- Email: [email protected]
Effects of protein synthesis inhibition on kindling in the mouse
EXPERIMENTAL
Effects
DONALD Department Department and
68,
NEUROLOGY
of Protein
P. CAIN,
Division
Rrcrit,ed
(1980)
Synthesis Inhibition in the Mouse
on Kindling
MICHAEL E. CORCORAN.AND WILLIAM
of Ps.whology. of Psychology, British
409-419
University CJnivrrsit~
of Neurological Colutnhiu. 311/y
Sciences.
Vatxorcver, 24.
of Wrsrrm of Victoria.
1979:
Foculry
British revision
Onfario. Victoria
Ontario Cohtrnhiu
of‘ Medicine.
Colut~~hia rrc~ri~~cd
Lordon. British V6T
Noi’emher
A. STAINES’
University I W5. 30.
N6A 5C2: V8W 2V2
of
Cnnatla I979
Mice treated with anisomycin during amygdaloid kindling failed to develop generalized seizures. Anisomycin failed to suppress fully developed seizures in kindled mice, indicating that its prophylactic effects are not secondary to a general anticonvulsant action. These results are consistent with the hypothesis that the central neuroplastic changes underlying kindling require the ongoing synthesis of proteins.
INTRODUCTION The development of generalized convulsions through repeated lowintensity electrical stimulation of the brain-the kindling phenomenon (ll)-appears to depend on neuroplastic changes in the stimulated tissue. The evidence for this comes from studies showing that kindling results in a facilitation of both epileptiform and nonepileptiform activity in the kindled neural circuits (5, 23, 24). The facilitation is permanent, manifesting itself after a period of 1 year or more without stimulation following initial kindling (29). Moreover, the changes in brain function that accompany kindling are not restricted to the region around the stimulating electrode, but are quite widespread (11, 21). More recent research showed that the facilitative Abbreviations: CHX-cycloheximide. ANI-anisomycin. TCA-trichloroacetic acid, AD-afterdischarge. ’ Supported in part by grants from the Natural Sciences and Engineering Research Council of Canada awarded to D.P.C. and from the Medical Research Council of Canada awarded to M.E.C.
0014-4886/80/060409-11$02.00/O Copyright All rights
C 1980 by Academic Prss. Inc of reproduction in any form reserved
410
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CORCORAN.
AND
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changes occurred in both the stimulated site and in distant sites strongly interconnected neuroanatomically with it (24). These findings, coupled with the striking intermittency of stimulation required for kindling (11,22), were the basis for suggestions that the neural changes underlying kindling may be similar to those underlying memory formation (10, 13, 16,20). In particular, the possibility exists that during the interstimulation interval, which is optimally in the range of 2 h or more, some growth process or other form of neural reorganization occurs that is necessary for kindling. It this is so, it would be reasonable to expect that protein synthesis is importantly involved, as was suggested for long-term memory formation. This possibility was approached recently in two studies, both of which assessed the effect on kindling of protein synthesis inhibition produced by injection of cycloheximide (CHX). In the first study (17) it was found that the progressive prolongation of epileptiform afterdischarge (AD) and the formation of spontaneous epileptiform potentials in the forebrain of the paralyzed bullfrog was significantly inhibited by CHX. However, as was pointed out [Racine, discussion in (17)], it is not clear from that work whether CHX blocked the development of the seizure response (the kindling process itself) or merely the expression of the seizure manifestations. In the second study (19) it was reported that CHX was effective in blocking AD in the rabbit amygdala when administered systemically 4 h prior to stimulation, and was thus effective in prophylactically preventing kindling through the suppression of AD-generating mechanisms. The drug was also found to be effective in blocking established kindled convulsions when given 3 to 4 h prior to stimulation. This interval is somewhat longer than the 30-min interval required to reach peak protein synthesis inhibition levels in mouse brain after systemic injection of CHX (2). On the other hand, CHX was found to be completely ineffective in preventing the progressive development of AD and behavioral convulsions when administered either 15 (intraventricularly) or 30 min (i.p.) prior to stimulation. Those results suggest that although protein synthesis inhibition by CHX in the rabbit is effective in preventing the expression of AD or fully kindled convulsions when the brain stimulation is applied some hours after the injection, it is ineffective in preventing the development of kindled convulsions at shorter intervals. However, conclusive interpretation of these results is precluded by a lack of information about the time course of the CHX-induced inhibition of protein synthesis in the rabbit. Thus, the question of whether or not kindling can proceed during inhibition of protein synthesis remains open. We felt that the optimal experimental approach to the question should include the following
PROTEIN
SYNTHESIS
INHIBITION
IN KINDLING
411
features: The drug used should yield a profound inhibition of protein synthesis at a dose that is not toxic, even when administered repeatedly. The inhibitory effect of the drug should be known for the species used, and should be verified during the course of the actual experiment. The stimulation should be applied at or near the peak of protein synthesis inhibition. Ideally, the drug should act on the hypothesized mechanism of neural reorganization, and not merely act to block convulsion development through elevation of the threshold current necessary for evoking AD, or through the suppression of AD or convulsion expression. Accordingly, a control procedure should be used in which the experimental and control groups, after an initial series of stimulations under the drug and control saline solution respectively, would be treated to the other condition and then restimulated. This would indicate, in the case of the experimental group, whether any disruptive effect of the drug on kindling was due to a selective blockade of the neuroplastic changes underlying seizure development or due merely to a blockade of seizure expression. It would indicate in the case of the control group whether the drug has a seizure-suppressant action on fully kindled convulsions. Having selected these requirements, we chose to work with the Swiss-Webster mouse, and we selected anisomycin as the inhibitor of protein synthesis. Published information and pilot work indicated that essentially all requirements could be met with this combination. METHOD The subjects were 67 adult male Swiss-Webster mice that weighed approximately 40 g at the time of surgery and were housed individually in stainless-steel cages. Lab chow and water were available at all times. The animals were anesthetized with pentobarbital and received implantation of one bipolar electrode in the amygdala and a ground in the frontal sinus. The mouse has a very thin skull, and good retention of the implant is therefore a problem, particularly during a series of generalized convulsions. The details of an implantation procedure designed to insure good retention of the implant under these circumstances were published elsewhere (3). The electrode coordinates were determined with the aid of the mouse brain atlas of Montemurro and Dukelow (15), and were anterior +2.5 mm, lateral 3.3 mm, and 4.9 mm below the skull surface, with the upper incisor bar at the level of the interaural line. Electrodes were constructed of twisted stainless-steel Teflon-coated wire 127 pm in diameter soldered to individual miniature connector pins. Testing began 2 weeks after surgery. Shielded leads connected the animal to a stimulator and polygraph. The electroencephalogram was taken
