Sprouting fibers gain access to circuitry transsynaptically altered by kindling

EXPERIMENTAL

NEUROLOGY

64,

469-481 (1979)

Sprouting Fibers Gain Access to Circuitry Transsynaptically Altered by Kindling JOHN A. MESSENHEIMER, Deparfments

ofNeurosurgery,

ERIC W. HARRIS,

AND OSWALD

Physiology, and Neurology, Medicine, Charlottesville, Virginia Received

November

University 22908

STEWARD’

of Virginia

School

of

20, 1978

In intact rats, “kindling” stimulation of the entorhinal cortex (EC) resulted, after an average of 23 daily stimulations, in the appearance of generalized convulsions. When the primary site of kindling in the EC was electrolytically destroyed and 14 days were permitted for the contralateral EC to sprout in response to the lesion, kindling stimulation of this surviving EC evoked fully developed motor seizures in 9 of 10 animals with theJirsr or second stimulation. However, if the primary site of kindling was not destroyed, kindling via the contralateral EC required an average of more than five stimulations. Furthermore, if sprouting was induced by a unilateral EC lesion prior to any kindling stimulation, kindling via the surviving “sprouted” EC contralateral to the lesion proceeded at a rate not significantly different from normal. Finally, the essentially immediate expression of seizure-evoking capabilities via the EC contralateral to a lesion was not observed if kindling via the secondary site was initiated 1 day after a primary site lesion, at a time prior to the completion of sprouting. These results were consistent with the hypothesis that EC kindlingresults in transsynaptic alterations either in the immediate targets ofthe EC (e.g., the dentate gyrus) or further “downstream” synaptically, and that destruction of the primary site of kindling results in the sprouting of projections which gain access to the altered circuitry.

Abbreviations: EC-entorhinal cortex, DG-dentate gyrus, EPSP-excitatory postsynaptic potential, EEG-electroencephalogram. ’ This research supported in part by National Science Foundation Research grant BNS 76-17750 to O.S. These results are to be included in a dissertation to be submitted by E.W.H. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Physiology, University of Virginia. A preliminary report has appeared elsewhere (9). Dr. Messenheimer’s present address is Department of Neurology, University of North Carolina, Chapel Hill, NC 27514. The authors thank Mr. Neil Liebowitz and Ms. Marian Wulffor their technical assistance. Please address reprint requests to Dr. 0. Steward, Department of Neurosurgery, University of Virginia School of Medicine, Charlottesville, VA 22908. 469 0014-4886/79/060469-13$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

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INTRODUCTION In recent years, the kindling phenomenon (6) has attracted interest as a model for studying persistent changes in cell-to-cell interactions which come about as a consequence of modifying functional activity along defined pathways. Although a variety of examples of kindling-like effects have now been described, the basic phenomenon involves periodic, low-intensity electrical stimulation of certain brain sites, which is initially without observable behavioral effects but which results in the gradual development of generalized convulsions (5,6, 12,15- 17). The resultant susceptibility to convulsions in response to the stimulus persists even if the animal is left unstimulated for months (3, 6). Although kindling has been recognized as a useful model for studying long-term plasticity and has even been discussed as a model of memory storage (5), the mechanisms underlying the changes in susceptibility to the stimulation remain obscure. Several authors reported an increase in evoked-response amplitude in regions which are synaptically activated during kindling stimulation, and those increases were interpreted as reflecting changes in synaptic efficacy (3,5, 16). It is not clear, however, if these changes reflect “presynaptic” alterations in pathways arising from the site of the kindling stimulation, or rather result from transsynaptic alterations in regions which receive either direct or indirect synaptic input from the kindled structures. One way to examine possible transsynaptic effects would be to destroy the site of initial kindling and activate its synaptic targets using another afferent system which is anatomically convergent with the initial kindling site. The entorhinal region of the rat affords a unique opportunity in this regard. In the rat, each entorhinal cortex (EC) projects heavily to the ipsilateral dentate gyrus (DG) but only sparsely to the contralateral DG (7, 18, 21, 22). However, after unilateral entorhinal lesions, which massively deafferent the ipsilateral DG, there occurs within 2 weeks a partial reinnervation of the DG as a result of sprouting of surviving afferent systems (11). Included among the afferent fibers which sprout is the sparse projection from the contralateral EC (7) which reinnervates some of the dendritic territory in the DG which was previously occupied by the ipsilateral entorhinal projection system (19, 23). In the present study, we used these lesion-induced modifications of connectivity to study long-lived transsynaptic alterations which might occur as a consequence of kindling. If kindling via EC stimulation induces transsynaptic changes either in the DG or further “downstream,” then the modifications underlying kindling should survive the destruction of the primary site of kindling. The presynaptic structures which were directly activated by the kindling stimulation (the projections from the “kindled”

