Nimodipine enhances new learning after hippocampal damage

EXPERIMENTAL NEUROLOGY 1 0 9 , 279-285 (1990)

Nimodipine Enhances New Learning after Hippocampal Damage S T A N L E Y F I N G E R , L E O N A R D G R E E N , M I C H A E L E . T A R N O F F , K E I T H D. M O R T M A N , AND A N D E R S A N D E R S E N

Department of Psychology, Washington University, St. Louis, Missouri 63130

R a t s w e r e t r a i n e d to l e v e r p r e s s a n d t h e n w e r e g i v e n e i t h e r b i l a t e r a l l e s i o n s o f t h e h i p p o c a m p u s or c o n t r o l operations. Half of the rats in each group received oral nimodipine, a calcium entry blocker, while the remaining rats received vehicle, over a 14-day period that began the evening of surgery. The rats were studied on a DRL 20-s schedule of reinforcement (differential reinforcement of low rates of responding) that required t h e m to w i t h h o l d a r e s p o n s e f o r at l e a s t 2 0 s a f t e r t h e i r l a s t l e v e r p r e s s i n o r d e r to e a r n a r e w a r d . R a t s w i t h lesions that did not receive the drug performed poorly on the DRL 20-s schedule. In contrast, rats sustaining the same hippocampal lesions but given the drug s h o w e d s c o r e s t h a t w e r e v i r t u a l l y e q u i v a l e n t to t h o s e o f the sham-operated control animals. Similar trends were observed when the rats were then tested on a DRL 40-s schedule of reinforcement. These findings suggest t h a t n i m o d i p i n e m a y a t t e n u a t e t h e e f f e c t s o f a c u t e , focal brain lesions on new learning of even difficult behavi o r a l a n d c o g n i t i v e t a s k s . © 1990 AcademicP..... Inc.

INTRODUCTION Nimodipine (isopropyl- ( 2 - m e t h o x y e t h y l ) - 1,4-dihydro-2,6,-dimethyl-4- (3-nitrophenyl)-3,5-pyridinedicarboxylate) is a calcium ent r y blocker t h a t has cerebrovasodilatory and anti-ischemic properties at doses t h a t have little or no effect on peripheral circulation (26). Because it is especially pot e nt in dilating blood vessels in injured parts of the brain (9, 14, 32), and reduces neuronal calcium overloading which can result in increased cell death after injury or with disease (14, 27, 28), nimodipine may be useful for treating the symptoms of some central nervous system disorders. Data directly s u p p o r t i n g this contention have come from l a b o r a t o r y animal studies a nd h u m a n clinical trials of focal and global ischemia (14, 29, 30) and subarachnoid hemorrhage (1, 4). T h e findings in these behavioral studies are consistent with those showing differences in pathoanatomical correlates, such as reduced infarct size, in animals given nim'odipine soon after arterial occlusion (10, 20). A r ecen t l a b o r a t o r y animal study with nimodipine also suggests th at it may be helpful in reducing symp279

tomatology after acute brain lesions. LeVere et al. (18) dem onst rat ed t hat rats with large posterior cortical lesions relearned a preoperatively acquired brightness discrimination with fewer errors when treated with nimodipine 2 weeks after surgery. Nimodipine has been observed to improve associative learning as well as a host of o t h e r behaviors (e.g., grooming, locomotion, exploration) in aged animals (6, 11, 24, 25). It is possible t h a t age-related declines in learning and memory may be due, at least in part, to changes in the responsivity of hippocampal neurons to calcium. T h e integrity of this part of the brain has been correlated with some types of learning (19), and hippocampal cells appear to be very sensitive to changes in calcium homeostasis associated with aging and dementias (15, 16, 17). Some hippocampal neurons also appear to be especially vulnerable to the effects of acute ischemia (28). Significantly, it was shown in a recent study t hat nimodipine can help protect CA1 hippocampal neurons after arterial occlusion (20). T h e present experiment was conducted to learn more about the effects of nimodipine on sparing and recovery of function in subjects with acute, focal brain damage. Unlike previous studies, it was designed to assess the behavioral effects of bilateral lesions of the hippocampus itself. Because hippocampal lesions are thought to affect memory for time estimation and the ability to withhold responses, performance on a DRL schedule of reinforcement (differential reinforcement of low rates of responding) was assessed. On a D RL operant task, a response is reinforced only if it occurs after a minimum period of time has elapsed since the previous response. Pressing too soon resets the clock and consequently furt her delays the time before a reinforcer can be obtained. For example, on a DRL 20-s schedule, at least 20 s must elapse between lever presses for a reinforcer to be delivered. Theoretically, if an animal has impaired temporal memory, or cannot inhibit a tendency to respond, performance will be poor on D RL tasks. Evidence t h a t damage to the hippocampus can in fact lead to deficits on D RL tasks comes from a num b e r of lesion studies, most using 20 s or shorter intervals (3, 5, 12, 21-23; for delays up to 60 s, see 8). In the present investigation, animals given nimodipine or just vehicle after bilateral hippocampal lesions were tested on both 0014-4886/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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D R L 20-s and 40-s tasks to learn more a b o u t the p o t e n tial of the calcium e n t r y blocker, nimodipine, to a t t e n u ate deficits on new learning tasks. METHODS