412
CAIN,
CORCORAN,
AND
STAINES
before and after stimulation to obtain a prestimulation baseline and to record the AD. For kindling, l-s electrical stimulation was provided by a constant current, 60-Hz sine wave source, with the initial stimulation intensity set at 20 ,uA. If this was not sufficient to evoke AD, the intensity was gradually raised until an AD was observed and was presented at that level thereafter. Thus all animals were stimulated at their individual AD “threshold”, arbitrarily defined as the lowest intensity of stimulation sufficient to evoke AD. After threshold determination the animals were divided into four groups, two experimental and two control, roughly equated for AD threshold. All animals were stimulated at 48-h intervals until a fully generalized convulsion was evoked or 14 stimulations had been given. Anisomycin (ANI) was supplied without charge by the Charles Pfizer Company, Groton, Connecticut. Because the inhibitory effect of AN1 is essentially unrelated to dose in the range of 0.5 to 3.0 mg/mouse (8), all injections in the present study were 0.5 mg/mouse in 0.25 ml saline. The solution of AN1 was prepared at a concentration of 2.0 mg/ml according to the method of Flood et al. (8) and the final acidity was adjusted topH 6 to 7. The two experimental groups received AN1 subcutaneously in the dorsal thoracic region, and the two control groups received similar injections of physiological saline. One each of the experimental and control groups received a single injection of the appropriate solution 15 min prior to each electrical stimulation (AN1 and saline groups, respectively). The other experimental and control groups received an injection 15 min prior to, and another injection 1.5 h after each stimulation (AN1 + AN1 and saline + saline groups). Thus, high levels of protein synthesis inhibition were maintained in both experimental groups at the time of stimulation. For the AN1 group the inhibition was approximately 70% for at least 1.75 h after each stimulation, and for the AN1 + AN1 group the inhibition was approximately 70% for at least 3.5 h after each stimulation (7, 9). This allowed a determination of the effect of varying the length of protein synthesis inhibition on kindling rate. After the initial series of kindling stimulations and a 3-day interval, a cross-over rekindling procedure was begun, in which the ANI injections were discontinued in the experimental groups and were initiated in the control groups. All features of the cross-over procedure were identical to those of the initial stimulation series, except that the saline and saline + saline groups now received one and two injections of ANI, respectively, in association with each stimulation, and the AN1 and AN1 + AN1 groups received saline injections. Stimulation was again applied at 48-h intervals until each animal displayed a fully generalized convulsion.
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IN KINDLING
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At the end of testing all animals were anesthetized and perfused with formol-saline, and the brain was removed, frozen, and sectioned at 40 pm for verification of the electrode placements. All animals discussed below had electrode placements in the amygdala. Inhibition of protein synthesis by ANI was determined by comparing the incorporation of [3H]leucine into the trichloroacetic acid (TCA)-insoluble fraction in ANI- and saline-injected mice. Three additional groups of naive adult male mice were used: animals in the acute AN1 group (N = 5) received a single injection of 0.5 mg AN1 as described previously, animals in the chronic AN1 group (N = 9) received 14 ANI injections at 48-h intervals, and animals in the saline group (N = 8) received a single control injection of physiological saline. [“Hlleucine (60 Ciimmol L14,.5-ZH]leucine, New England Nuclear, Boston, Massachusetts, 10 Ci/animal) was injected in 20 ~1 physiological saline into the tail vein 15 min after the single AN1 or control saline injection or 15 min after the last chronic AN1 injection. The mice were killed 30 min later by decapatation, and the forebrain was sampled bilaterally. weighed, and immediately frozen at -90°C until extracted. The brains were homogenized in 1.5 ml distilled water at 4°C and an equal volume of 10% (w/v) TCA was added. After centrifugation and two additional rehomogenizations in 5% TCA, the precipitate was rehomogenized and washed in 99% ethanol. The pellet obtained after the ethanol wash was solubilized overnight in 2.5 ml Soluene-100 (Packard), transferred to a scintillation vial with an equal volume of ACS (Amersham) and brought to neutral pH with 1 ml 1 .O N acetic acid. The supernatants were counted as 1 ml samples in 10 ml ACS. Samples were counted in a Nuclear Chicago scintillation counter and were corrected for counting efficiency. The degree of incorporation of [“Hlleucine into protein was calculated by determining for each forebrain hemisphere the ratio of [“Hlleucine incorporated into the TCA-insoluble protein fraction to the total [“Hlleucine found in the sample and multiplying by 100. Thus, incorporation equals protein precipitate disintergrations per minute/(protein precipitate disintegration per minute + supernatant disintegrations per minute) X 100. Values obtained for the two hemispheres were averaged, and these values were averaged for each group. The values obtained for the ANI-injected groups were then expressed as a percentage of that obtained for the saline control group. RESULTS Initial Kindling. The basic phenomena of kindling in the mouse are similar to those seen in the rat. The mouse passes through a series of seizure stages that are comparable to stages l-5 as described by Racine (21). The
CAIN,
CORCORAN,
AND
STAINES
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SYNTHESIS
INHIBITION
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IN KINDLING
convulsions are tonic-clonic in form and culminate in generalization (stage 5) characterized by rearing with forelimb clonus and facial automatisms, falling, and associated polyspike AD in the stimulated amygdala. A typical seizure record from a control animal appears in Fig. 1B. Table 1 presents the main results of the experiment. Because the saline and saline + saline groups did not differ on any of the measures (Mann-Whitney U, P > 0.05, two-tailed), their data were pooled. The saline control animals kindled in a mean of 8.4 ADS. In contrast, kindling in the ANI-treated animals was almost completely blocked during the 14 stimulations tested under the drug. Of 10 animals in the ANI group, one kindled after 12 ADS and the others attained a mean of stage 1.9 after the 14 stimulations. Of nine animals in the ANI + AN1 group one kindled after 12 ADS and the remainder attained a mean of stage 2.6 after the 14 stimulations. The administration of AN1 did not disturb background neural activity or fragment or disrupt the AD in these animals. Rather, the AD did not increase in duration and complexity as did the AD in the saline control animals. This is reflected in a shorter final seizure duration for the initial kindling phase (Table 1). In other respects the AD, as well as the clinical manifestations, appeared normal in the ANI-treated animals (see Fig. 1A). Cross-Oiler Rekindling. The number of ADS required to evoke a stage 5 convulsion during cross-over rekindling are summarized in Table 1. The kindled saline control animals showed almost immediate rekindling under ANI. requiring a mean of 1.6 ADS. AN1 appeared to have no disruptive or seizure-suppressant effect on either the AD or convulsion manifestations during rekindling. TABLE Initial
Kindling
Treatment Saline AN1 AN1 + AN1
and Cross-Over
N 20 10 9
Afterdischarge threshold (PA) 31.8 ? 3.3 29.9 k 2.5 30.6 lr 2.1
h Of 10 animals, 1 kindled 1.9 after 14 stimulations. r Of 9 animals, 1 kindled 2.6 after 14 stimulations.
