Brain Research Bulletin, Vol. 2, pp. 41-48, 1977. Copyright 0 ANKHO All rights of reproduction in any form reserved. Printed in the U.S.A.
Behavioral Correlates of Denervation and Reinnervation of the Hippocampal Formation of the Rat: Open Field Activity and Cue Utilization Following Bilateral Entorhinal Cortex Lesions OSWALD
Surgery and Physiology, University of Virginia School of Medicine Charlottesville, VA 22901 (Received
STEWARD, O., J. LOESCHE AND W. C. HORTON. Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: open field activity and cue utilization following bilateral entorhinal cortex lesions. BRAIN RES. BULL. 2(l) 41-48, 1977. - Bilateral lesions of the entorhinal cortex (E.C.) of the rat result in persistent deficits in both spontaneous and reinforced alternation. The present study analyzes the nature of this impairment. To determine if changes in exploratory activity accompanied the deficits in alternation, open field activity was measured daily from 2-22 days following bilateral E.C. lesions. Such lesions resulted in a pronounced transient increase in open field activity which peaked between 5 and 7 days postlesion, but subsequently decreased to near preoperative levels at approximately 11 days postlesion. Alternation performance was also analyzed, to determine which cues are utilized to make the alternation, and whether cue utilization is affected by bilateral E.C. lesions. Utilizing a plus (+) maze, animals readily learned to alternate goal arms, but even with extensive training, failed to learn to alternate turns (left and right). However, the ability to identify the two goal arms in a nonalternation situation (which does not require short term recall of the preceding trial) was not permanently impaired by bilateral E.C. lesions. Since bilateral E.C. lesions do not result in persistent deficits in the ability to identify the two goal arms, but do disrupt alternation performance, we hypothesize that the deficit in alternation might reflect an inability to recall which arm was chosen on preceding trials. The implications of these results for an understanding of the behavioral consequences of postlesion reorganization of neuronal circuitry are discussed. Entorhinal
THE entorhinal cortex (E.C.) of the rat provides the major extrinsic synaptic input to the hippocampal formation, via the circuit which relays through the dentate gyrus [ 1,
[ 181 , and
[ 111. [ 15, 33, 34, 35, 391 , the
This role of the entorhinal cortex in providing the link between the neocortex and the hippocampus has long been recognized [29, 36, 371, yet relatively few studies have examined the behavioral consequences of E.C. lesions [8, 22, 23, 381. Such investigations are of particular importance, however, since damage to the E.C. results in a dramatic proliferation of several surviving afferent systems in the denervated dentate gyrus. For example, in response to unilateral E.C. lesions, the normally c;parse projection from the contralateral entorhinal area to the dentate gyrus
be mediated through the reinnervation of the dentate gyrus by the surviving contralateral E.C. [ 141. Because of this possible relationship between postlesion afferent proliferation and behavioral recovery, the nature of the behavioral deficit is of special interest. The present study analyzes the persistent deficits in alternation behavior following bilateral E.C. lesions. Since the reinnervation of the dentate gyrus by the contralateral E.C. is precluded by bilateral lesions, a favorable situation also exists in which to analyze the potential behavioral correlates of reinnervation of the dentate gyrus by the surviving septal, commissural, and associational afferents. While deficits in spontaneous alternation, such as those which occur following bilateral E.C. lesions  could result from changes in exploratory activity, habituation to novelty, etc. (see [3, 4, 51 ) such changes are unlikely to account for deficits in the reinforced alternation tasks [ 141. This does not, however, preclude the possibility that changes in exploratory activity might also occur following E.C. lesions. To examine this question, open field behavior was analyzed from 2-22 days following bilateral E.C. lesions. The components of the reinforced alternation task itself were also analyzed. Alternation performance in a T-maze requires, (1) an ability to distinguish between the two arms, or between responses (left or right), and (2) presumably an ability to recall the preceding choice (although the possibility cannot be excluded that animals alternate by avoiding the most recent odor trail). We first attempt to determine the general class of cues utilized by the animals in reinforced alternation, specifically whether animals alternate on the basis of turns (responses) or on the basis of place cues which identify the individual arms of the maze. Subsequently, the ability to utilize these cues after bilateral E.C. lesions is analyzed. GENERAL