Subjects F e m a l e albino rats (Charles River CD strain) weighing a p p r o x i m a t e l y 205 g at the s t a r t of t h e e x p e r i m e n t served as subjects. T h e animals were h o u s e d in groups of t h r e e in clear plastic cages and were exposed to a 12-h l i g h t / d a r k cycle. R a t chow was available ad libitum, b u t w a t e r was r e s t r i c t e d to 1 h / d a y a n d was available following each daily session.

Apparatus T h e rats were studied in t h r e e identical C o u l b o u r n Instruments operant chambers measuring approxim a t e l y 30 cm long by 25 cm wide by 29 cm high. T h e c h a m b e r s were enclosed in light- a n d s o u n d - a t t e n u a t i n g boxes. A 3.5-cm-wide m e t a l response lever e x t e n d e d 2.0 cm f r o m the f r o n t p a n e l a n d was located 2.4 cm f r o m the left wall and 2.5 c m f r o m the floor. P r e s s e s on the lever p r o d u c e d a feedback click by o p e r a t i n g a 28-V relay. A liquid d i p p e r was l o c a t e d on t h e c e n t e r o f t h e f r o n t panel, 5.0 cm to the right of the lever a n d 1.0 cm from the floor. R e i n f o r c e m e n t consisted of 5 s of access to a dipper c o n t a i n i n g 0.1 cc of saccharin-flavored w a t e r (1 g saccharin per liter of water). T h e c h a m b e r was illumin a t e d by a 7-W houselight h o u s e d in a m e t a l case t h a t d e f l e c t e d t h e light d o w n w a r d . T h e light was e x t i n guished during r e i n f o r c e m e n t , at which time the dipper a r e a was i l l u m i n a t e d . V e n t i l a t i o n a n d m a s k i n g noise were p r o v i d e d by an e x h a u s t fan. Scheduling and recording were c o n t r o l l e d b y e l e c t r o m e c h a n i c a l e q u i p m e n t located in an a d j a c e n t room.

Preoperative Training E a c h rat was t r a i n e d to press the lever for saccharin solution. T r a i n i n g o n t h e c o n t i n u o u s r e i n f o r c e m e n t schedule c o n t i n u e d until c o n s i s t e n t lever pressing occurred, usually within 2-4 days.

Surgery T h e rats were o p e r a t e d u p o n within 7 days a f t e r the c o m p l e t i o n of lever press training. T h e y were food deprived for a p p r o x i m a t e l y 18 h before surgery a n d were a n e s t h e t i z e d with sodium p e n t o b a r b i t a l (55 mg/kg, ip). T h e fur on the top of the h e a d was shaved and the rats were placed in a stereotaxic a p p a r a t u s with the h e a d held horizontally. After a midline incision was m a d e to expose the skull, a small hole was drilled above the anterior h i p p o c a m p u s on each side of t h e brain in those rats t h a t were to receive lesions. A 0 . 2 - m m - d i a m e t e r stain-

less steel electrode was positioned 3.0 m m p o s t e r i o r to b r e g m a a n d +2.0 m m f r o m t h e m i d l i n e surface. T h e electrode was lowered t h r o u g h holes drilled in the skull to a d e p t h of 3.2 m m below the dura. A 1.5 mA dc c u r r e n t was p a s s e d for 45 s on each side of the brain with t h e electrode stripped of insulation 0.5 m m f r o m the tip. T h e ear served as the cathode, a n d anodal c u r r e n t was p a s s e d t h r o u g h the electrode. After the electrode was retracted, G e l f o a m was fitted into the holes in the skull a n d t h e skin was s u t u r e d with silk thread. R a t s r a n d o m l y a s s i g n e d t o t h e c o n t r o l g r o u p s received the same a n e s t h e t i c a n d h a d the fascia cut without holes being drilled in the skull. T h e i r w o u n d s were s u t u r e d with silk t h r e a d a n d t h e y were t r e a t e d identically to the animals t h a t received electrolytic lesions of the a n t e r i o r hippocampus. All animals were given 7 days to recover from the surgery before D R L testing began.