Rekindling
1 in Saline-
Initial kindling: afterdischarges to stage 5 8.4 -c 0.5 -b -1’
in 12 afterdischarges, in 12 afterdischarges,
and Anisomycin-Treated
Mice”
Final seizure duration (s)
Cross-over rekindling: afterdischarges to stage 5
28.8 2 2.6 19.3 2 1.7 17.7 i- 1.2
1.6 r 0.2 5.5 2 1.5 8.1 -c 1.9
and the other
9 attained
a mean
of stage
and the other
8 attained
a mean
of stage
416
CAIN,
CORCORAN,
AND
STAINES
The AN1 and AN1 + AN1 groups required 5.5 and 8.1 ADS, respectively, to rekindle. Adding these figures to the initial AD evoked during AD threshold determination yields a total of 6.5 and 9.1 ADS required to kindle these groups. The latter figures do not differ from the 8.4 ADS required to kindle the saline control animals (P > 0.05, two-tailed), nor do they differ from one another (P > 0.05, two-tailed). Biochemical Assays. The effect of the AN1 injections was to reduce the incorporation of [“Hlleucine into protein to 32.0% of control values in the acute AN1 group and to 3 1.2% of control values in the chronic AN1 group (Mann-Whitney U, both P < 0.001, two-tailed). DISCUSSION Both ANI-treated groups showed little or no tendency to develop kindled convulsions despite being stimulated 14 times, which is greater than the mean number of sessions required to kindle the control animals and is above the range of control values as well. All ANI-treated mice developed generalized convulsions during rekindling, indicating that there was no long-lasting debilitating effect of the drug on brain mechanisms necessary for kindling. The cross-over treatment of the control animals with AN1 after development of kindled convulsions showed that AN1 is essentially free of anticonvulsant effects, because these animals showed almost immediate rekindling while under the drug. Thus, we conclude that kindling is substantially blocked during ANI-induced inhibition of protein synthesis and that the blockage is not due to an anticonvulsant effect of the drug. These results agree with findings reported by Kessler et al. (paper presented at the 1977 meeting of the Eastern Association of Electroencephalographers), who examined the effects of CHX on kindling in frogs, and by Jonec et al. (12) who examined the effects of AN1 on kindling in rats. For reasons that are not immediately apparent, the anticonvulsant effect of protein synthesis inhibition described by Ogata (19) was not confirmed. Two ANI-treated mice kindled after 12 ADS, which is somewhat longer than the 8.4 ADS required by the control animals. Those mice did not differ from the other experimental animals in terms of electrode placement of any other known variable, and we cannot explain why these animals kindled and the other experimental animals did not. The reduction in incorporation of ]“H]leucine into protein to 32% of control values in the ANI-treated animals approximates similar measures obtained in previous studies (9). This verifies the efficacy of AN1 as an inhibitor of protein synthesis in mice, and indicates that there was no attenuation of the inhibiting properties of AN1 as a result of the 14 repeated
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IN KINDLING
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injections. Although with the method used this difference could be attributed to a differential labeling of the protein synthesis precursor pool, the vastly more plausible interpretation is that it represents a reliable measure of the degree of inhibition of protein synthesis produced by ANI. Two successive injections of ANI yielded no greater blockage of kindling than a single injection. This may be evidence of a ceiling effect of AN1 on kindling because the single-injection group showed little or no convulsion development while under the drug. It should be emphasized that the conclusion that ongoing synthesis of central proteins is necessary for kindling is not necessarily warranted by the results presented here. It is possible that the link between protein synthksis and kindling is not direct, but rather that AN1 disrupted some other process that is crucial for kindling. For example, it was shown that another protein synthesis inhibitor, cycloheximide, has inhibitory effects on the activity of certain brain enzymes, such as tyrosine hydroxylase and acetylcholinesterase (6, 30). Inhibition of the activity of these enzyme proteins would be expected to affect the concentration or function of the relevant neurotransmitters, and these side effects of inhibitors of protein synthesis might be causally related to the effects on kindling observed here. Inhibition of the activity of tyrosine hydroxylase or acetylcholinesterase would be expected to facilitate kindling (1, 4, 28), however, suggesting that the mechanism by which AN1 blocks kindling probably does not involve those enzymes. But an effect of anisomycin on any of a number of other brain enzyme or other systems could conceivably affect kindling, and must be kept in mind when interpreting those results. In addition, it is possible that the disruption of kindling produced by AN1 is related to the peripheral effects of the drug and not to inhibition of protein synthesis in the brain. It was reported, for example, that administration of CHX disrupts the synthesis of adrenal steroid hormones, and that this peripheral effect might mediate the amnesic effects of CHX (18). It is unlikely that ANI-induced modulation of adrenal function accounts for the present results, because kindling proceeds without major change after adrenalectomy (14). But other peripheral effects of AN1 could be responsible for the present findings, and further work is required to confirm the central origin of these effects. For the present, therefore, we can conclude only that the observed results are consistent with the hypothesis that ongoing synthesis of central proteins is required for kindling. Previous attempts to find structural changes in kindled brain have either failed (24) or yielded inconclusive results (10). In the latter study it was reported that there was an increase in axon terminal size in kindled compared with control amygdala, but that extraneous factors could not be ruled out as a cause of the difference. More recently Racine and Zaide (25)
