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Recovery of function after neurotoxic damage to the hippocampal CA3 region: importance of postoperative recovery interval and task experience
B E H A V I O R A L A N D N E U R A L BIOLOGY
33, 453-464 (1981)
Recovery of Function after Neurotoxic Damage to the Hippocampal CA3 Region: Importance of Postoperative Recovery Interval and Task Experience GAIL E . HANDELMANN AND DAVID S. OLTON l
Department of Psychology, The Johns Hopkins University, Baltimore, Mao'land 21218 Recovery of function often follows damage to the hippocampal system: behavior that is severely disrupted immediately following the damage gradually becomes more normal. The present experiment examines the extent to which the length of the postoperative recovery interval and the amount of postoperative behavioral testing can influence the rate of recovery exhibited by rats following damage to the CA3 subfield of the hippocampus. Rats were trained in a rewarded alternation task and then given injections of either kainic acid (experimental) or saline (control) into the hippocampal CA3 region. Half the rats in each group began postoperative testing 5 days after surgery; the rest began 30 days after surgery. Of the rats that began retesting 5 days postoperatively, half received one daily test session and half received two daily test sessions. The same was true of the rats that began retesting 30 days postoperatively. All experimental groups initially performed worse than controls, but all eventually relearned the task. The worst performance was shown by the group tested once a day beginning 5 days after surgery. The rate of recovery was increased significantly by either increasing the amount of task experience or by lengthening the postoperative recovery interval. These results indicate that recovery of function after damage to the hippocampal CA3 region can be facilitated by both postoperative recovery time and task-specific experience.
Following damage to a neural system in the adult brain, behavior may be disrupted for a period of time but then return to normal (Stein, Rosen, & Butters, 1974). During the interval between the neural trauma and recovery, at least three variables may influence the return of normal behavior. The first is the passage of time, which provides the opportunity for neurological changes to occur as a function of the time elapsed following the brain damage. These changes may comprise either a reThis research was supported by research grant MH 24123 to D. S. O. from the National Institute of Mental Health. G. E. H. was supported by a predoctoral fellowship from the National Science Foundation. The authors thank C. Anderson, E. Breitinger, K. Kokobun, E. Phillips, and T. Rumbarger for their assistance in conducting the experiment, M. Weigel for typing the manuscript, and M. Evans, K. Fowler, and S. Mitchell for their comments on this manuscript. 453 0163-1047/81 / 120453-12502.00/0
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HANDELMANN AND OLTON
organization of neural circuits or a recovery from transient dysfunction (Finger, 1978). For example, when sequential lesions of a specific brain region were performed, the length of time between the two lesions determined the ultimate degree of behavioral recovery (Stewart & Ades, 1951; Meyer, Isaac, & Maher, 1958). These studies indicate that some time-related event occurring after the first lesion attenuated the behavioral impairment caused by the second. A second variable is general experience and interaction with the environment, which may enhance recovery of behavior. For example, housing adult rats with cortical lesions postoperatively in "enriched" environments, containing conspecifics and many sources of sensory stimulation, facilitated performance of maze tasks relative to rats housed postoperatively in standard conditions (Will & Rosenzweig, 1976). Therefore, even experience not directly related to the behavioral task in which recovery of function is amended may alter the rate of their recovery. A third variable influencing behavioral recovery of function may be task-specific experience, or practice. Braun (1966) found that the recovery of visual placing behavior in rats after neocortical lesions was facilitated by massed practice in the task. This practice may alter the neurological system that was damaged by the lesion or may allow the animal to develop a new strategy using other undamaged parts of the brain. After a given type of brain damage, therefore, two important questions arise: Will some degree of recovery occur in the absence of task-specific experience? Can task-specific behavioral experience affect the rate of recovery? In this experiment, both the amount of experience in performing a task and the amount of time elapsed between surgery and the beginning of testing were varied to determine their effect on the rate of recovery after damage to a discrete part of the hippocampus, the CA3 subfield. In a previous experiment, destruction of the CA3 pyramidal cells in the hippocampus by kainic acid produced a severe disruption of choice accuracy when rats were tested for the retention of a radial maze task. After about 30 days of testing, however, these same rats relearned the task and performed it at criterion levels (Handelmann & Olton, 1981). In the present investigation, rats with lesions of the CA3 subfield were tested postoperatively on a different spatial maze task, rewarded alternation in a T maze. Postoperatively, they were given one of two different recovery intervals (5 or 30 days) and one of two different amounts of test experience (one test session per day or two test sessions per day). To the extent that added practice in the task facilitates recovery of function, rats given two daily test sessions should demonstrate recovery faster than rats given one daily test session. To the extent that timerelated endogenous factors and nonspecific experience facilitate recovery
RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
455
of function, rats tested after a 30-day interval should demonstrate rec o v e r y faster than rats tested after a 5-day interval.
METHODS Subjects Fifty-six male S p r a g u e - D a w l e y rats weighing 250-300 g at the start of testing were housed in individual cages with free access to water. T h e y were kept in a colony room with a 10-hr dark/14-hr light cycle. Behavioral tests were conducted during the light portion of the cycle. Prior to beginning training on the task, each rat was gradually deprived of food until it reached 85% of its ad lib body weight and was maintained at this level plus 5 g for each week of testing while being trained on the maze task.
Apparatus The apparatus was an elevated T maze constructed from unpainted wood. The runaways were 6 cm wide and had a Masonite strip, I cm high, fastened to each side. A guillotine door was located on the stem of the T, dividing the stem into a start area and a runway leading to the choice point, the point at which the stem and the two arms of the T met. The stem of the T was 30 cm long, and each arm was 47.5 cm long from the choice point. At the end of each arm, a hole about 1 cm deep served as a food cup to hold the reinforcer, a 190-mg food pellet (P. J. N o y e s Co., Lancaster, N.H.). The maze was placed near a wall in a well-lit room with many extramaze stimuli.