Drug Administration R a t s in the lesion and control groups were r a n d o m l y divided into drug a n d vehicle conditions after completion of surgery. Since nimodipine is insoluble, it was a d m i n i s t e r e d as a suspension via intragastric i n t u b a t i o n (7). T h e drug was s u s p e n d e d (15 mg/kg) in 7.5% aqueous methylcellulose solution a n d a plastic n e o n a t a l feeding t u b e was used for intragastric injection (size 4FG; P o r t e x , H y t h e , K e n t , UK). Once the rats were used to being handled, t h e y readily a c c e p t e d the feeding t u b e a n d i n t u b a t i o n t o o k place rapidly. T h e rats in the control group received equivalent a m o u n t s of methylcellulose (approxim a t e l y 3.0 ml) w i t h o u t drug. B o t h groups of rats were i n t u b a t e d for 14 days, the final 7 of which o v e r l a p p e d with the first 7 days of D R L testing. I n t u b a t i o n began the night the surgeries were p e r f o r m e d . T h e r e a f t e r , i n t u b a t i o n t o o k place early in the m o r n i n g a n d p r e c e d e d D R L testing.

DRL Testing W h e n t h e rats were r e t u r n e d to the o p e r a n t c h a m b e r s 7 days after surgery, t h e y were placed directly on the D R L 20-s s c h e d u l e of r e i n f o r c e m e n t . L e v e r p r e s s e s were now r e i n f o r c e d only if at least 20 s h a d elapsed since t h e previous lever press. A lever press during the 20-s period reset the timer. T h e rats r e m a i n e d on the D R L 20-s schedule for 30 days after which the schedule was c h a n g e d to D R L 40-s for a n o t h e r 30 days. T h e rats were studied 7 days a week, with each session ending 40 min after the first lever press. T o t a l n u m b e r of lever presses e m i t t e d a n d r e i n f o r c e m e n t s o b t a i n e d were r e c o r d e d . R e s p o n s e r a t e s ( r e s p o n s e s / m i n ) , responses per r e i n f o r c e m e n t , a n d efficiency ratios (number of r e i n f o r c e m e n t s o b t a i n e d divided b y the n u m b e r of r e i n f o r c e m e n t s possible) were c o m p u t e d for each rat on

NIMODIPINE ENHANCES NEW LEARNING each day of testing. Statistical analyses were based on the mean scores for each rat on each block of 3 days.

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addition, total capillary length in the spared CA1 region and in the triangular septal nucleus anterior to the lesion also did not differ between the two lesion groups.

Histology

T h e rats were sacrificed with sodium pentobarbital (100 mg/kg, ip) upon completion of testing. T h e y were perfused with 0.9% saline followed by a 50:50 mixture of 10% formalin and India ink injected through the aorta. T h e India ink was used to stain blood vessels in order to see if there would be more blood vessels in the septum and remaining hippocampus in the rats t r e a t e d with nimodipine (7). T h e brains were removed and put on a freezing microtome after fixation in formalin solution. Sections were cut at 50 #m and every fifth section was saved and stained with cresyl violet. T h e sections were studied with a light microscope and drawings were made of the lesions. Blood vessels in the triangular septal nucleus, anterior to the lesion, and the hilus of the dentate fascia, 0.2 mm posterior to the lesion, were examined by projecting slides onto drawing paper for tracing. Stained blood vessels within a fixed area were measured and lengths were totaled for statistical analyses. After eliminating two rats with poor lesions, t h e r e were 30 acceptable subjects. Group numbers were as follows: (a) H i p p o c a m p a l Lesion × Drug (HI-Drug), N = 10; (b) Hippocampal Lesion X Vehicle (HI-No Drug), N = 10; (c) Sham Operation X Drug, N = 5; and (d) Sham Operation X Vehicle, N = 5. RESULTS Hippocampal Lesions

No gross anatomical differences were apparent between the lesions of the rats t h a t received nimodipine and those t h a t received vehicle alone. Figure 1 shows the maximal cross-sectional extent of the lesions of nine rats chosen at random from the two lesion conditions. In both groups, the typical lesion covered the entire dorsal region of the CA1 and CA4 fields, severely damaged the dentate gyrus, and involved the fimbria. In about half of the animals in each group, the lesion also included the CA2 and CA3 fields. Rostrally, the lesions extended to the hippocampus proper where they transected the ventral hippocampal commissure but did not directly damage the septum. Caudally, the lesions extended to the point where the hippocampus be nt vertically. Damage to th e corpus callosum was seen only around the electrode tracts, and in some instances there was some damage above the corpus callosum associated with the electrode tracts, but this occured in both lesion groups. T h e r e were no differences b e t w e e n t he two lesion groups in n u mb er of neurons in samples of the hilus of the dentate fascia 0.2 m m posterior to the lesions. In