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EC) would be replaced, however, by a system (the sprouted connections from the contralateral side) naive to the direct application of the kindling stimulus. If the sprouting projections from the contralateral EC gain access to circuitry which had been transsynaptically modified during the course of primary kindling, then appropriate activation of the lesion-induced crossed entorhinal projections might precipitate a kindled convulsion. This possibility was examined in the present study by kindling convulsions in rats via indwelling electrodes in one EC, destroying this primary kindling site, and, after sufficient time to permit sprouting, analyzing the time course of kindling via stimulation of the surviving contralateral EC. METHODS All animals were male Sprague-Dawley-derived rats weighing 250 to 550 g. Bipolar stimulating-recording electrodes made of twisted strands of Teflon-coated stainless-steel wire were implanted bilaterally in the entorhinal region under stereotoxic guidance (coordinates were 4.2 mm lateral to midline, 1.5 mm anterior to the transverse sinus, and 3.9 mm deep to the skull). The wires were insulated except at the tips, and the tips were separated by approximately 0.5 mm. The electrodes were fixed to the skull with NuWeld dental cement anchored with six machine screws, and the wires were fitted with male Amphenol microplugs for connecting with the stimulating and recording devices. Fourteen postoperative days passed after implantation before beginning the kindling stimulation. For three animals in a pilot study, the stimulation consisted of an approximately 2-s, 60-Hz train of 50-PA rectangular biphasic pulses delivered via small metal strips held against the microplugs. In the remaining cases the stimulation was delivered through female microplugs soldered to Belden four-conductor shielded phono pick-up cable via a switching box which permitted rapid connection of the animal to either the stimulator or a Grass Model 6 electroencephalograph. The stimulation consisted of an approximately l-s, 60-Hz train of I-ms diphasic constant-current pulses generated by a WPI Model 601 bipolar stimulator. The stimulus intensity was set just above threshold for afterdischarges in the EC as follows: Kindling stimulation was begun at 500 PA on the first day. If no after discharge was evoked, as determined by examining the electroencephalogram (EEG) recorded via the stimulating electrodes immediately after the stimulation, the intensity was increased in steps of 100 /LA until an afterdischarge was evoked, or until an intensity of 1000 ,uA was reached. If no afterdischarge was evoked at 1000 PA on the first day, this intensity was tried for only 3 more days. If no afterdischarge was evoked, the animal was discarded. If an afterdischarge was evoked with the 500-PA stimulus, the intensity was decreased in IOO-PA steps for