Procedure Behavioral Task Shaping. Each rat was given 15-30 min to explore the maze during each of 3 consecutive days. During the first day, about 15 food pellets were scattered throughout the maze. During the 2 subsequent days, pellets were located only at the ends of the arms. Testing. The testing procedure is illustrated schematically in Fig. 1. At the beginning of each trial, one pellet of food was placed in each food cup. F o r the f o r c e d run, a block of wood, 13 cm high, was placed at the entrance to one of the arms. The rat was placed in the start area and the guillotine door raised, allowing the rat to run to the choice point and then down the open arm. After it had eaten the food pellet, the rat was picked up and placed again in the start area with the guillotine door lowered. F o r the choice run, the block of wood was removed so that both arms were accessible. Food, however, was present only in the arm not visited during the first run. After the rat had been in the start area for about 5 sec, the guillotine door was raised and the rat allowed to run to the end of one arm. A correct choice was choosing the arm containing
456
HANDELMANN AND OLTON FORCED RUN
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FIG. 1. Behavioral task. For the forced run, one arm of the T m a z e was blocked by a barrier (black bar) so that the rat was guided down the other arm to obtain a food pellet (black dot). For the choice run, the barrier was r e m o v e d and a food pellet was present in the previously blocked arm. To receive both pellets, the rat therefore had to alternate arms c h o s e n within each trial. The arm used for the forced run was varied randomly for the eight trials of each test session with the constraint that each arm be used for four trials.
the food pellet. An incorrect choice was choosing the arm previously visited during that trial, the arm which no longer contained a food pellet. The rat was then removed from the maze and placed in his home cage. Each day, one test session of eight trials was given within a period of about 40 min, making the intertrial interval about 5 min. During each test session, both the left arm and the right arm were each used four times for the forced run. The order of the left and right runs was randomized and varied daily.
General Procedure Preoperative. After shaping, each rat was tested in the rewarded alternation task for at least 6 days and to a criterion of seven correct choices in a test session for 2 consecutive days. After reaching this criterion, the rats were fed ad lib for 2 days and not tested. Surgical. Each rat was anesthetized with Chloropent (Fort Dodge Laboratories, Fort Dodge, Indiana, 3.0 cc/kg intraperitoneally) and administered Bicillin (Wyeth, Philadelphia, Penna., 0.05 cc, intramuscularly). When anesthetized, the rat was placed in a stereotaxic apparatus with the incisor bar set 2.5 mm below the interaural line. The scalp was incised and retracted. Four small holes were drilled through the skull to allow injections into the hippocampus. The coordinates for the anterior placements were: 3.5 mm posterior to bregma, 4.0 mm lateral to the sagittal suture, and 3.3 mm ventral to the surface of the skull, with the needle angled medially 22° from the vertical in the coronal plane. The coordinates for the posterior placements were: 6.5 mm posterior to bregma, 5.0 mm lateral to the sagittal suture, and 6.3 mm ventral to the surface of the skull with the needle vertical.
R E C O V E R Y F O L L O W I N G H I P P O C A M P A L CA3 L E S I O N S
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All rats received bilateral injections of either kainic acid or saline. A 5-~1 syringe fitted with a 31-gage needle (Hamilton, Reno, Nev.) was used for the injections. Rats receiving kainic acid injections (experimental group) were injected with 0.8 nmol of kainic acid (Sigma, St. Louis, Mo.) dissolved in 0.4 ~1 of phosphate-buffered saline (pH 7.4) at each placement during a 5-min interval. Rats receiving saline injections (control group) were given 0.4 Ixl of phosphate-buffered saline at each placement, injected during a 5-min interval. Postoperative. After surgery, the rats were randomly assigned to four experimental treatments. Seven rats with kainic acid injections and seven with saline injections were assigned to each treatment. The treatments differed in the length of the postoperative recovery interval and in the amount of experience received on the maze, making a two-by-two design with two recovery intervals and two amounts of experience (see Table 1). The abbreviations for the groups indicate (1) the number of days between surgery and the start of testing and (2) the number of test sessions per day. Two of the groups of rats received a 5-day recovery interval after surgery and then resumed testing. Of these two groups, one received one test session daily (Group 5D-IT); the other received two daily test sessions, at least 3 hr apart (Group 5D-2T). The second two groups received a 30-day recovery interval after surgery. Of these two groups, one received one test session daily (Group 30D-1T); the other received two daily test sessions, at least 3 hr apart (Group 30D-2T). All rats were tested every day for at least 30 days and until they reached a criterion of five consecutive test sessions with at least seven correct choices in each session. The rats were then killed and their brains taken for histology. Histology. All rats were anesthetized with ether and perfused with 0.9% saline followed by 10% formalin in saline; the brains were removed and stored in 30% sucrose-formalin for at least 5 days. They were then frozen and cut coronally in sections 20 Ixm thick. Every fifth section was stained with cresyl violet. TABLE 1 Performance o f Rewarded Spatial Alternation Task Expressed in Terms o f the Mean N u m b e r of Days (--- SEM) to Begin Criterion 5-Day postoperative recovery interval
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The brain sections were examined with the light microscope, and rats found to have cell damage in the CA1 subfield, dentate fascia, or subiculum were excluded from the analysis. The extent of loss of CA3 pyramidal cells was quantified by counting the remaining CA3 pyramidal cells in the hippocampus on one side at five levels. These levels corresponded to levels A4890, A3430, A3180, A2580, and A1950 of K6nig and Klippel (1967). In most rats, the damage appeared to be bilaterally symmetrical, but for the occasional brain in which the extent of damage on each side was not equivalent, the side with the lesser amount of damage was counted. In the control rats, the mean number of CA3 pyramidal cells was determined and this value was taken as 100%. The mean number of cells remaining at each level in rats receiving kainic acid injections was determined and then expressed as a percentage of the control values (Handelmann & Olton, 1981).