418
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AND
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provided the first convincing evidence that kindling involves the growth of synaptic contacts. In a well-controlled, blind study, they reported that cortical kindling resulted in a nonsignificant increase in the number of synaptic terminals and a significant increase in the size of synaptic terminals on dendritic spines in kindled neocortical tissue. A number of other recent studies utilizing electrical stimulation in potentiation or conditioning paradigms also reported increases in the size or numbers of dendritic spine contacts (26, 27). Therefore, if inhibition of protein synthesis by AN1 does directly block kindling by inhibiting neural growth during the kindling process, it may do so by inhibiting growth of dendritic spine synaptic contacts. REFERENCES 1. ARNOLD, P. S., R. J. RACINE. AND R. A. WISE. 1973. Effects of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat. E~J. Neural. 40: 457-470. 2. BARONDES, S. H. 1970. Is the amnesic effect of cycloheximide due to specific interference with a processs in memory storage? Pages 545-553 in A. LAJTHA, Ed., Protein Metabolism of the Nervous System. Plenum, New York. 3. CAIN, D. P., AND S. DEKERGOMMEAUX. 1979. Electrode implantation in small rodents for kindling and long term brain stimulation. Physiol. Behat,. 22: 799-801. 4. CORCORAN, M. E., H. C. FIBIGER, J. A. MCCALJGHRAN, AND J. A. WADA. 1974. Potentiation of amygdaloid kindling and metrazol-induced seizures by 6-hydroxydopamine in rats. Exp. Neurol. 45: 118-133. 5. DOUGLAS, R. M., AND G. V. GODDARD. 1975. Long-term protentiation of the perforant path-granule cell synapse in the rat hippocampus. Bruin Res. 86: 205-215. 6. FLEXNER, L. B., R. G. SEROTA, AND R. H. GOODMAN. 1973. Cycloheximide and acetoxycycloheximide: inhibition of tyrosine hydroxylase activity and amnesic effects. Proc. Natl. Acad. Sci. U.S.A. 70: 354-356. 7. FLOOD, J. F., E. L. BENNETT, A. E. ORME, AND M. R. ROSENZWEIG. 1975. Relation of memory formation to controlled amounts of brain protein synthesis. Physiol. Behav. 15: 97-102. 8. FLOOD, J. F., E. L. BENNETT, M. R. ROSENZWEIG, AND A. E. ORME. 1973. Theinfluence of duration of protein synthesis inhibition on memory. Physiol. Behav. 10: 555-562. 9. FLOOD, J. F., M. R. ROSENZWEIG, E. L. BENNETT, AND A. E. ORME. 1974. Comparison of the effects of anisomycin on memory across six strains of mice. Behara. Biol. 10: 147- 160. 10. GODDARD; G. V., AND R. M. DOUGLAS. 1975. Does the engram of kindling model the engram of normal long term memory? Can. .I. Neural. Sci. 2: 385-394. 11. GODDARD, G. V.. D. C. MCINTYRE, AND C. K. LEECH. 1969. A permanent change in brain function resulting from daily electrical stimulation. &I. Neural. 25: 295-330. 12. JONEC, V., S. HOLMS, D. MASUOKA, AND C. G. WASTERLAIN. 1977. Animsomycin delays amygdaloid kindling in rats. Sot. Neurosci. Abstr. 3: 141. 13. LEECH, C. K., AND D. C. MCINTYRE. 1976. Kindling rates in inbred mice: an analog to learning? Behav. Biol. 16: 439-452. 14. MCINTYRE, D. C.. AND P. D. WANN. 1978. Adrenalectomy: protection from kindled convulsion induced dissociation in rats. Physiol. Behav. 20: 469-474.
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SYNTHESIS
INHIBITION
IN KINDLING
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15. MONTEMURRO, D. G., AND R. H. DUKELOW. 1972. A Sterrotoric Atlns c!f‘ the Dirnc~ephtrlor~ nrld Ralrrtrd Structrrrc.r t;f tilt, Mortse. Futura, New York. 16. MORRELL, F. 1973. Goddard’s kindling phenomenon: a new model of the “mirror focus.” Pages 207-223 in H. C. SABELLI. Ed., C/1emic,trl Modulcrtior~ of Brc~inFunctior~. Raven. New York. 17. MORR~LL, F.. N. TSURU. T. J. HO~PPNER. D. MORGAN. AND W. H. HARRISON. 1975. Secondary epileptogenesis in frog forebrain: effect of inhibition of protein synthesis. Con. J. Neural. Sc,i. 2: 407-416. 18. NAKAJIMA. S. 1975. Amnesic effect of cycloheximide in the mouse mediated by adrenocortical hormones. J. Camp. Physiol. Ps~c~~w/. 88: 378-385. 19. OGATA. N. 1977. Effects of cycloheximide on experimental epilepsy induced by daily amygdaloid stimulation in rabbits. Epilepsicr 18: 101-108. 20. RACINE. R. J. 1972. Modification of seizure activity by electrical stimulation. I. After-discharge threshold. Elec,r~oenc,rph. Cliff. Neuropkysiol. 32: 269-279. 21. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Elecfrorr~crpk. C/in. Ncr,rop/ysiol. 32: 78 l-294. 22. RACINE, R. J.. W. M. BURNHAM. J. G. GARTNER, AND D. LEVITAN. 1973. Rates ofmotor seizure development in rats subjected to electrical brain stimulation: Strain and interstimulation interval effects. E14(.f~o~ll(.r~/?. C/in. Nwroplz~sid. 35: 553-556. 23. RACINE. R. J., J. G. GARTNER. AND W. M. BURNHAM. 1972. Epileptiform activity and neural plasticitiy in limbic structures. Brtrirz Rrs. 47: 262-268. 24. RACINE. R. J.. L. TUFF. AND J. ZAIDE. 1975. Kindling, unit discharge patterns and neural plasticity. Ctrn. J. Neural. Sci. 2: 395-405. 25. RACINE. R. J.. AND J. ZAIDE. 1978. A further investigation into the mechanisms underlying the kindling phenomenon. Pages 457-493 in K. E. LIVINGSTON AND 0. HORNYKIEWICZ. Eds.. Littihic Mecircrtris~n,s, Plenum, New York. 26. RUTLEDGE, L. T., C. WRIGHT. AND J. Duncan. 1974. Morphological changes in pyramidal cells of mammalian neocortex associated with increased use. E.rp. Neorol.
44: 209-228. 27. VAN HARREVELD.
A.. AND E. FIFKOVA. 1975. Swelling of dendritic spines in the fascia dentata after stimulation of the perforant fibers as a mechanism of post-tetanic potentiation. Erp. Nerrrol. 49: 739-749. 28. Vosu. H.. AND R. A. WISE. 1975. Cholinergic seizure kindling in the rat: Comparison of caudate. amygdala. and hippocampus. Be/la\*. Eiol. 13: 491-495. 29. WADA, J. A.. M. SATO, AND M. E. CORCORAN. 1974. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsiu 15: 465-478. 30. ZECH, R., AND G. F. DOMAGK. 1975. Puromycin and cycloheximide as inhibitors of human brain acetylcholinesterase. Brcrin RCS. 86: 339-342.