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subsequent daily stimulations (to not less than 50 PA), or until no afterdischarge was elicited, in which case the lowest previously effective intensity was used for 3 days before again reducing stimulation intensity. In this way, by successive reductions in stimulus intensity, the minimal effective stimulus was continuously determined and used for successive stimulations. The rate of kindling was defined by the number of daily stimulations (which effectively induced afterdischarges) required to elicit the first generalized convulsion. Daily stimulations which were ineffective in eliciting afterdischarges were not included in the count. Eighty-seven animals were prepared, but a total of 61 were discarded because of loss of headplugs prior to the completion of all testing (N = 3 l), failure to elicit afterdischarges or kindled convulsions (N = 2), or unusual reactions to the stimulation such as immediate violent motor responses (N = 20). Eight additional animals were discarded on the basis of the histology (inappropriate electrode position, incomplete lesion, etc.). The 26 animals which were included in the study aggregated into four groups. Experimental. Kindle-Lesion-Kindle (KLK) group (N = 10). The animals were implanted bilaterally, and daily kindling stimulation was delivered 5 days per week to one side (primary kindling). After a total of five generalized convulsions of class 5 motor seizure type (14) were elicited, the primary kindling site was destroyed by passing I-mA DC anodal current for 60 s through each pole of the stimulating electrode, with a rectal electrode serving as cathode. Fourteen days passed following the lesions to permit the development of the lesion-induced projections before kindling stimulation was initiated in the intact contralateral entorhinal area. The number of afterdischarge-evoking stimuli delivered prior to the appearance of the first generalized convulsion for stimulation of the contralateral entorhinal area served as the index of the rate of secondary-site kindling in this group. Transfer Control. Kindle-Kindle (KK) group (N = 6). To determine whether or not kindling via one EC has an effect on secondary-site kindling [transfer effect (6,12)], animals were treated identically to the experimental KLK group except that, after primary kindling and the elicitation of five generalized convulsions, the primary site was left intact. Fourteen days later, secondary-site kindling was initiated. The number of stimuli delivered until the first generalized convulsion for kindling in the contralateral EC was the index of the rate of normal transfer kindling. Lesion Control. Lesion-Kindle (LK) group (N = 5). To determine whether lesion-induced sprouting alone altered the rate of development of kindling after stimulation of the structure giving rise to lesion-induced projections, the entorhinal region was destroyed unilaterally as previously described (10) at the time of electrode implantation. After a 14-day

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postoperative interval, kindling stimulation of the surviving entorhinal region was begun. Kindle-Lesion-Kindle (Immediate). KLKi group (N = 5). To further evaluate the significance of sprouting for changes in the rate of kindling, animals were prepared as for the KLK group, except that the stimulation of the secondary site was begun prior to the completion of sprouting. After primary kindling and ablation of one EC, secondary kindling via the contralateral EC was begun 1 day postoperatively rather than 14 days postoperatively. Histology. After completion of the experiment the animals were killed with an overdose of sodium pentobarbital (Nembutal) and perfused transcardially with 10% formalin-0.9% saline. The brains were removed, postfixed overnight in the perfusion solution, and embedded in egg yolk (4) for subsequent sectioning in the horizontal plane on a freezing microtome at 40 pm. Every third section was processed for cresyl violet staining for examining electrode and lesion locations, and every sixth section was processed for the histochemical demonstration of acetylcholinesterase (14) to examine the extent of the denervation in the dentate gyrus (13). RESULTS Of the animals (N = 22) which were kindled prior to any other manipulation (primary kindling in KLK, KK, and KLKi groups) the average number of daily stimulations required to evoke the first generalized convulsion was 23.4. The positions of stimulating electrodes in the entorhinal cortex are illustrated in Fig. 2. These are the positions of the electrodes used for stimulating the contralateral EC, but these should be representative of the positions of the electrodes used for primary kindling as well. There were no obvious differences in stimulating electrode placement between the various groups. Although varying considerably in their dorsoventral position, most electrodes were in the medial portion of the entorhinal cortex, usually partly in the angular bundle, often on the border between entorhinal and subicular regions. The animals which received primary kindling were divided into the KLK group (N = lo), the KK group (N = 6), and the KLKi group (N = 6). The average number of stimulations required to evoke the first generalized kindled convulsion via the primary site for each of these groups is seen in Fig. 1. Kindle -Lesion -Kindle. Whereas an average of 22.1 daily stimulations was required for primary-site kindling in this group, an average of only 1.8 stimulations was required to elicit the first generalized convulsion via stimulation of the surviving contralateral entorhinal area. Nine of ten animals exhibited kindled generalized convulsions with the first or second