RESULTS Histology F o u r rats were found to have neuronal loss in the CA1 subfield and were excluded from the histological and behavioral analyses. Two of these rats were from Group 30D-1T, one was from Group 5D-2T, and one was from Group 30D-2T. The extent of pyramidal cell loss in the CA3 region, as determined by cell counts at five levels of the hippocampus, is summarized in Fig. 2.
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RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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Substantial cell loss occurred throughout the extent of the hippocampus. CA3 pyramidal cells were virtually eliminated in the temporal and septal poles. Any remaining cells were usually located about midway through the length of the hippocampus, at about the level of A3180. Other cells in the CAI, CA4, and dentate fascia appeared intact, as illustrated in Fig. 3. A detailed anatomical analysis of the intra- and extrahippocampal effects of kainic acid injections into the CA3 region has been presented elsewhere (Handelmann & Olton, 1981). Behavioral Test
Rats receiving saline injections performed well after surgery. As illustrated in Table 1, all of the control groups required a mean of between 2 and 3 days to begin criterion performance. Neither the length of the postoperative recovery interval nor the number of daily test sessions significantly affected the amount of time required to reach the criterion. All rats receiving kainic acid injections demonstrated impaired choice accuracy at the start of behavioral testing. As illustrated in Fig. 4, the mean number of correct responses in the first block of test sessions for all these groups was less than 5.4, and the rats required more days to
F16. 3. Coronal section of the hippocampus after injections of kainic acid into the CA3 subfield (cresyl violet stain). Note the absence of the CA3 pyramidal cells. Other cell fields remained intact.
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begin criterion performance than did the control rats (two-way ANOVA; F(1,42) = 100.9, p < .001). The worst performance was shown by Group 5D-1T, which required a mean of 27.7 days to begin criterion, more days than any other group receiving kainic acid injections (Neuman-Keuls, p < .05). Increasing the number of test sessions given each day decreased the number of days required to begin criterion. Rats tested twice a day beginning 5 days after surgery (Group 5D-2T) required a mean of only 13 days to begin criterion, a value significantly less than the 27.7 days of Group 5D-2T (Neuman-Keuls, p < .05). Increasing the length of time between surgery and retesting also decreased the number of days to criterion. Rats tested once a day beginning 30 days after surgery (Group 30D-1T) required a mean of only 16.6 days to begin criterion, a value significantly less than the 27.7 days of Group 5 D - I T (Neuman-Keuls, p < .05). Rats receiving two test sessions and a 30-day recovery interval (Group 30D-2T) required a mean of 15.2 days to begin criterion, a value significantly less than that required by Group 5D-1T (Neuman-Keuls, p < .05) but not significantly different from that of either Group 5D-2T or 30D-1T. The effects of added task experience and lengthened recovery interval were therefore not additive in decreasing the number of days to criterion.
RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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As shown in Fig. 4, all rats were tested during the seventh block of 5 test days, allowing a comparison of choice accuracy of all groups. The amount of prior test experience had a strong influence on choice accuracy during this time. The group that had received 50 test sessions (5D-2T) chose more accurately than the group that had received 25 test sessions (5D-1T), and this group chose more accurately than either of the two groups that had received 0 test sessions (30D-1T and 30D-2T).
DISCUSSION Rats were trained to perform a rewarded alternation task on a T maze and then given injections of kainic acid to destroy the CA3 pyramidal cells. When testing resumed, these rats exhibited a severe impairment in choice accuracy. Performance improved with continued testing, however, and the choice accuracy of all rats eventually returned to criterion levels. The rate of recovery of function, as indicated by choice accuracy in the T maze, was enhanced by two variables. The first was the amount of postoperative behavioral testing, which affected choice accuracy in two ways. First, in terms of the number of days to reach criterion performance, rats tested twice a day beginning 5 days after surgery showed a faster recovery than rats tested once a day. Thus, additional test experience increased the rate of recovery for this group. Second, in terms of the number of correct responses during the seventh block of 5 days, rats tested either once or twice a day for the preceding 25 days chose more accurately than rats not tested at all during that time. Thus, some test experience (once or twice a day) was better than no test experience. A second variable unrelated to task experience was also influential. Rats given a 30-day recovery interval (30D-1T) reached criterion performance more rapidly than rats given only a 5-day recovery interval (5D-1T). Thus, during the first 30 days after surgery, some amelioration of the rats' impairment in choice accuracy occurred independently of experience in the task. Although the 30-day recovery interval, as compared to the 5-day recovery interval, decreased the number of days to reach criterion performance, it did not affect initial choice accuracy. At the start of testing, rats with the longer recovery interval chose no more accurately than did rats with the shorter recovery interval at the start of their testing. Thus, the recovery period selectively influenced the rate of improvement in the task rather than the initial level of performance. This pattern of results suggests that the recovery interval facilitated the rats' ability to relearn the task on the basis of their postoperative test experience rather than because of some spontaneous recall of their preoperative learning.