Effects
DONALD Department Department and
68,
NEUROLOGY
of Protein
P. CAIN,
Division
Rrcrit,ed
(1980)
Synthesis Inhibition in the Mouse
on Kindling
MICHAEL E. CORCORAN.AND WILLIAM
of Ps.whology. of Psychology, British
409-419
University CJnivrrsit~
of Neurological Colutnhiu. 311/y
Sciences.
Vatxorcver, 24.
of Wrsrrm of Victoria.
1979:
Foculry
British revision
Onfario. Victoria
Ontario Cohtrnhiu
of‘ Medicine.
Colut~~hia rrc~ri~~cd
Lordon. British V6T
Noi’emher
A. STAINES’
University I W5. 30.
N6A 5C2: V8W 2V2
of
Cnnatla I979
Mice treated with anisomycin during amygdaloid kindling failed to develop generalized seizures. Anisomycin failed to suppress fully developed seizures in kindled mice, indicating that its prophylactic effects are not secondary to a general anticonvulsant action. These results are consistent with the hypothesis that the central neuroplastic changes underlying kindling require the ongoing synthesis of proteins.
INTRODUCTION The development of generalized convulsions through repeated lowintensity electrical stimulation of the brain-the kindling phenomenon (ll)-appears to depend on neuroplastic changes in the stimulated tissue. The evidence for this comes from studies showing that kindling results in a facilitation of both epileptiform and nonepileptiform activity in the kindled neural circuits (5, 23, 24). The facilitation is permanent, manifesting itself after a period of 1 year or more without stimulation following initial kindling (29). Moreover, the changes in brain function that accompany kindling are not restricted to the region around the stimulating electrode, but are quite widespread (11, 21). More recent research showed that the facilitative Abbreviations: CHX-cycloheximide. ANI-anisomycin. TCA-trichloroacetic acid, AD-afterdischarge. ’ Supported in part by grants from the Natural Sciences and Engineering Research Council of Canada awarded to D.P.C. and from the Medical Research Council of Canada awarded to M.E.C.
0014-4886/80/060409-11$02.00/O Copyright All rights
C 1980 by Academic Prss. Inc of reproduction in any form reserved
410
CAIN,
CORCORAN.
AND
STAINES
changes occurred in both the stimulated site and in distant sites strongly interconnected neuroanatomically with it (24). These findings, coupled with the striking intermittency of stimulation required for kindling (11,22), were the basis for suggestions that the neural changes underlying kindling may be similar to those underlying memory formation (10, 13, 16,20). In particular, the possibility exists that during the interstimulation interval, which is optimally in the range of 2 h or more, some growth process or other form of neural reorganization occurs that is necessary for kindling. It this is so, it would be reasonable to expect that protein synthesis is importantly involved, as was suggested for long-term memory formation. This possibility was approached recently in two studies, both of which assessed the effect on kindling of protein synthesis inhibition produced by injection of cycloheximide (CHX). In the first study (17) it was found that the progressive prolongation of epileptiform afterdischarge (AD) and the formation of spontaneous epileptiform potentials in the forebrain of the paralyzed bullfrog was significantly inhibited by CHX. However, as was pointed out [Racine, discussion in (17)], it is not clear from that work whether CHX blocked the development of the seizure response (the kindling process itself) or merely the expression of the seizure manifestations. In the second study (19) it was reported that CHX was effective in blocking AD in the rabbit amygdala when administered systemically 4 h prior to stimulation, and was thus effective in prophylactically preventing kindling through the suppression of AD-generating mechanisms. The drug was also found to be effective in blocking established kindled convulsions when given 3 to 4 h prior to stimulation. This interval is somewhat longer than the 30-min interval required to reach peak protein synthesis inhibition levels in mouse brain after systemic injection of CHX (2). On the other hand, CHX was found to be completely ineffective in preventing the progressive development of AD and behavioral convulsions when administered either 15 (intraventricularly) or 30 min (i.p.) prior to stimulation. Those results suggest that although protein synthesis inhibition by CHX in the rabbit is effective in preventing the expression of AD or fully kindled convulsions when the brain stimulation is applied some hours after the injection, it is ineffective in preventing the development of kindled convulsions at shorter intervals. However, conclusive interpretation of these results is precluded by a lack of information about the time course of the CHX-induced inhibition of protein synthesis in the rabbit. Thus, the question of whether or not kindling can proceed during inhibition of protein synthesis remains open. We felt that the optimal experimental approach to the question should include the following
PROTEIN
SYNTHESIS
INHIBITION
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features: The drug used should yield a profound inhibition of protein synthesis at a dose that is not toxic, even when administered repeatedly. The inhibitory effect of the drug should be known for the species used, and should be verified during the course of the actual experiment. The stimulation should be applied at or near the peak of protein synthesis inhibition. Ideally, the drug should act on the hypothesized mechanism of neural reorganization, and not merely act to block convulsion development through elevation of the threshold current necessary for evoking AD, or through the suppression of AD or convulsion expression. Accordingly, a control procedure should be used in which the experimental and control groups, after an initial series of stimulations under the drug and control saline solution respectively, would be treated to the other condition and then restimulated. This would indicate, in the case of the experimental group, whether any disruptive effect of the drug on kindling was due to a selective blockade of the neuroplastic changes underlying seizure development or due merely to a blockade of seizure expression. It would indicate in the case of the control group whether the drug has a seizure-suppressant action on fully kindled convulsions. Having selected these requirements, we chose to work with the Swiss-Webster mouse, and we selected anisomycin as the inhibitor of protein synthesis. Published information and pilot work indicated that essentially all requirements could be met with this combination. METHOD The subjects were 67 adult male Swiss-Webster mice that weighed approximately 40 g at the time of surgery and were housed individually in stainless-steel cages. Lab chow and water were available at all times. The animals were anesthetized with pentobarbital and received implantation of one bipolar electrode in the amygdala and a ground in the frontal sinus. The mouse has a very thin skull, and good retention of the implant is therefore a problem, particularly during a series of generalized convulsions. The details of an implantation procedure designed to insure good retention of the implant under these circumstances were published elsewhere (3). The electrode coordinates were determined with the aid of the mouse brain atlas of Montemurro and Dukelow (15), and were anterior +2.5 mm, lateral 3.3 mm, and 4.9 mm below the skull surface, with the upper incisor bar at the level of the interaural line. Electrodes were constructed of twisted stainless-steel Teflon-coated wire 127 pm in diameter soldered to individual miniature connector pins. Testing began 2 weeks after surgery. Shielded leads connected the animal to a stimulator and polygraph. The electroencephalogram was taken