Animals Experimental animals were 36 male Sprague-Dawley derived rats obtained from Flow Labs, which ranged in age from 90- 120 days at the start of the experiments. For the open field studies, animals were housed in groups of three and provided food and water ad lib. The remainder of the animals were food-deprived to 80% of their initial weight (initial weight ranged from 250-350 g) and were maintained at the 80% level for the duration of the experiments. The colony room in which all animals were housed, was on a 12 hour light-dark cycle (lights on at 7 a.m.). Surgery Anaesthesia was induced by intraperitoneal injections of sodium pentobarbital (Nembutal) at a dosage of 50 mg/kg. The entorhinal cortical region was destroyed electrolytically as described previously [ 141. For the sham operations, holes were drilled bilaterally in the skull but the brain was not penetrated by the electrode. After the completion of the lesions, the skull was filled with gel foam, the scalp sutured, and the animals were given an intramuscular injection of penicilin (Wycillin, Wyeth Labs.). Histology On completion of behavioral testing the animals sacrificed with an overdose of sodium pentobarbital,
perfused transcardially with 10% formalin. The brains were embedded in egg yolk, and sectioned at 33 ,urn. Alternate sections were stained with Cresyl Violet. EXPERIMENT
A total of 9 animals were tested in a 24 x 24 in. open field, divided into 36 squares. The floor of the open field was bounded by plywood walls 40 in. high, with a screened viewing port on one side. The apparatus was placed in a darkened room, and the interior of the open field was illuminated by a 15 W bulb, 40 in. above the floor. Open field activity was measured daily over a total of 7 min., between 1200 and 1700 hr. After 9 days of preoperative testing, which served to establish baseline activity levels, 5 animals received bilateral entorhinal cortical lesions, and 4 animals received sham operations. Testing began on Day 22. Subsequently, the group of sham operates received unilateral entorhinal lesions, and testing continued for this group from postlesion Days l-6, and Days 13 - 18. RESULTS
Histology As illustrated by the examples in Fig. 1 the lesions of the present experiment included virtually the entire entorhinal cortex, including medial (MEC) and lateral (LEC) divisions (areas 28a, and 28b respectively). In addition, at most dorso-ventral levels the parasubiculum (Pas) and presubiculum (PrS) were damaged. Portions of these areas were spared to a variable extent in ventral sites (for example, Level VI of Fig. 1). At most dorso-ventral levels, the lesions also interrupted the angular bundle, which carries the projections from the entorhinal area to the hippocampal formation. In all cases of the present study, the lesions extended slightly into the neocortex adjacent to the entorhinal area, particularly in dorsal sections, and in approximately 50% of the cases, the caudal portions of the subiculum were damaged at some dorso-ventral levels. There was also some evidence of damage to the caudal dentate gyrus in one animal, although this involvement was very slight, and did not involve the cell layers (stratum granulare) of the dentate. No differences in postoperative behavior were observed between the animals with the largest and smallest lesions. The lesions in Experiment 2 were quite comparable to those of Experiment 1, and thus are not described separately. Open Field Activity As illustrated by Fig. 2, bilateral E.C. lesions resulted in pronounced increases in open field activity which were maximal from 5-7 days postlesion. After Day 7 postlesion, the activity levels began to decrease and by 11 days postlesion had reached an apparently stable level which was still slightly elevated with respect to either the sham operated group, or the preoperative activity of the bilaterally operated group. The differences between the sham and bilaterally operated groups were significant from the first postlesion testing day through Day 10, (t values ranged from t = 2.37, pO.l in the case of the animals trained to make a particular turn, and t= 1.77, p>O.O5 in the case of the group trained to choose a particular arm). While a categorical statement on the absence of deficits cannot be made in view of the limited number of animals (particularly in view of the slight transient deficit in the case of the animals trained to choose a particular goal arm, see Fig. 6B), it is clear that if bilateral E.C. lesions do result in deficits in cue utilization, these deficits are transient and do not compare with the persistent deficits in alternation performance.