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Because both test experience and postoperative recovery time each facilitated performance by themselves, the rats that had the benefit of both together (30D-2T) might have been expected to show the best performance. Such was not the case. The number of trials taken by this group to reach criterion was not substantially different from that of either of the other two groups with facilitated performance (5D-2T and 30D-1T). These results indicate that the effects of the two variables were not additive in influencing recovery and suggest that some factor may have limited the rate at which the processes underlying the behavioral recovery could be completed. The data analysis was presented in terms of the number of days to reach criterion performance rather than the number of test sessions for two reasons. First, we felt that the most important information in recovery.was performance at a particular time following surgery. Second, the data provide a readily interpretable pattern of results. If the analysis is conducted in terms of the number of test sessions to reach criterion performance, the most rapid learning is found for the 30D-1T group; the learning of the other three groups was slower and similar for all groups. Thus, the longer postoperative recovery interval (30D-IT as compared to 5D-1T) still facilitated performance. The effects of experience, however, are more difficult to interpret. On the one hand, experience did have some effect; during the seventh block of 5 days, the choice accuracy of the two groups that began testing after a 5-day postoperative recovery period (and thus had received 25 days of testing prior to that block) was higher than the choice accuracy of the two groups that began testing after a 30-day postoperative recovery period (and thus had received no testing prior to that block). On the other hand, neither of the groups tested twice a day (5D-2T and 30D-2T) reached criterion performance more rapidly than the comparable group tested once a day (5D-IT and 30D-IT, respectively). These analyses support the same qualitative interpretations as before--both the length of the postoperative recovery interval and the amount of postoperative test experience influenced the rate of recoverymbut suggest a different quantitative one with respect to test experiencensome test experience was better than no test experience, but more test experience (two test sessions each day) did not produce a further enhancement. Further parametric studies should be able to delineate more accurately the relationship between the amount of test experience and the rate of behavioral recovery. Recovery of function following partial damage to the hippocampal formation has been demonstrated by previous research. For example, performance of a continuous alternation task was disrupted immediately after dorsal hippocampal lesions, but returned to normal after 60 days of testing (Dawson, Conrad, & Lynch, 1973; Isseroff, Leveton, Freeman, Lewis, & Stein, 1976). The ability to solve Hebb-Williams maze prob-
RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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lems was impaired immediately after damage to the perforant path, but recovered after 35 days of testing (Myhrer, 1975). Alternation behavior in a Y maze was disrupted by unilateral entorhinal cortex lesions, but the alternation behavior returned to normal within 10 days, a time course similar to that found for reinnervation of the dentate gyrus by the contralateral entorhinal area (Loesche & Steward, 1977; Scheff & Cotman, 1977). Thus, recovery of function is not unusual following partial hippocampal lesions, and the present experiment provides information about the variables that can influence the rate at which this recovery occurs. A variety of neural mechanisms may be responsible for the behavioral recovery observed here. For example, the mechanism may be a reorganization within the hippocampus to repair the circuit damaged by the removal of the CA3 cells. The hippocampus shows a high degree of anatomical plasticity following partial lesions (see Milner & Loy, 1980, for a recent summary), and sprouting may compensate for the absence of the CA3 cells. Alternatively, the behavioral recovery might be due to the elimination of a transient dysfunction, or diaschesis (Rosner, 1974). Activation of specific neurochemical systems may also enhance recovery (Luria, Naydim, Tsvetkova, & Vinarskaya, 1969; Braun, Meyer, & Meyer, 1966). Although the neurological mechanisms underlying the behavioral recovery following CA3 damage were not addressed by this experiment, the fact that the time course of this recovery can be readily manipulated should make it a useful model for the study of recovery of function. REFERENCES Braun, J. J. (1966). The neocortex and visual placing in rats. Brain Research, 1,381-394. Braun, J. J., Meyer, P. M., & Meyer, D. R, (1966). Sparing of a brightness habit in rats following visual decortication. Journal of Comparative and Physiological Psychology, 61, 79-82. Dawson, R. G., Conrad, L., & Lynch, G. (1973). Single and two-stage hippocampal lesions: A similar syndrome. Experimental Neurology, 40, 263-277. Finger, S. (Ed.). (1978). Recovepsyfrom Brain Damage. New York: Plenum. Handelmann, G. E., & Olton, D. S. (1981). Spatial memory following damage to the hippocampal CA3 pyramidal cells with kainic acid: Impairment and recovery with preoperative training. Brain Research, 21, 41-58. Isseroff, A., Leveton, L., Freeman, G., Lewis, M. E., & Stein, D. G. (1976). The limits of behavioral recovery from serial lesions of the hippocampus. Experimental Neurology, 53, 339-354. K6nig, J. F. R., & Klippel, R. A. (1967). The Rat Brain. Huntington, N.Y.: R. E. Kreiger. Loesche, J., & Steward, O. (1977). Behavioral correlates of denervation and reinnervation of the hippocampal formation in the rat: Recovery of alternation performance following unilateral entorhinal cortex lesions. Brain Research Bulletin, 2, 31-39. Luria, A. R., Naydin, V. L., Tsvetkova, L. S., & Vinarskaya, E. N. (1969). Restoration of higher cortical function following local brain damage. In P. J. Vinkin & G. W. Brugh (Eds.). Handbook of Clinical Neurology. New York: Wiley. Meyer, D. R., Isaac, W., & Maher, B. (1958). The role of stimulation in spontaneous reorganization of visual habits. Journal of Comparative and Physiological Psychology, 51, 5-16.
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Milner, T., & Loy, R. (1980). A delayed sprouting response to partial hippocampal deafferentation: Time course of sympathetic ingrowth following fimbrial lesions. Brain Research, 197, 339-379. Myhrer, T. (1975). Maze performance in rats with hippocampal perforant path lesions: Some aspects of functional recovery. Physiology and Behavior, 15, 433-437. Rosner, B. S. (1974). Recovery of function and localization of function in historical perspective. In D. G. Stein, J. J. Rosen, & N. Butters (Eds.), Plasticity and Recovery of Function in the Nervous System. New York: Academic Press. Scheff, S. W., & Cotman', C. W. (1977). Recovery of spontaneous alternation following lesions of the entorhinal cortex in adult rats: Possible correlation to axon sprouting. Behavioral Biology, 21, 286-293. Stein, D. G., Rosen, J. J., & Butters, N. (Eds.) (1974). Plasticity and Recovery of Function in the Nervous System. New York: Academic Press. Stewart, J. W., & Ades, H. W. (1951). The time factor in reintegration of a learned habit after temporal lobe lesions in the monkey (Macaca mulatta). Journal of Comparative and Physiological Psychology, 44, 479-486. Will, B. E., & Rosenzweig, M. R. (1976). Effets de l'environnement sur la r6cup6ration fonctionelle apr~s 16sions c6r6brales chez les rats adultes. Biological Behavior, 1, 5-16.