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before and after stimulation to obtain a prestimulation baseline and to record the AD. For kindling, l-s electrical stimulation was provided by a constant current, 60-Hz sine wave source, with the initial stimulation intensity set at 20 ,uA. If this was not sufficient to evoke AD, the intensity was gradually raised until an AD was observed and was presented at that level thereafter. Thus all animals were stimulated at their individual AD “threshold”, arbitrarily defined as the lowest intensity of stimulation sufficient to evoke AD. After threshold determination the animals were divided into four groups, two experimental and two control, roughly equated for AD threshold. All animals were stimulated at 48-h intervals until a fully generalized convulsion was evoked or 14 stimulations had been given. Anisomycin (ANI) was supplied without charge by the Charles Pfizer Company, Groton, Connecticut. Because the inhibitory effect of AN1 is essentially unrelated to dose in the range of 0.5 to 3.0 mg/mouse (8), all injections in the present study were 0.5 mg/mouse in 0.25 ml saline. The solution of AN1 was prepared at a concentration of 2.0 mg/ml according to the method of Flood et al. (8) and the final acidity was adjusted topH 6 to 7. The two experimental groups received AN1 subcutaneously in the dorsal thoracic region, and the two control groups received similar injections of physiological saline. One each of the experimental and control groups received a single injection of the appropriate solution 15 min prior to each electrical stimulation (AN1 and saline groups, respectively). The other experimental and control groups received an injection 15 min prior to, and another injection 1.5 h after each stimulation (AN1 + AN1 and saline + saline groups). Thus, high levels of protein synthesis inhibition were maintained in both experimental groups at the time of stimulation. For the AN1 group the inhibition was approximately 70% for at least 1.75 h after each stimulation, and for the AN1 + AN1 group the inhibition was approximately 70% for at least 3.5 h after each stimulation (7, 9). This allowed a determination of the effect of varying the length of protein synthesis inhibition on kindling rate. After the initial series of kindling stimulations and a 3-day interval, a cross-over rekindling procedure was begun, in which the ANI injections were discontinued in the experimental groups and were initiated in the control groups. All features of the cross-over procedure were identical to those of the initial stimulation series, except that the saline and saline + saline groups now received one and two injections of ANI, respectively, in association with each stimulation, and the AN1 and AN1 + AN1 groups received saline injections. Stimulation was again applied at 48-h intervals until each animal displayed a fully generalized convulsion.
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At the end of testing all animals were anesthetized and perfused with formol-saline, and the brain was removed, frozen, and sectioned at 40 pm for verification of the electrode placements. All animals discussed below had electrode placements in the amygdala. Inhibition of protein synthesis by ANI was determined by comparing the incorporation of [3H]leucine into the trichloroacetic acid (TCA)-insoluble fraction in ANI- and saline-injected mice. Three additional groups of naive adult male mice were used: animals in the acute AN1 group (N = 5) received a single injection of 0.5 mg AN1 as described previously, animals in the chronic AN1 group (N = 9) received 14 ANI injections at 48-h intervals, and animals in the saline group (N = 8) received a single control injection of physiological saline. [“Hlleucine (60 Ciimmol L14,.5-ZH]leucine, New England Nuclear, Boston, Massachusetts, 10 Ci/animal) was injected in 20 ~1 physiological saline into the tail vein 15 min after the single AN1 or control saline injection or 15 min after the last chronic AN1 injection. The mice were killed 30 min later by decapatation, and the forebrain was sampled bilaterally. weighed, and immediately frozen at -90°C until extracted. The brains were homogenized in 1.5 ml distilled water at 4°C and an equal volume of 10% (w/v) TCA was added. After centrifugation and two additional rehomogenizations in 5% TCA, the precipitate was rehomogenized and washed in 99% ethanol. The pellet obtained after the ethanol wash was solubilized overnight in 2.5 ml Soluene-100 (Packard), transferred to a scintillation vial with an equal volume of ACS (Amersham) and brought to neutral pH with 1 ml 1 .O N acetic acid. The supernatants were counted as 1 ml samples in 10 ml ACS. Samples were counted in a Nuclear Chicago scintillation counter and were corrected for counting efficiency. The degree of incorporation of [“Hlleucine into protein was calculated by determining for each forebrain hemisphere the ratio of [“Hlleucine incorporated into the TCA-insoluble protein fraction to the total [“Hlleucine found in the sample and multiplying by 100. Thus, incorporation equals protein precipitate disintergrations per minute/(protein precipitate disintegration per minute + supernatant disintegrations per minute) X 100. Values obtained for the two hemispheres were averaged, and these values were averaged for each group. The values obtained for the ANI-injected groups were then expressed as a percentage of that obtained for the saline control group. RESULTS Initial Kindling. The basic phenomena of kindling in the mouse are similar to those seen in the rat. The mouse passes through a series of seizure stages that are comparable to stages l-5 as described by Racine (21). The
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convulsions are tonic-clonic in form and culminate in generalization (stage 5) characterized by rearing with forelimb clonus and facial automatisms, falling, and associated polyspike AD in the stimulated amygdala. A typical seizure record from a control animal appears in Fig. 1B. Table 1 presents the main results of the experiment. Because the saline and saline + saline groups did not differ on any of the measures (Mann-Whitney U, P > 0.05, two-tailed), their data were pooled. The saline control animals kindled in a mean of 8.4 ADS. In contrast, kindling in the ANI-treated animals was almost completely blocked during the 14 stimulations tested under the drug. Of 10 animals in the ANI group, one kindled after 12 ADS and the others attained a mean of stage 1.9 after the 14 stimulations. Of nine animals in the ANI + AN1 group one kindled after 12 ADS and the remainder attained a mean of stage 2.6 after the 14 stimulations. The administration of AN1 did not disturb background neural activity or fragment or disrupt the AD in these animals. Rather, the AD did not increase in duration and complexity as did the AD in the saline control animals. This is reflected in a shorter final seizure duration for the initial kindling phase (Table 1). In other respects the AD, as well as the clinical manifestations, appeared normal in the ANI-treated animals (see Fig. 1A). Cross-Oiler Rekindling. The number of ADS required to evoke a stage 5 convulsion during cross-over rekindling are summarized in Table 1. The kindled saline control animals showed almost immediate rekindling under ANI. requiring a mean of 1.6 ADS. AN1 appeared to have no disruptive or seizure-suppressant effect on either the AD or convulsion manifestations during rekindling. TABLE Initial
Kindling
Treatment Saline AN1 AN1 + AN1
and Cross-Over
N 20 10 9
Afterdischarge threshold (PA) 31.8 ? 3.3 29.9 k 2.5 30.6 lr 2.1
h Of 10 animals, 1 kindled 1.9 after 14 stimulations. r Of 9 animals, 1 kindled 2.6 after 14 stimulations.