The present results suggest that animals trained in the reinforced alternation paradigm prefer to utilize the same sorts of cues as are apparently utilized by rats in spontaneous alternation situations . Thus, rats can readily learn to alternate their choices of goal arms, but cannot readily alternate turns. Indeed, in the present study, animals were unable to learn to alternate their turns even with extensive training. These results suggest that reinforced alternation, like spontaneous alternation, is accomplished by identifying the particular goal arms on the basis of some place cues. In the nonalternation task, while the performance was not persistently affected by bilateral E.C. lesions, there was some evidence for slight and transient deficits. In this regard, it is interesting to note the apparently greater deficit in the case of animals trained on the goal arm discrimination despite the fact that the goal arm discrimination is learned more rapidly than the turn discrimination. This result is reminiscent of DeCastro’s observations on performance in a plus maze following fornix lesions, where again goal arm discrimination was more susceptible to lesions than was the turn discrimination. DeCastro thus suggests that lesions of the fornix result in a impairment in the ability to utilize spatial information, and O’Keefe et al. utilizing a com[241, arrived at a similar interpretation pletely different task. The results of the present study suggest that the goal arm discrimination might also be more susceptible to lesions of the E.C. than the turn discrimination, although the deficits appear transient. Because, however, DeCastro [ 21 examined postlesion acquisition of the discrimination, while the present study tested postlesion retention, a precise comparison of the effects of the two kinds of lesions on performance in the plus maze will require further investigation.
The present results suggest that the severe disruption of alternation behavior following bilateral lesions of the entorhinal cortex (E.C.) is not a consequence of changes in avoid stimulus re-exposure,” the motivation to “explore, etc (see [S]). This conclusion is supported by the fact that EC. lesions affect spontaneous and reinforced alternation comparably [ 141, and is reinforced by the present observations that the severe long lasting deficits in alternation performance are not even paralleled by profound changes in exploratory activity as measured by open field behavior. Rather, the present results suggest an impaired ability to
perform in the alternation task, rather than any change in the motivation to alternate. The present results furthermore provide some insight into the nature of the impairment. Alternation performance requires, (1) the discrimination of the two goal arms (either on the basis of the turn made to enter the arm, or some external cues, and (2) presumably the short-term recollection of the arm chosen on the preceding trial. While alternation could theoretically involve avoiding the most recent odor trail, (a strategy which does not require short term recall of the preceding choice) this strategy is probably not utilized, since rats alternate for 11 massed trials with short intertrial intervals. Furthermore, Douglas’ [ 31 experiments with spontaneous alternation suggest that olfactory cues play only a minor role, while the major role is apparently played by nonolfactory cues, particularly vestibular cues. The use of any strategy other than an odor trace would necessitate short-term recall of the preceding choice. The present results demonstrate that following bilateral E.C. lesions, rats are still capable of distinguishing between the two goal arms (as long as alternation is not required), or distinguishing between turns (which is an inadequate cue for alternation behavior). Assuming that the cues utilized during alternation performance are either the same or comparable to those utilized in the nonalternation situation, the results suggest that impaired alternation performance following bilateral E.C. lesions is not a result of an impaired ability to utilize cues to distinguish between the appropriate choices. Since the other aspect of alternation performance is the short-term recall of the arm chosen on the preceding trial, the results are consistent with the interpretation that the impaired alternation performance might reflect an inability to recall which arm was chosen on the preceding trial. Whether this deficit is specific to the maze alternation tasks, or is generalized to all tasks which require short term memory will require further investigation. A particularly interesting aspect of the present observations is the time course of the changes in open field activity following bilateral E.C. lesions. Activity levels were increased following the lesions, reaching a peak between 5 and 7 days postlesion. However, after postlesion Day 7, the activity levels began to decrease, returning to near normal levels by 11 days postlesion. The return to near normal levels of activity between 7 and 10 days postlesion corresponds approximately to the time during which the surviving afferents in the dentate gyrus proliferate (see . This correlation suggests a relationship between the reorganization of surviving afferents and the recovery of near normal levels of activity. If the recovery to near-normal