33, 453-464 (1981)
Recovery of Function after Neurotoxic Damage to the Hippocampal CA3 Region: Importance of Postoperative Recovery Interval and Task Experience GAIL E . HANDELMANN AND DAVID S. OLTON l
Department of Psychology, The Johns Hopkins University, Baltimore, Mao'land 21218 Recovery of function often follows damage to the hippocampal system: behavior that is severely disrupted immediately following the damage gradually becomes more normal. The present experiment examines the extent to which the length of the postoperative recovery interval and the amount of postoperative behavioral testing can influence the rate of recovery exhibited by rats following damage to the CA3 subfield of the hippocampus. Rats were trained in a rewarded alternation task and then given injections of either kainic acid (experimental) or saline (control) into the hippocampal CA3 region. Half the rats in each group began postoperative testing 5 days after surgery; the rest began 30 days after surgery. Of the rats that began retesting 5 days postoperatively, half received one daily test session and half received two daily test sessions. The same was true of the rats that began retesting 30 days postoperatively. All experimental groups initially performed worse than controls, but all eventually relearned the task. The worst performance was shown by the group tested once a day beginning 5 days after surgery. The rate of recovery was increased significantly by either increasing the amount of task experience or by lengthening the postoperative recovery interval. These results indicate that recovery of function after damage to the hippocampal CA3 region can be facilitated by both postoperative recovery time and task-specific experience.
Following damage to a neural system in the adult brain, behavior may be disrupted for a period of time but then return to normal (Stein, Rosen, & Butters, 1974). During the interval between the neural trauma and recovery, at least three variables may influence the return of normal behavior. The first is the passage of time, which provides the opportunity for neurological changes to occur as a function of the time elapsed following the brain damage. These changes may comprise either a reThis research was supported by research grant MH 24123 to D. S. O. from the National Institute of Mental Health. G. E. H. was supported by a predoctoral fellowship from the National Science Foundation. The authors thank C. Anderson, E. Breitinger, K. Kokobun, E. Phillips, and T. Rumbarger for their assistance in conducting the experiment, M. Weigel for typing the manuscript, and M. Evans, K. Fowler, and S. Mitchell for their comments on this manuscript. 453 0163-1047/81 / 120453-12502.00/0
454
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organization of neural circuits or a recovery from transient dysfunction (Finger, 1978). For example, when sequential lesions of a specific brain region were performed, the length of time between the two lesions determined the ultimate degree of behavioral recovery (Stewart & Ades, 1951; Meyer, Isaac, & Maher, 1958). These studies indicate that some time-related event occurring after the first lesion attenuated the behavioral impairment caused by the second. A second variable is general experience and interaction with the environment, which may enhance recovery of behavior. For example, housing adult rats with cortical lesions postoperatively in "enriched" environments, containing conspecifics and many sources of sensory stimulation, facilitated performance of maze tasks relative to rats housed postoperatively in standard conditions (Will & Rosenzweig, 1976). Therefore, even experience not directly related to the behavioral task in which recovery of function is amended may alter the rate of their recovery. A third variable influencing behavioral recovery of function may be task-specific experience, or practice. Braun (1966) found that the recovery of visual placing behavior in rats after neocortical lesions was facilitated by massed practice in the task. This practice may alter the neurological system that was damaged by the lesion or may allow the animal to develop a new strategy using other undamaged parts of the brain. After a given type of brain damage, therefore, two important questions arise: Will some degree of recovery occur in the absence of task-specific experience? Can task-specific behavioral experience affect the rate of recovery? In this experiment, both the amount of experience in performing a task and the amount of time elapsed between surgery and the beginning of testing were varied to determine their effect on the rate of recovery after damage to a discrete part of the hippocampus, the CA3 subfield. In a previous experiment, destruction of the CA3 pyramidal cells in the hippocampus by kainic acid produced a severe disruption of choice accuracy when rats were tested for the retention of a radial maze task. After about 30 days of testing, however, these same rats relearned the task and performed it at criterion levels (Handelmann & Olton, 1981). In the present investigation, rats with lesions of the CA3 subfield were tested postoperatively on a different spatial maze task, rewarded alternation in a T maze. Postoperatively, they were given one of two different recovery intervals (5 or 30 days) and one of two different amounts of test experience (one test session per day or two test sessions per day). To the extent that added practice in the task facilitates recovery of function, rats given two daily test sessions should demonstrate recovery faster than rats given one daily test session. To the extent that timerelated endogenous factors and nonspecific experience facilitate recovery
RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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of function, rats tested after a 30-day interval should demonstrate rec o v e r y faster than rats tested after a 5-day interval.
METHODS Subjects Fifty-six male S p r a g u e - D a w l e y rats weighing 250-300 g at the start of testing were housed in individual cages with free access to water. T h e y were kept in a colony room with a 10-hr dark/14-hr light cycle. Behavioral tests were conducted during the light portion of the cycle. Prior to beginning training on the task, each rat was gradually deprived of food until it reached 85% of its ad lib body weight and was maintained at this level plus 5 g for each week of testing while being trained on the maze task.
Apparatus The apparatus was an elevated T maze constructed from unpainted wood. The runaways were 6 cm wide and had a Masonite strip, I cm high, fastened to each side. A guillotine door was located on the stem of the T, dividing the stem into a start area and a runway leading to the choice point, the point at which the stem and the two arms of the T met. The stem of the T was 30 cm long, and each arm was 47.5 cm long from the choice point. At the end of each arm, a hole about 1 cm deep served as a food cup to hold the reinforcer, a 190-mg food pellet (P. J. N o y e s Co., Lancaster, N.H.). The maze was placed near a wall in a well-lit room with many extramaze stimuli.