Rekindling
1 in Saline-
Initial kindling: afterdischarges to stage 5 8.4 -c 0.5 -b -1’
in 12 afterdischarges, in 12 afterdischarges,
and Anisomycin-Treated
Mice”
Final seizure duration (s)
Cross-over rekindling: afterdischarges to stage 5
28.8 2 2.6 19.3 2 1.7 17.7 i- 1.2
1.6 r 0.2 5.5 2 1.5 8.1 -c 1.9
and the other
9 attained
a mean
of stage
and the other
8 attained
a mean
of stage
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The AN1 and AN1 + AN1 groups required 5.5 and 8.1 ADS, respectively, to rekindle. Adding these figures to the initial AD evoked during AD threshold determination yields a total of 6.5 and 9.1 ADS required to kindle these groups. The latter figures do not differ from the 8.4 ADS required to kindle the saline control animals (P > 0.05, two-tailed), nor do they differ from one another (P > 0.05, two-tailed). Biochemical Assays. The effect of the AN1 injections was to reduce the incorporation of [“Hlleucine into protein to 32.0% of control values in the acute AN1 group and to 3 1.2% of control values in the chronic AN1 group (Mann-Whitney U, both P < 0.001, two-tailed). DISCUSSION Both ANI-treated groups showed little or no tendency to develop kindled convulsions despite being stimulated 14 times, which is greater than the mean number of sessions required to kindle the control animals and is above the range of control values as well. All ANI-treated mice developed generalized convulsions during rekindling, indicating that there was no long-lasting debilitating effect of the drug on brain mechanisms necessary for kindling. The cross-over treatment of the control animals with AN1 after development of kindled convulsions showed that AN1 is essentially free of anticonvulsant effects, because these animals showed almost immediate rekindling while under the drug. Thus, we conclude that kindling is substantially blocked during ANI-induced inhibition of protein synthesis and that the blockage is not due to an anticonvulsant effect of the drug. These results agree with findings reported by Kessler et al. (paper presented at the 1977 meeting of the Eastern Association of Electroencephalographers), who examined the effects of CHX on kindling in frogs, and by Jonec et al. (12) who examined the effects of AN1 on kindling in rats. For reasons that are not immediately apparent, the anticonvulsant effect of protein synthesis inhibition described by Ogata (19) was not confirmed. Two ANI-treated mice kindled after 12 ADS, which is somewhat longer than the 8.4 ADS required by the control animals. Those mice did not differ from the other experimental animals in terms of electrode placement of any other known variable, and we cannot explain why these animals kindled and the other experimental animals did not. The reduction in incorporation of ]“H]leucine into protein to 32% of control values in the ANI-treated animals approximates similar measures obtained in previous studies (9). This verifies the efficacy of AN1 as an inhibitor of protein synthesis in mice, and indicates that there was no attenuation of the inhibiting properties of AN1 as a result of the 14 repeated
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injections. Although with the method used this difference could be attributed to a differential labeling of the protein synthesis precursor pool, the vastly more plausible interpretation is that it represents a reliable measure of the degree of inhibition of protein synthesis produced by ANI. Two successive injections of ANI yielded no greater blockage of kindling than a single injection. This may be evidence of a ceiling effect of AN1 on kindling because the single-injection group showed little or no convulsion development while under the drug. It should be emphasized that the conclusion that ongoing synthesis of central proteins is necessary for kindling is not necessarily warranted by the results presented here. It is possible that the link between protein synthksis and kindling is not direct, but rather that AN1 disrupted some other process that is crucial for kindling. For example, it was shown that another protein synthesis inhibitor, cycloheximide, has inhibitory effects on the activity of certain brain enzymes, such as tyrosine hydroxylase and acetylcholinesterase (6, 30). Inhibition of the activity of these enzyme proteins would be expected to affect the concentration or function of the relevant neurotransmitters, and these side effects of inhibitors of protein synthesis might be causally related to the effects on kindling observed here. Inhibition of the activity of tyrosine hydroxylase or acetylcholinesterase would be expected to facilitate kindling (1, 4, 28), however, suggesting that the mechanism by which AN1 blocks kindling probably does not involve those enzymes. But an effect of anisomycin on any of a number of other brain enzyme or other systems could conceivably affect kindling, and must be kept in mind when interpreting those results. In addition, it is possible that the disruption of kindling produced by AN1 is related to the peripheral effects of the drug and not to inhibition of protein synthesis in the brain. It was reported, for example, that administration of CHX disrupts the synthesis of adrenal steroid hormones, and that this peripheral effect might mediate the amnesic effects of CHX (18). It is unlikely that ANI-induced modulation of adrenal function accounts for the present results, because kindling proceeds without major change after adrenalectomy (14). But other peripheral effects of AN1 could be responsible for the present findings, and further work is required to confirm the central origin of these effects. For the present, therefore, we can conclude only that the observed results are consistent with the hypothesis that ongoing synthesis of central proteins is required for kindling. Previous attempts to find structural changes in kindled brain have either failed (24) or yielded inconclusive results (10). In the latter study it was reported that there was an increase in axon terminal size in kindled compared with control amygdala, but that extraneous factors could not be ruled out as a cause of the difference. More recently Racine and Zaide (25)