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FIG. 1. Comparison of rates of kindling for each group. The vertical scale is the number of of kindling stimulation required until the appearance of the first generalized convulsion for each kindling site within each group of animals. The brackets denote 21 SD. Open bars-Primary kindling in normal animals, striped bars-postlesion kindling, stippled bars-secondary (transfer) kindling. The treatment for each group of animals is indicated in abbreviated form below the bar graphs. The levels of significance for each comparison are indicated below the initials for each group (unequal N, one-tailed t-test). days

stimulation of the surviving EC, with 6 of the 10 animals convulsing with the first stimulation, and only 1 animal required more than two stimulations to evoke the first generalized convulsion. The extent of typical lesions produced by passing current through the stimulating electrodes at the primary site of kindling is illustrated in Fig. 2. Lesions were more extensive dorsally than ventrally, always involved the medial entorhinal cortex, and usually spared the lateral entorhinal cortex. The medial border of the angular bundle and subiculum was often damaged. Intensification of acetylcholinesterase stain (13) and an increased number of small dense cells (11) as seen in cresylviolet-stained sections, in the molecular layer in the DG ipsilateral to the EC lesion, was noted in every case, indicating successful partial deafferentation by destruction or interruption of the pathway from EC to the DG. There did not seem to be any correlation between the size of the lesion and the rate of kindling via the contralateral EC. One animal in the KLK group, not included in this figure or the statistical treatment, had a lesion comparable to those of the other animals, but the secondary kindling electrode was placed in the intermediate (somewhat more lateral) entorhinal cortex (1, 18), in a position clearly different from the others shown in Fig. 2A. The rate of secondary kindling

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FIG. 2. Electrode and lesion positions are illustrated on tracings of horizontal sections through the rat brain separated by approximately 0.33 mm. The contralateral entorhinal cortex (EC) electrode placements for all groups are indicated in A (W, KLK; Cl, KK; IXi, LK; 0, KLK,), and the extent of one ofthe largest (broken line) and smallest (hatched area) lesions (ipsilateral EC) in the KLK group is shown in B.

in this animal (8 days) was also quite different (4 SDS) from the mean for the other KLK animals; this may have been due to differences in the distribution along the DG granule cell dendrites of fibers from medial and more lateral EC (18). Kindle-Kindle. In contrast to the KLK group, animals which received secondary kindling stimulation with the primary site intact required an average of 5.2 daily stimulations to evoke the first generalized convulsion via the secondary site. Also unlike the KLK animals, only two of the six animals convulsed with the first or second stimulation and only one animal had a generalized convulsion with the first stimulation. The difference in the average number of daily stimulations required to evoke generalized convulsions via the contralateral side between the KLK and the KK groups was significant (t = 2.98, P < 0.01; see Fig. 1). It should be noted that the rate of kindling via stimulation of the secondary site in the KK group could be spuriously low as a result of the exclusion of two animals who lost their headplugs prior to the completion of secondary-site kindling. Neither of those animals had generalized convulsions prior to headplug

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loss, but they had received 3 and 20 stimulations, respectively. If the assumption is made that these numbers represent the minimum number of required stimulations, and these numbers are then included, the average number of daily stimulations for secondary-site kindling in KK animals becomes 6.8. Thus, we feel that our estimates of the number of afterdischarges required for secondary-site kindling above err if anything by being too small. Lesion -Kindle. In contrast to the dramatic effect seen following lesions involving structures which had received kindling stimulation, unilateral lesions of the EC with no prior kindling (LK) did not significantly alter the rate of kindling. The number of daily stimulations required to evoke the first generalized convulsion was 23.5 (see Fig. l), a value which is not significantly different from the average number of stimulations required to evoke the first generalized convulsion in intact animals (t = 0.158, P > 0.4). Kindle -Lesion