Procedure Behavioral Task Shaping. Each rat was given 15-30 min to explore the maze during each of 3 consecutive days. During the first day, about 15 food pellets were scattered throughout the maze. During the 2 subsequent days, pellets were located only at the ends of the arms. Testing. The testing procedure is illustrated schematically in Fig. 1. At the beginning of each trial, one pellet of food was placed in each food cup. F o r the f o r c e d run, a block of wood, 13 cm high, was placed at the entrance to one of the arms. The rat was placed in the start area and the guillotine door raised, allowing the rat to run to the choice point and then down the open arm. After it had eaten the food pellet, the rat was picked up and placed again in the start area with the guillotine door lowered. F o r the choice run, the block of wood was removed so that both arms were accessible. Food, however, was present only in the arm not visited during the first run. After the rat had been in the start area for about 5 sec, the guillotine door was raised and the rat allowed to run to the end of one arm. A correct choice was choosing the arm containing
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HANDELMANN AND OLTON FORCED RUN
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FIG. 1. Behavioral task. For the forced run, one arm of the T m a z e was blocked by a barrier (black bar) so that the rat was guided down the other arm to obtain a food pellet (black dot). For the choice run, the barrier was r e m o v e d and a food pellet was present in the previously blocked arm. To receive both pellets, the rat therefore had to alternate arms c h o s e n within each trial. The arm used for the forced run was varied randomly for the eight trials of each test session with the constraint that each arm be used for four trials.
the food pellet. An incorrect choice was choosing the arm previously visited during that trial, the arm which no longer contained a food pellet. The rat was then removed from the maze and placed in his home cage. Each day, one test session of eight trials was given within a period of about 40 min, making the intertrial interval about 5 min. During each test session, both the left arm and the right arm were each used four times for the forced run. The order of the left and right runs was randomized and varied daily.
General Procedure Preoperative. After shaping, each rat was tested in the rewarded alternation task for at least 6 days and to a criterion of seven correct choices in a test session for 2 consecutive days. After reaching this criterion, the rats were fed ad lib for 2 days and not tested. Surgical. Each rat was anesthetized with Chloropent (Fort Dodge Laboratories, Fort Dodge, Indiana, 3.0 cc/kg intraperitoneally) and administered Bicillin (Wyeth, Philadelphia, Penna., 0.05 cc, intramuscularly). When anesthetized, the rat was placed in a stereotaxic apparatus with the incisor bar set 2.5 mm below the interaural line. The scalp was incised and retracted. Four small holes were drilled through the skull to allow injections into the hippocampus. The coordinates for the anterior placements were: 3.5 mm posterior to bregma, 4.0 mm lateral to the sagittal suture, and 3.3 mm ventral to the surface of the skull, with the needle angled medially 22° from the vertical in the coronal plane. The coordinates for the posterior placements were: 6.5 mm posterior to bregma, 5.0 mm lateral to the sagittal suture, and 6.3 mm ventral to the surface of the skull with the needle vertical.
R E C O V E R Y F O L L O W I N G H I P P O C A M P A L CA3 L E S I O N S
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All rats received bilateral injections of either kainic acid or saline. A 5-~1 syringe fitted with a 31-gage needle (Hamilton, Reno, Nev.) was used for the injections. Rats receiving kainic acid injections (experimental group) were injected with 0.8 nmol of kainic acid (Sigma, St. Louis, Mo.) dissolved in 0.4 ~1 of phosphate-buffered saline (pH 7.4) at each placement during a 5-min interval. Rats receiving saline injections (control group) were given 0.4 Ixl of phosphate-buffered saline at each placement, injected during a 5-min interval. Postoperative. After surgery, the rats were randomly assigned to four experimental treatments. Seven rats with kainic acid injections and seven with saline injections were assigned to each treatment. The treatments differed in the length of the postoperative recovery interval and in the amount of experience received on the maze, making a two-by-two design with two recovery intervals and two amounts of experience (see Table 1). The abbreviations for the groups indicate (1) the number of days between surgery and the start of testing and (2) the number of test sessions per day. Two of the groups of rats received a 5-day recovery interval after surgery and then resumed testing. Of these two groups, one received one test session daily (Group 5D-IT); the other received two daily test sessions, at least 3 hr apart (Group 5D-2T). The second two groups received a 30-day recovery interval after surgery. Of these two groups, one received one test session daily (Group 30D-1T); the other received two daily test sessions, at least 3 hr apart (Group 30D-2T). All rats were tested every day for at least 30 days and until they reached a criterion of five consecutive test sessions with at least seven correct choices in each session. The rats were then killed and their brains taken for histology. Histology. All rats were anesthetized with ether and perfused with 0.9% saline followed by 10% formalin in saline; the brains were removed and stored in 30% sucrose-formalin for at least 5 days. They were then frozen and cut coronally in sections 20 Ixm thick. Every fifth section was stained with cresyl violet. TABLE 1 Performance o f Rewarded Spatial Alternation Task Expressed in Terms o f the Mean N u m b e r of Days (--- SEM) to Begin Criterion 5-Day postoperative recovery interval
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The brain sections were examined with the light microscope, and rats found to have cell damage in the CA1 subfield, dentate fascia, or subiculum were excluded from the analysis. The extent of loss of CA3 pyramidal cells was quantified by counting the remaining CA3 pyramidal cells in the hippocampus on one side at five levels. These levels corresponded to levels A4890, A3430, A3180, A2580, and A1950 of K6nig and Klippel (1967). In most rats, the damage appeared to be bilaterally symmetrical, but for the occasional brain in which the extent of damage on each side was not equivalent, the side with the lesser amount of damage was counted. In the control rats, the mean number of CA3 pyramidal cells was determined and this value was taken as 100%. The mean number of cells remaining at each level in rats receiving kainic acid injections was determined and then expressed as a percentage of the control values (Handelmann & Olton, 1981).