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provided the first convincing evidence that kindling involves the growth of synaptic contacts. In a well-controlled, blind study, they reported that cortical kindling resulted in a nonsignificant increase in the number of synaptic terminals and a significant increase in the size of synaptic terminals on dendritic spines in kindled neocortical tissue. A number of other recent studies utilizing electrical stimulation in potentiation or conditioning paradigms also reported increases in the size or numbers of dendritic spine contacts (26, 27). Therefore, if inhibition of protein synthesis by AN1 does directly block kindling by inhibiting neural growth during the kindling process, it may do so by inhibiting growth of dendritic spine synaptic contacts. REFERENCES 1. ARNOLD, P. S., R. J. RACINE. AND R. A. WISE. 1973. Effects of atropine, reserpine, 6-hydroxydopamine, and handling on seizure development in the rat. E~J. Neural. 40: 457-470. 2. BARONDES, S. H. 1970. Is the amnesic effect of cycloheximide due to specific interference with a processs in memory storage? Pages 545-553 in A. LAJTHA, Ed., Protein Metabolism of the Nervous System. Plenum, New York. 3. CAIN, D. P., AND S. DEKERGOMMEAUX. 1979. Electrode implantation in small rodents for kindling and long term brain stimulation. Physiol. Behat,. 22: 799-801. 4. CORCORAN, M. E., H. C. FIBIGER, J. A. MCCALJGHRAN, AND J. A. WADA. 1974. Potentiation of amygdaloid kindling and metrazol-induced seizures by 6-hydroxydopamine in rats. Exp. Neurol. 45: 118-133. 5. DOUGLAS, R. M., AND G. V. GODDARD. 1975. Long-term protentiation of the perforant path-granule cell synapse in the rat hippocampus. Bruin Res. 86: 205-215. 6. FLEXNER, L. B., R. G. SEROTA, AND R. H. GOODMAN. 1973. Cycloheximide and acetoxycycloheximide: inhibition of tyrosine hydroxylase activity and amnesic effects. Proc. Natl. Acad. Sci. U.S.A. 70: 354-356. 7. FLOOD, J. F., E. L. BENNETT, A. E. ORME, AND M. R. ROSENZWEIG. 1975. Relation of memory formation to controlled amounts of brain protein synthesis. Physiol. Behav. 15: 97-102. 8. FLOOD, J. F., E. L. BENNETT, M. R. ROSENZWEIG, AND A. E. ORME. 1973. Theinfluence of duration of protein synthesis inhibition on memory. Physiol. Behav. 10: 555-562. 9. FLOOD, J. F., M. R. ROSENZWEIG, E. L. BENNETT, AND A. E. ORME. 1974. Comparison of the effects of anisomycin on memory across six strains of mice. Behara. Biol. 10: 147- 160. 10. GODDARD; G. V., AND R. M. DOUGLAS. 1975. Does the engram of kindling model the engram of normal long term memory? Can. .I. Neural. Sci. 2: 385-394. 11. GODDARD, G. V.. D. C. MCINTYRE, AND C. K. LEECH. 1969. A permanent change in brain function resulting from daily electrical stimulation. &I. Neural. 25: 295-330. 12. JONEC, V., S. HOLMS, D. MASUOKA, AND C. G. WASTERLAIN. 1977. Animsomycin delays amygdaloid kindling in rats. Sot. Neurosci. Abstr. 3: 141. 13. LEECH, C. K., AND D. C. MCINTYRE. 1976. Kindling rates in inbred mice: an analog to learning? Behav. Biol. 16: 439-452. 14. MCINTYRE, D. C.. AND P. D. WANN. 1978. Adrenalectomy: protection from kindled convulsion induced dissociation in rats. Physiol. Behav. 20: 469-474.
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15. MONTEMURRO, D. G., AND R. H. DUKELOW. 1972. A Sterrotoric Atlns c!f‘ the Dirnc~ephtrlor~ nrld Ralrrtrd Structrrrc.r t;f tilt, Mortse. Futura, New York. 16. MORRELL, F. 1973. Goddard’s kindling phenomenon: a new model of the “mirror focus.” Pages 207-223 in H. C. SABELLI. Ed., C/1emic,trl Modulcrtior~ of Brc~inFunctior~. Raven. New York. 17. MORR~LL, F.. N. TSURU. T. J. HO~PPNER. D. MORGAN. AND W. H. HARRISON. 1975. Secondary epileptogenesis in frog forebrain: effect of inhibition of protein synthesis. Con. J. Neural. Sc,i. 2: 407-416. 18. NAKAJIMA. S. 1975. Amnesic effect of cycloheximide in the mouse mediated by adrenocortical hormones. J. Camp. Physiol. Ps~c~~w/. 88: 378-385. 19. OGATA. N. 1977. Effects of cycloheximide on experimental epilepsy induced by daily amygdaloid stimulation in rabbits. Epilepsicr 18: 101-108. 20. RACINE. R. J. 1972. Modification of seizure activity by electrical stimulation. I. After-discharge threshold. Elec,r~oenc,rph. Cliff. Neuropkysiol. 32: 269-279. 21. RACINE, R. J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Elecfrorr~crpk. C/in. Ncr,rop/ysiol. 32: 78 l-294. 22. RACINE, R. J.. W. M. BURNHAM. J. G. GARTNER, AND D. LEVITAN. 1973. Rates ofmotor seizure development in rats subjected to electrical brain stimulation: Strain and interstimulation interval effects. E14(.f~o~ll(.r~/?. C/in. Nwroplz~sid. 35: 553-556. 23. RACINE. R. J., J. G. GARTNER. AND W. M. BURNHAM. 1972. Epileptiform activity and neural plasticitiy in limbic structures. Brtrirz Rrs. 47: 262-268. 24. RACINE. R. J.. L. TUFF. AND J. ZAIDE. 1975. Kindling, unit discharge patterns and neural plasticity. Ctrn. J. Neural. Sci. 2: 395-405. 25. RACINE. R. J.. AND J. ZAIDE. 1978. A further investigation into the mechanisms underlying the kindling phenomenon. Pages 457-493 in K. E. LIVINGSTON AND 0. HORNYKIEWICZ. Eds.. Littihic Mecircrtris~n,s, Plenum, New York. 26. RUTLEDGE, L. T., C. WRIGHT. AND J. Duncan. 1974. Morphological changes in pyramidal cells of mammalian neocortex associated with increased use. E.rp. Neorol.
44: 209-228. 27. VAN HARREVELD.
A.. AND E. FIFKOVA. 1975. Swelling of dendritic spines in the fascia dentata after stimulation of the perforant fibers as a mechanism of post-tetanic potentiation. Erp. Nerrrol. 49: 739-749. 28. Vosu. H.. AND R. A. WISE. 1975. Cholinergic seizure kindling in the rat: Comparison of caudate. amygdala. and hippocampus. Be/la\*. Eiol. 13: 491-495. 29. WADA, J. A.. M. SATO, AND M. E. CORCORAN. 1974. Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats. Epilepsiu 15: 465-478. 30. ZECH, R., AND G. F. DOMAGK. 1975. Puromycin and cycloheximide as inhibitors of human brain acetylcholinesterase. Brcrin RCS. 86: 339-342.