-Kindle (Immediate). The preceding results indicated that secondary-site kindling was accelerated if the primary EC site was destroyed and sufficient time (14 days) allowed for sprouting of the contralateral EC projections. To determine whether or not a similar acceleration would occur after lesions if sprouting were not permitted (by not allowing a sufficient postlesion interval), secondary-site kindling was begun on the day after the lesion rather than 14 days postlesion. This group required an average of 8.3 daily stimulations of the EC contralateral to a lesion to evoke the first generalized convulsion (see Fig. l), a value significantly greater than the number required for the KLK group (I = 4.34, P < 0.001). Because sprouting had not occurred by the time secondary-site stimulation was begun, it might be expected that the rate of secondary-site kindling would be comparable to that for the KK group. In fact, the average number of stimulations required for the KLKi group was larger than the average required for the KK group (8.3 vs 5.2), although this difference was not statistically significant (t = 1.46, P > 0.05).

DISCUSSION Lesion-induced changes in synaptic relationships were used to alter the degree of connectivity between a structure (the dentate gyrus) which had been synaptically activated during the process of primary kindling and another site (the contralateral entorhinal cortex) which was naive to the direct application of the kindling stimulation. Associated with this change in connectivity is the acquisition by the surviving contralateral EC of a capability for evoking generalized convulsions with even the first kindling stimulation. If the primary site is not destroyed, however (as in the KK

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group), thus not initiating sprouting, or if insufficient time is allowed for sprouting to occur prior to the initiation of secondary-site kindling (as in the KLKi group), then the apparent immediately efficacious “accessing” of seizure-evoking circuitry is not observed. These observations suggest that the ability to evoke generalized convulsions via contralateral EC stimulation for the KLK animals occurs as a consequence of sprouting of projections from the secondary EC kindling site into the dentate gyrus normally innervated by the primary EC kindling site. These results point directly to the occurrence of some transsynaptic alterations, either in the dentate gyrus which was activated by the primary kindling site or possibly which led to the establishment of seizure-evoking further “downstream,” circuitry. Furthermore, these alterations survived destruction of the primary kindling site, and can be activated by surviving circuitry, resulting again in the elicitation of a behavioral convulsion. This interpretation is based on the assumption that the activity evoked by the kindling stimulation traveled through the dentate gyrus and hippocampal formation en route to whatever structures are involved in the generation of behavioral convulsions. The evidence in support of this assumption is at this time circumstantial. First, the dentate gyrus is the primary synaptic target of the entorhinal cortex (1, 18) and the entorhinal electrode placements in this study were chosen on the basis of our electrophysiological experience regarding the optimal electrode placement for evoking monosynaptic responses in the dentate gyrus. Furthermore, preliminary observations suggested that cutting the major hippocampal output pathway (the fomix) can eliminate, at least for a time, the seizures evoked by EC stimulation (unpublished observations). Finally, the ability of the EC contralateral to the primary kindling site to evoke generalized convulsions with the first few stimulations was correlated with sprouting of the projections from this secondary site, and was not observed if sprouting was not initiated (by failing to destroy the primary site), or was disallowed (by not allowing sufficient time postlesion for sprouting to occur). The most parsimonious interpretation at present is that the activity evoked by kindling stimulation traveled through the dentate gyrus. If the activity evoked by the kindling stimulation did pass through the dentate gyrus, the question arises as to whether the transsynaptic alterations which enable seizure elicitation occur at this site, beyond, or both. Kindling stimulation of the EC was shown to result in alterations in synaptic transmission in the entorhinal projection system to the dentate gyrus (3, 4), suggesting this monosynaptic pathway as a substrate for the transsynaptic alterations. However, our measure of kindling effects (behavioral convulsions) is based on activity obviously many synapses removed from the dentate gyms, and the route of the activity giving rise to

