant. sham-operated
(mean + SEM) of synaptic discs. At the completion of kindling, both the dendritic shaft synapses and the dendritic spine synapses were significantly lower in density as compared to the control groups. In the rats that were housed for 100days after the completion of kindling and in the rats that received subsequent stimulation, the densities of synapses were significantly lower than the control values. However, there was no statistical difference in the densities of synapses between these rats and the rats killed when the kindling had been completed. These results obtained by the unfolding correction (1) indicated that the reductions in numbers of dendritic synapses and the persistence of the reduced densities in the MAN of the kindled rats were likely ascribed to the results of kindling but not of severe biasedness ofthe counting methods. DISCUSSION
The present results clearly indicate that the density of dendritic synapses was decreased in the MAN of the kindled rats. This decrease was not due to tissue damage because the reduction was seen in the MAN contralateral to the sites of stimulation. The occurrence of~ductions in numbers of dendritic shaft and spine synapses in the MAN coincides with our previous observations of decreased numbers of synapses in the same region of rats after kindling stimulation of the BLA, the septum, or the corpus callosum (15). Because the appearance of epileptiform neural activity, independent of the spike discharges occurring in the stimulation site, suggested that transfer of the kindling effects from the sites of stimulation to the ipsi- and contralateral sides of the MAN had occurred ( 1S), the decrease in number of synapses was considered to be correlated with the formation of the secondary focus in the MAN. The most important among the present findings is the observation that, once the kindling had been completed, the re-
duced densities of dendritic shaft and of dendritic spine synapses were preserved in the MAN. Elimination of a certain population of synapses may result in reorganization of the remaining neuronal circuits in the MAN. Once the reduction in numbers of dendritic synapses had occurred, enhancement ofthe remodeling and recovery of the connectivity was not encountered in the MAN, the newly acquired connectivity of the MAN being ap parently stable. Such preservation of the remodeled circuits obviously has implications with respect to the classical observation that cessation of kindling stimulation does not result in disap pearance of the acquired electrical and behavioral responses (5,22,29). Recent advance in stereology has revealed that the disector method that is based on making counts in two parallel sections offers the possibility of unbiased estimates of the numerical density of biological particles including synapses (26). Another correction technique, the unfolding method, can give essentially similar values for the numerical density of synapses to those estimated by the disector method, even though the unfolding method is still biased (1). Because serial ultrathin sections a known distance apart were not obtained, the unfolding correction (1) was applied in the present study. Because the somatic synapses in the MAN were not influenced by the kindling stimulation, it appears that, in the MAN, the kindling remodeled the synaptic connectivity primarily on the dendrites. The possibility remains that the loss of dendritic synapses is attributable to a decrease in or remodeling of axonal ~mi~cations in the MAN. The BLA in the present study included a part of the basomedial and the basal nuclei and these regions send axons to the MAN ipsi- and contralateral directions (8,16,20). These int~yg~oid inputs that formed synaptic connections in the MAN presumably played a role in the kindling and the reduction of synapses in the MAN.
634
OKADA ET AL. TABLE I
NUMBERS
OF SOMATIC
SYNAPSES
Gr0lJps
No. of Neurons
Unoperated Kindled
296 300
ON NEURONS
IN THE MEDIAL
AMYGDALOID
Circumferential Length (pm)/Neuron
NUCLEUS
IN KINDLED
No. of Synapses
36.9 + 1.2* 38.3 -+ 1.3
AND UNOPERATED
No. of Synapses/40 pm Circumference
No. of Synapses/Neuron
418 415
RATS
1.5 t 0.2* 1.7 + 0.3
1.5 c 0.2* I .7 + 0.2
* Mean f SEM of results from six rats in a given group. The differences, as examined by the anaylsis of variance, are not significant between the two groups.
With regard to hippocampal kindling that was produced by stimulation of the perforant path in the rat, the active zones of the presynaptic terminals of the perforant path axons were enlarged in the dentate gyrus (2). A similar type of kindling induced a marked decrease in number of spine synapses that were made by the perforant path axons on the granule cells of the dentate gyms (3), and it also caused a long-lasting regrowth of the axon of the granule cells (27). In the hippocampal neurons of Mongolian gerbils that exhibited spontaneous seizures, the density of dendritic spines on which the axons of the granule cells synapsed was reduced as compared to that in seizure-resistant animals (18). These findings in the hippocampus suggest the involvement of multiple neural substrates in the selective synaptic remodeling that occurs in the MAN. Involvement of gamma-aminobutyric acid (GABA) in epileptogenesis had been discussed repeatedly (6,7,9,17,23-25). A decrease in GABA-specific immunoreactivity was particularly obvious in the interneurons and terminals in the hippocampus of kindled rats (27). Although no information about interneurons
was provided by our earlier morphological classification of the MAN neurons (14) previous observations that numerous synapses of the MAN survived complete deatferentation (13) indicate that either the interneurons or the axon collaterals of some neurons make intrinsic synaptic connections in the MAN. Neurons that contain GABA are constituents of the MAN in the rat (12). Levels of GABA-synthesizing enzymes (6,9) and of receptors for GABA (9) were apparently decreased in the amygdala of BLA-kindled rats. Taken all together, these results indicate that some GABAergic neurons, in addition to the peptidergic (24,25,28) and aminergic (11,24) elements suggested by others (11,24,25,28), participate in the kindling-induced remodeling of the neurocircuits in the MAN. ACKNOWLEDGEMENTS
We thank to M. Sato in Tohoku University who generously provided the nichrome wire that was used for the electrodes, Y. Kojima for her valuable suggestions in operation of the computerized image analyzer, and Y. Tajima and M. Ishida for their excellent technical assistance.
TABLE 2 LENGTH OF SYNAPTIC DISCS AND NUMERICAL DENSITY OF DENDRITIC SHAFT SYNAPSES AND OF DENDRITIC SPINE SYNAPSES IN THE MEDIAL AMYGDALOID NUCLEUS IN KINDLED RATS Dendritic Spine Synapses
Dendritic Shaft Synapses
Length of Discs (nm)
Unoperated [5] Sham-operated 30 days (51 130 days [5] Kindled [7] Kindled and housed [6] Kindled and stimulated [6]
419 f 389 * 403 + 361 f 395 + 390 f
6.2* 6.8 10.9 5.5 7.0 6.9
Numerical
Numerical Density/ 1,000 pm’
Length of Discs (nm)
Density/ I ,ooO pm3
4.6 + 0.09
339 rt 5.0
4.8 t 0.04
4.4 * 3.8 + 2.8 ? 3.2 + 3.0 f
347 + 309 + 314 f 338 + 341 +
4.5 * 5.1 f 3.2 + 3.1 f 3.1 +
0.07 0.11 0.04 0.05 0.06
6.1 4.3 4.8 5.0 5.9
0.09 0.11 0.06 0.04 0.06
Numbers in brackets = number of rats. Unoperated rats, sham-operated rats that were housed for 30 and 130 days, the rats killed at the completion of the kindling (kindled), the rats that were housed for 100 days after the completion of kindling (kindled and housed), and the rats that received additional stimulation for 25 days subsequent to the completion of kindling (kindled and stimulated) were studied. The stimulation was applied to the right BLA and the left MAN was observed. As for the numerical density, the differences between the control groups and the kindled groups, as examined by the analysis of variance followed by Duncan’s multiple range test, are significant (P i 0.05). * Mean +- SEM.
SYNAPTIC REMODELING IN AMYGDALOID
KINDLING
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