RESULTS Histology F o u r rats were found to have neuronal loss in the CA1 subfield and were excluded from the histological and behavioral analyses. Two of these rats were from Group 30D-1T, one was from Group 5D-2T, and one was from Group 30D-2T. The extent of pyramidal cell loss in the CA3 region, as determined by cell counts at five levels of the hippocampus, is summarized in Fig. 2.
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RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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Substantial cell loss occurred throughout the extent of the hippocampus. CA3 pyramidal cells were virtually eliminated in the temporal and septal poles. Any remaining cells were usually located about midway through the length of the hippocampus, at about the level of A3180. Other cells in the CAI, CA4, and dentate fascia appeared intact, as illustrated in Fig. 3. A detailed anatomical analysis of the intra- and extrahippocampal effects of kainic acid injections into the CA3 region has been presented elsewhere (Handelmann & Olton, 1981). Behavioral Test
Rats receiving saline injections performed well after surgery. As illustrated in Table 1, all of the control groups required a mean of between 2 and 3 days to begin criterion performance. Neither the length of the postoperative recovery interval nor the number of daily test sessions significantly affected the amount of time required to reach the criterion. All rats receiving kainic acid injections demonstrated impaired choice accuracy at the start of behavioral testing. As illustrated in Fig. 4, the mean number of correct responses in the first block of test sessions for all these groups was less than 5.4, and the rats required more days to
F16. 3. Coronal section of the hippocampus after injections of kainic acid into the CA3 subfield (cresyl violet stain). Note the absence of the CA3 pyramidal cells. Other cell fields remained intact.
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begin criterion performance than did the control rats (two-way ANOVA; F(1,42) = 100.9, p < .001). The worst performance was shown by Group 5D-1T, which required a mean of 27.7 days to begin criterion, more days than any other group receiving kainic acid injections (Neuman-Keuls, p < .05). Increasing the number of test sessions given each day decreased the number of days required to begin criterion. Rats tested twice a day beginning 5 days after surgery (Group 5D-2T) required a mean of only 13 days to begin criterion, a value significantly less than the 27.7 days of Group 5D-2T (Neuman-Keuls, p < .05). Increasing the length of time between surgery and retesting also decreased the number of days to criterion. Rats tested once a day beginning 30 days after surgery (Group 30D-1T) required a mean of only 16.6 days to begin criterion, a value significantly less than the 27.7 days of Group 5 D - I T (Neuman-Keuls, p < .05). Rats receiving two test sessions and a 30-day recovery interval (Group 30D-2T) required a mean of 15.2 days to begin criterion, a value significantly less than that required by Group 5D-1T (Neuman-Keuls, p < .05) but not significantly different from that of either Group 5D-2T or 30D-1T. The effects of added task experience and lengthened recovery interval were therefore not additive in decreasing the number of days to criterion.
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As shown in Fig. 4, all rats were tested during the seventh block of 5 test days, allowing a comparison of choice accuracy of all groups. The amount of prior test experience had a strong influence on choice accuracy during this time. The group that had received 50 test sessions (5D-2T) chose more accurately than the group that had received 25 test sessions (5D-1T), and this group chose more accurately than either of the two groups that had received 0 test sessions (30D-1T and 30D-2T).
DISCUSSION Rats were trained to perform a rewarded alternation task on a T maze and then given injections of kainic acid to destroy the CA3 pyramidal cells. When testing resumed, these rats exhibited a severe impairment in choice accuracy. Performance improved with continued testing, however, and the choice accuracy of all rats eventually returned to criterion levels. The rate of recovery of function, as indicated by choice accuracy in the T maze, was enhanced by two variables. The first was the amount of postoperative behavioral testing, which affected choice accuracy in two ways. First, in terms of the number of days to reach criterion performance, rats tested twice a day beginning 5 days after surgery showed a faster recovery than rats tested once a day. Thus, additional test experience increased the rate of recovery for this group. Second, in terms of the number of correct responses during the seventh block of 5 days, rats tested either once or twice a day for the preceding 25 days chose more accurately than rats not tested at all during that time. Thus, some test experience (once or twice a day) was better than no test experience. A second variable unrelated to task experience was also influential. Rats given a 30-day recovery interval (30D-1T) reached criterion performance more rapidly than rats given only a 5-day recovery interval (5D-1T). Thus, during the first 30 days after surgery, some amelioration of the rats' impairment in choice accuracy occurred independently of experience in the task. Although the 30-day recovery interval, as compared to the 5-day recovery interval, decreased the number of days to reach criterion performance, it did not affect initial choice accuracy. At the start of testing, rats with the longer recovery interval chose no more accurately than did rats with the shorter recovery interval at the start of their testing. Thus, the recovery period selectively influenced the rate of improvement in the task rather than the initial level of performance. This pattern of results suggests that the recovery interval facilitated the rats' ability to relearn the task on the basis of their postoperative test experience rather than because of some spontaneous recall of their preoperative learning.