levels of activity following E.C. lesions is related to the reorganization of surviving afferents in the hippocampal formation, the implications would be immense. It has been assumed that postlesion afferent reorganization at best would have no behavioral effect, and at worst, would contribute to further behavioral dysfunction, owing to a scrambling of normal connections [ 19, 251. In a previous possible case where behavioral recovery accompanied reinnervation [ 14) the reinnervating fibers originated from the contralateral homologue of the damaged structure [ 321 , a situation which makes a relationship between reinnervation and recovery at least interpretable. In the present study, however, becasue the lesions of the E.C. were bilateral, the reinnervation is restricted to afferents (septal, commissural, associational, etc. [ 151,
which bear no obvious relationship with the afferents which had been destroyed by the lesion. If there is a relationship between reinnervation and the recovery of near normal levels of activity in the open field, then it is perhaps conceivable that some dysfunctions which result from lesions can be partially compensated for by reinnervation of denervated cells by any afferent, regardless of the similarity between lesion-induced and the normal afferents which had been destroyed. For example, perhaps a sufficient number of afferent inputs are required for a neuron to maintain or to prevent denervation super“excitatory tone”, sensitivity. In view of this hypothesis, it is interesting to note that recent correlative evidence suggests that reflex recovery following damage to the spinal cord might also be mediated by sprouting [ 9, 10, 2 11 . While the correlations between the time course of reinnervation, and the time course of recovery are enticing, it is clear that other types of lesions can produce changes in activity with similar time courses. For example, bilateral destruction of the hippocampus proper results in transient increases in locomotor activity which are quite reminiscent in the present study , including the of those described
return to near normal levels of activity (this time at approximately 12 days postlesion). If the return of near normal activity levels following bilateral hippocampal lesions is also related to afferent reorganization, it is clear that this reorganization is occurring outside the hippocampal formation. A potential site is the medial septal nucleus, which undergoes extensive afferent reorganization following hippocampal lesions [ 25, 271. Until more is known however, about the extent and time course of afferent reorganization which might occur following lesions of structures other than the E.C., the possible relationships between behavioral changes and afferent reorganization will remain speculative.
Supported in part by an Alfred Sloan Foundation Grant No. 75-2-7 to the University of Virginia, by USPHS Research Grant No. 1 R01 NS12333, and by National Science Foundation Grant No. BNS76-17750 to 0. Steward. Special thanks go to Kathy Steward, who initiated the behavioral experiments in this lab, and whose preliminary experiments provided the impetus for the present work.
REFERENCES Blackstad, T. Commissural connections of the hippocampal region in the rat, with special reference to their mode of terminati0n.J. camp. Neurol. 105: 417-537, 1956. DeCastro, J. M. A selective spatial discrimination deficit after fornicotomy in the rat. Behav. Biol. 12: 373-382, 1974. Douglas, R. J. Cues for spontaneous alternation. J. camp. physiol. Psychol. 62: 171-183, 1966. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. Douglas, R. J. The development of hippocampal function: Implications for theory and for therapy. In: 7’he Hippocampus, Vol. 2, edited by R. L. Isaacson and R. H. Pribram. New York: Academic Press, 1975. lesions and 6. Douglas, R. J. and R. L. Isaacson. Hippocampal activity. Psychon. Sci. 1: 187-l 88, 1964. I. Ely, D. L., E. G. Greene and J. P. Henry. Minicomputer monitored social behavior of mice with hippocampus lesions. Behav. Biol. 16: l-29, 1976. responding and hyperphagia follow8. Entingh, D. Perseverative ing entorhinal lesions in cats. J. camp. physiol. Psychol. 75: 50-58, 1971. 9 Goldberger, M. E. Recovery of movement after CNS lesions in monkeys. In: Plasticity and Recovery of Function in the Central Nervous System, edited by D. G. Stein, J. J. Rosen and N. Butters. New York: Academic Press, 1974. M. E. and M. Murray. Restitution of function and 10. Goldberger, collateral sprouting in the cat spinal cord: The deafferented animal. J. romp. Neural. 158: 37-54, 1974. 11. Hjorth-Simonsen, A. Hippocampal efferents to the ipsilateral entorhinal area: An experimental study in the rat. J. camp. Neurol. 142: 417-438, 1971. 12. Kimble, D. P. The effects of bilateral hippocampal lesions in rats. J. camp. physiol. Psychol. 56: 273-283, 1963. 13. Kim, C., H. Choi, J. K. Kim, H. K. Chang, R. S. Park and I. Y. Kang. General behavioral activity and its component patterns in hippocampectomized rats. Brain Res. 19: 379-394, 1970. 14. Loesche, J. and 0. Steward. 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, 1977. 15. Lynch. G. and C. Cotman. The hippocampus as a model for studying anatomical plasticity in the adult brain. In: The Hippocampus, edited by R. L. Isaacson and R. H. Pribram. New York: Academic Press, 1975. 1.