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the motor seizures is not yet known. Thus, it is possible that the kindling-induced transsynaptic alterations occur many synapses beyond the dentate gyrus, and that sprouting creates circuits which can activate these “downstream” sites in a way comparable to the activation resulting from stimulation of the primary site prior to its destruction. Although the ability of the contralateral EC to evoke generalized convulsions with the first stimulation occurred if the primary entorhinal kindling site had been destroyed, significant enhancement of the rate of kindling via this secondary site was also observed in animals in which the primary site was not destroyed (the KK group). Part of the transfer in the KK group could occur as a result of inadvertent damage to the EC resulting from implantation of the indwelling stimulating electrodes (which would presumably induce some sprouting of contralateral projections), although we could detect no evidence of denervation or the attendant sprouting in any of the KK animals. Alternatively, this transfer effect could be due to the normal, sparse crossed pathway to the dentate gyrus, or the bilaterally symmetrical entorhinal projections to regio superior of the hippocampus proper (1, 18), or to a convergence of hippocampal outputs somewhere beyond the dentate gyrus. Whatever the mechanism underlying the transfer, all these interpretations are based on the concept of the ultimate convergence of projections from the primary site and the secondary site onto some structure which had been transsynaptically modified by the kindling stimulation. Thus, as a principle we suggest that transfer may be due in part to the convergence of projections from a primary and some secondary sites. Furthermore, we suggest that when the degree of convergence is increased by some manipulation (for example, one which induces sprouting), the transfer will be enhanced accordingly. Although sprouting-induced increases in connectivity from an EC to structures which presumably have been transsynaptically altered enhanced the rate of kindling via stimulation of the EC, merely increasing the projections from one EC prior to any kindling (the LK group) failed to enhance the rate of kindling via this EC. Because evidence based on stimulation of the amygdala and hippocampus (17) suggests that simultaneous bilateral application of kindling stimulation to homologous sites will accelerate the rate of kindling, it might be expected that increasing the degree of bilaterality of the projection from the entorhinal cortex to the dentate gyri might facilitate kindling. Certainly there are problems in drawing parallels between the simultaneous bilateral stimulation of homologous brain sites and the stimulation of a single site with bilateral projections. Nevertheless, if we choose to interpret the results as indicating that the lesion-induced pathway can evoke generalized convulsions via synaptic activation of “kindled targets” (KLK group) but cannot

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accelerate the rate of development of kindling in unstimulated animals (LK group), there are existing electrophysiologic data which might explain such effects. Investigations designed to compare the characteristics of synaptic transmission in the lesion-induced crossed and the normal ipsilateral entorhinal projection systems demonstrated that the lesion-induced crossed pathway exhibited seemingly normal operational characteristics in response to low-frequency activation (8, 19) but somewhat abnormal characteristics in response to more complex modes of stimulation (20,22). Activation of the normal ipsilateral projection system with paired pulses (20), or high-frequency trains (2-4,22), will result in dramatic potentiation of both the excitatory postsynaptic potential (EPSP) and population spike components of the evoked response recorded in the dentate gyrus. Although apparently normal potentiation of the EPSP with both paired pulse (20) and high-frequency (22) stimulation are observed for the lesion-induced crossed pathway, potentiation of the population spike is not comparable to that observed for the normal ipsilateral pathway (22). The failure to observe population spike potentiation along the lesion-induced crossed pathway may provide a hint as to why sprouting fails to enhance primary kindling (LK). If kindling depends on enhancing the granule cell population output, then the absence of enhancement of granule cell discharge with kindling stimulation of the sprouted system might preclude any contribution to the enhancement of the rate of kindling of a “sprouted” EC. Nonetheless, the sprouted connections, which are electrophysiologitally quite potent (8, 19, 20, 22), may prove adequate for evoking generalized convulsions in the dentate gyrus which has been transsynaptitally altered by previous kindling stimulation. This is all consistent with the interpretation of Douglas and Goddard (3) that kindling via entorhinal cortical stimulation may be related to long-term potentiation of synaptic responses in the dentate gyrus, but that the maintenance of a kindled response is accompanied by maintained increases in the size of the synaptic component of the evoked population response only, and not the postsynaptic cell discharge component (the population spike). Although the preceding discussion indicates the complexity of the problems involved in interpreting our findings, in agreement with previous work (3,5,6, 12,16), our results suggest a useful set of working hypotheses as a guide to future experimentation: (i) Kindling stimulation results in transsynaptic alterations. (ii) These alterations result in the generation of circuitry which, when appropriately activated, can precipitate a behavioral convulsion. (iii) This transsynaptically altered circuitry survives destruction of the primary site of kindling. (iv) “Transfer” (enhancement of kindling at secondary sites) depends in part on the degree of convergence of