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Because both test experience and postoperative recovery time each facilitated performance by themselves, the rats that had the benefit of both together (30D-2T) might have been expected to show the best performance. Such was not the case. The number of trials taken by this group to reach criterion was not substantially different from that of either of the other two groups with facilitated performance (5D-2T and 30D-1T). These results indicate that the effects of the two variables were not additive in influencing recovery and suggest that some factor may have limited the rate at which the processes underlying the behavioral recovery could be completed. The data analysis was presented in terms of the number of days to reach criterion performance rather than the number of test sessions for two reasons. First, we felt that the most important information in recovery.was performance at a particular time following surgery. Second, the data provide a readily interpretable pattern of results. If the analysis is conducted in terms of the number of test sessions to reach criterion performance, the most rapid learning is found for the 30D-1T group; the learning of the other three groups was slower and similar for all groups. Thus, the longer postoperative recovery interval (30D-IT as compared to 5D-1T) still facilitated performance. The effects of experience, however, are more difficult to interpret. On the one hand, experience did have some effect; during the seventh block of 5 days, the choice accuracy of the two groups that began testing after a 5-day postoperative recovery period (and thus had received 25 days of testing prior to that block) was higher than the choice accuracy of the two groups that began testing after a 30-day postoperative recovery period (and thus had received no testing prior to that block). On the other hand, neither of the groups tested twice a day (5D-2T and 30D-2T) reached criterion performance more rapidly than the comparable group tested once a day (5D-IT and 30D-IT, respectively). These analyses support the same qualitative interpretations as before--both the length of the postoperative recovery interval and the amount of postoperative test experience influenced the rate of recoverymbut suggest a different quantitative one with respect to test experiencensome test experience was better than no test experience, but more test experience (two test sessions each day) did not produce a further enhancement. Further parametric studies should be able to delineate more accurately the relationship between the amount of test experience and the rate of behavioral recovery. Recovery of function following partial damage to the hippocampal formation has been demonstrated by previous research. For example, performance of a continuous alternation task was disrupted immediately after dorsal hippocampal lesions, but returned to normal after 60 days of testing (Dawson, Conrad, & Lynch, 1973; Isseroff, Leveton, Freeman, Lewis, & Stein, 1976). The ability to solve Hebb-Williams maze prob-
RECOVERY FOLLOWING HIPPOCAMPAL CA3 LESIONS
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lems was impaired immediately after damage to the perforant path, but recovered after 35 days of testing (Myhrer, 1975). Alternation behavior in a Y maze was disrupted by unilateral entorhinal cortex lesions, but the alternation behavior returned to normal within 10 days, a time course similar to that found for reinnervation of the dentate gyrus by the contralateral entorhinal area (Loesche & Steward, 1977; Scheff & Cotman, 1977). Thus, recovery of function is not unusual following partial hippocampal lesions, and the present experiment provides information about the variables that can influence the rate at which this recovery occurs. A variety of neural mechanisms may be responsible for the behavioral recovery observed here. For example, the mechanism may be a reorganization within the hippocampus to repair the circuit damaged by the removal of the CA3 cells. The hippocampus shows a high degree of anatomical plasticity following partial lesions (see Milner & Loy, 1980, for a recent summary), and sprouting may compensate for the absence of the CA3 cells. Alternatively, the behavioral recovery might be due to the elimination of a transient dysfunction, or diaschesis (Rosner, 1974). Activation of specific neurochemical systems may also enhance recovery (Luria, Naydim, Tsvetkova, & Vinarskaya, 1969; Braun, Meyer, & Meyer, 1966). Although the neurological mechanisms underlying the behavioral recovery following CA3 damage were not addressed by this experiment, the fact that the time course of this recovery can be readily manipulated should make it a useful model for the study of recovery of function. REFERENCES Braun, J. J. (1966). The neocortex and visual placing in rats. Brain Research, 1,381-394. Braun, J. J., Meyer, P. M., & Meyer, D. R, (1966). Sparing of a brightness habit in rats following visual decortication. Journal of Comparative and Physiological Psychology, 61, 79-82. Dawson, R. G., Conrad, L., & Lynch, G. (1973). Single and two-stage hippocampal lesions: A similar syndrome. Experimental Neurology, 40, 263-277. Finger, S. (Ed.). (1978). Recovepsyfrom Brain Damage. New York: Plenum. Handelmann, G. E., & Olton, D. S. (1981). Spatial memory following damage to the hippocampal CA3 pyramidal cells with kainic acid: Impairment and recovery with preoperative training. Brain Research, 21, 41-58. Isseroff, A., Leveton, L., Freeman, G., Lewis, M. E., & Stein, D. G. (1976). The limits of behavioral recovery from serial lesions of the hippocampus. Experimental Neurology, 53, 339-354. K6nig, J. F. R., & Klippel, R. A. (1967). The Rat Brain. Huntington, N.Y.: R. E. Kreiger. Loesche, J., & Steward, O. (1977). Behavioral correlates of denervation and reinnervation of the hippocampal formation in the rat: Recovery of alternation performance following unilateral entorhinal cortex lesions. Brain Research Bulletin, 2, 31-39. Luria, A. R., Naydin, V. L., Tsvetkova, L. S., & Vinarskaya, E. N. (1969). Restoration of higher cortical function following local brain damage. In P. J. Vinkin & G. W. Brugh (Eds.). Handbook of Clinical Neurology. New York: Wiley. Meyer, D. R., Isaac, W., & Maher, B. (1958). The role of stimulation in spontaneous reorganization of visual habits. Journal of Comparative and Physiological Psychology, 51, 5-16.
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Milner, T., & Loy, R. (1980). A delayed sprouting response to partial hippocampal deafferentation: Time course of sympathetic ingrowth following fimbrial lesions. Brain Research, 197, 339-379. Myhrer, T. (1975). Maze performance in rats with hippocampal perforant path lesions: Some aspects of functional recovery. Physiology and Behavior, 15, 433-437. Rosner, B. S. (1974). Recovery of function and localization of function in historical perspective. In D. G. Stein, J. J. Rosen, & N. Butters (Eds.), Plasticity and Recovery of Function in the Nervous System. New York: Academic Press. Scheff, S. W., & Cotman', C. W. (1977). Recovery of spontaneous alternation following lesions of the entorhinal cortex in adult rats: Possible correlation to axon sprouting. Behavioral Biology, 21, 286-293. Stein, D. G., Rosen, J. J., & Butters, N. (Eds.) (1974). Plasticity and Recovery of Function in the Nervous System. New York: Academic Press. Stewart, J. W., & Ades, H. W. (1951). The time factor in reintegration of a learned habit after temporal lobe lesions in the monkey (Macaca mulatta). Journal of Comparative and Physiological Psychology, 44, 479-486. Will, B. E., & Rosenzweig, M. R. (1976). Effets de l'environnement sur la r6cup6ration fonctionelle apr~s 16sions c6r6brales chez les rats adultes. Biological Behavior, 1, 5-16.