Lynch, G., C. Gall, G. Rose and C. Cotman. Changes in the distribution of the dentate gyrus associational system following unilateral or bilateral entorhinal lesions in the adult rat. Brain Res. 110: 57-71, 1976. 17. Lynch, G., D. A. Matthews, S. Mosko, T. Parks and C.W. Cotman. Induced acetylcholinesterase-rich layer in rat dentate gyrus following entorhinal lesions. Brain Res. 42: 31 l-318, 1972. G., B. Stanfield and C. Cotman. Developmental 18. Lynch, differences in postlesion axonal growth in the hippocampus. Brain Rex 59: 155-168, 1973. G.P., C. M. Austin, C. N. Liu and C. Y. Liu. 19. McCouch, Sprouting as a cause of spasticity. J. Neurophysiol. 21: 205-216, 1958. lesions 20. Means, L. W. and R. J. Douglas. Effects of hippocampal on cue utilization in spatial discrimination in rats. J. camp. physiol. Psychol. 73: 254-260, 1970. Restitution of function and 21. Murray, M. and M. E. Goldberger. collateral sprouting in cat spinal cord: The partially hemisected animal. J. camp. Neural. 158: 19-36, 1974. 22. Myhrer, T. Locomotor and avoidance behavior in rats with partial or total hippocampal perforant path sections. Physiol. Behav. 15: 217-224, 1975. 23. Myhrer, T. Maze performance in rats with hiuoocampal perforant path lesions: Some aspects of functiona; kecovery. Phvsiol. Behav. 15: 433-437. 1975. 24. O’Keefe, J., L. Nadel, S. Keightley and D. Kill. Fornix lesions selectively abolish place learning in the rat. Expl Neurol.48: 152-166, 1975. 25. Raisman, G. Neuronal plasticity in the septal nuclei of the adult rat. Brain Res. 14: 25-48, 1969. G., W. M. Cowan and T. P. S. Powell. The extrinsic 26. Raisman, afferent, commissural, and association fibers of the hippocampus. Brain 88: 963-996, 1965. 27. Raisman, G. and P. M. Field. A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei. Brain Rex 50: 241-264, 1973. 28. Ross, J. F., L. L. Walsh and S. P. Grossman. Some behavioral effects of entorhinal cortex lesions in the albino rat. J. camp. physiol. Psyclrol. 85: 70-81, 1973. 29. Ramon y Cajal, S. Histologic du Systkmc Nerveux de 1’Homme et des VertCbr&. T. 2, Paris, A. Maloine, 1911.
Smith, R. L., 0. Steward, C. Cotman and G. Lynch. Axon sprouting in the hippocampal formation and behavioral recovery following unilateral entorhinal cortex lesions. (Abs.). Third Annual Meeting of the Society for Neuroscience. San Diego, CA., 1973. Steward, 0. Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat. J. camp. Neural. 167: 285314, 1976. Steward, 0. Reinnervation of the dentate gyrus by homologous afferents following entorhinal cortical lesions in adult rats. Science, 194: 426-428, 1976. Steward, O., C. W. Cotman and G. Lynch. Growth of a new fiber projection in the brain of adult rats: Reinnervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions. Expl Bruin Rex 20: 45-66, 1974. Steward, O., C. Cotman and G. Lynch. A quantitative autoradiographic and electrophysiological study of the reinnervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions. Brain Res., 114: 181-200. 1976.
LOESCHE AND HORTON
Steward, 0. and J. Loesche. Quantitative autoradiographic analysis of the time course of proliferation of contralateral entorhinal efferents in the dentate gyrus denervated by ipsilateral entorhinal lesions. Brain Res., in press. Van Hoesen, G. W. and D. N. Pandya. Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. I. Temporal lobe afferents. Bruin Res. 95: l-24, 1975. Van Hoesen, G. W., D. N. Pandya and N. Butters. Cortical afferents to the entorhinal cortex of the rhesus monkey. Science 175: 1471-1473,1972. Van Hoesen, G. W., L. M. Wilson, J. M. MacDougall and J. C. Mitchell. Selective hippocampal complex deafferentation and deefferentation and avoidance behavior in rats. Physiol. Behau. 8: 873-879, 1972. West, J. R., S. Deadwyler, C. W. Cotman and G. Lynch. Time dependent changes in commissural field potentials in the dentate gyrus following lesions of the entorhinal cortex in adult rats. Brain Res. 97: 215-233, 1975.