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the projections from the secondary site upon the circuitry which was transsynaptically altered by the primary-site kindling; in particular, our results suggest that for kindling via the entorhinal cortex the transsynaptic alterations occur, at least in part, in the dentate gyrus, which receives direct projections from the primary site of kindling. (v) The degree of transfer can be enhanced by increasing access to the kindled circuitry as a result of sprouting following destruction of the primary kindling site. We hope that the extensive knowledge of the anatomy and electrophysiology of the dentate gyrus and hippocampal formation will provide a useful model system in which a detailed test of the various components of this working hypothesis will be possible. REFERENCES 1. BLACKSTAD, T. W. 1956. Commissural connections of the hippocampal region of the rat, with special reference to their mode of termination. J. Camp. Neural. 105: 417-537. 2. BLISS, T. V. P., AND T. LIMO. 1970. Plasticity in a monosynaptic cortical pathway. J. Physiol. (London) 287: 61P. 3. DOUGLAS, R. M., AND G. V. GODDARD. 1975. Long-term potentiation of the perforant path-granule cell synapse in the rat hippocampus. Brain Res. 86: 205-215. 4. EBBESSON, S. 0. E. 1970. The selective silver impregnation of degenerating axons and their synaptic endings in non-mammalian species. Pages 132- 161 in W. J. H. NAUTA AND S. 0. E. EBBESSON, Eds., Contemporary Research Methods in Neuronatomy. Springer-Verlag, Heidelberg. 5. GODDARD, G. V., AND R. M. DOUGLAS. 1975. Does the engram of kindling model the engram of normal long-term memory. J. Can. Sci. Neural. 2: 385-394. 6. GODDARD, G. V., D. C. MCINTYRE, AND C. K. LEECH. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neural. 25: 295-330. 7. GOLDOWITZ, D., W. F. WHITE, 0. STEWARD, C. COTMAN,AND G. LYNCH. 1975. Anatomical evidence for a projection from the entorhinal cortex to the contralateral dentate gyrus of the rat. Exp. Neurol. 41: 433-441. 8. HARRIS, E. W., S. S. LASHER, AND 0. STEWARD. 1978. Habituation-like decrements in transmission along the normal and lesion-induced temporodentate pathways in the rat. Brain Res. 151: 623-631. 9. HARRIS, E. W., J. MESSENHEIMER, AND 0. STEWARD. 1978. Seizures evoked by fibers from the contralateral entorhinal cortex which reinnervate the dentate gyrus after kindling and ablation of the ipsilateral entorhinal cortex. Sot. Neurosci. Abstr. 4: 473. 10. LOESCHE, J. AND 0. STEWARD. 1977. Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: Recovery of alternation performance following unilateral entorhinal cortex lesions. Brain Res. Bull. 2: 31-39. 11. LYNCH, G., G. ROSE, C. GALL, AND C. W. COTMAN. 1975. The response of the dentate gyrus to partial deafferentation. Pages 505-5 17 in M. SANTINI, Ed., Golgi Centennial Symposium Proceedings. Raven Press, New York. 12. MCINTYRE, D. C., AND G. V. GODDARD. 1973. Transfer, interference and spontaneous recovery of convulsions kindled from the rat amygdala. Electroenceph. Clin. Neurophysiol. 35: 533-543. 13. NADLER, J. V., C. W. COTMAN, AND G. S. LYNCH. 1977. Histochemical evidence of altered development of cholinergic fibers in the rat dentate gyrus following lesions. I.

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