D R L 20 s

T h e scores of the two sham-operated groups (treated with nimodipine or just vehicle) overlapped extensively. Repeated-measure analyses of variance (ANOVAs) for the three dependent measures failed to reveal any main effects IF's(1, 8) = 1.77, 0.35, and 0.95; P ' s > 0.05, ns] or drug condition by blocks interactions [F's(9, 72) = 0.72, 0.55, and 0.61; P's > 0.05, ns] between the two shamoperated groups. For this reason, these two groups were combined (Sham) for further analyses and pictorial representation of the results. T h e p e r f o r m a n c e s of t he t h r e e groups (Sham, HIDrug, H I-N o Drug) on the three dependent measures (response rate, responses/reinforcement, efficiency) are shown in Fig. 2. As can be seen, the scores of the HIDrug group were virtually equivalent to those of th e Sham group. In contrast, the HI-No Drug group had higher rates of responding, more responses per reinforcement, and lower efficiency scores t h a n the other two groups. T h e results from 3 X 10 (Groups X Blocks) repeatedmeasure ANOVAs on response rate, responses per reinforcement, and efficiency confirmed these observations. For each dependent variable, the difference among the three groups was significant at the 0.05 level or better IF's(2,27) = 3.4, 4.8, and 4.6]; the blocks effect was significant at the 0.001 level [F's(9,243) = 40.2, 29.7, and 33.0]; and the interaction of groups by blocks was significant at the 0.01 level [F's(18,243) = 2.97, 2.56, and 2.14]. Additional r e p e a t e d m e a s u r e ANOVAs were conduct ed to d e t e r m i n e t he sources of the groups and groups by blocks differences. In all cases, there were no main effects between the Sham and the HI-Drug groups [F's(1,18) = 1.33 or lower; P ' s > 0.05, ns], but there were significant differences between the Sham and the HINo Drug groups [F's(1,18) = 4.80 or higher; P ' s < 0.05]. T h e difference between the two lesion groups was significant on the responses per r e i n f o r c e m e n t IF(l,18) = 4.60; P < 0.05] and efficiency [F(1.18) = 6.28; P < 0.025] measures, but not for response rate [F(1,18) = 2.67; P > 0.05, ns]. For response rate, however, there was a significant interaction [F(18,162) = 2.57; P < 0.01] bet w een groups and blocks, with the H I - D r u g group performing better t han the HI-No Drug group on most blocks. D R L 40 s

On t he D R L 40-s schedule of r e i n f o r c e m e n t , repeated-measure ANOVAs on the three dependent variables (response rate, responses per reinforcement, efficiency) for the two sham-operated groups again failed to

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reveal a n y significant m a i n effects of g r o u p [F's(1,8) = 1.10, 0.94, 0.84; P ' s > 0.05, ns]. T h e only significant i n t e r a c t i o n of g r o u p s b y blocks was on only one b l o c k of trials on one of t h e m e a s u r e s . C o n s e q u e n t l y , t h e two s h a m - o p e r a t e d g r o u p s a g a i n were c o m b i n e d ( S h a m ) . T h e results for e a c h m e a s u r e of p e r f o r m a n c e on t h e D R L 40-s schedule are p r e s e n t e d in Fig. 3. T h e shift to t h e D R L 40-s t a s k led to a m a r k e d a n d s u s t a i n e d d r o p in r e s p o n s e rate. I n c o n t r a s t , t h e r e s p o n s e s p e r reinforcem e n t a n d efficiency m e a s u r e s s h o w e d a n initial decline followed b y a g r a d u a l r e t u r n to levels m o r e c o m p a r a b l e to t h o s e a c h i e v e d on t h e D R L 20-s schedule. T h e m e a n scores of t h e H I - N o D r u g g r o u p again did n o t o v e r l a p t h o s e of t h e S h a m or H I - D r u g g r o u p s on r e s p o n s e s p e r r e i n f o r c e m e n t or efficiency b u t t h e r e w a s n o t a b l e overlap in r e s p o n s e r a t e on t h e last four blocks of sessions b e t w e e n t h e H I - N o D r u g a n d t h e S h a m groups. A N O V A s c o m p a r i n g t h e t h r e e g r o u p s on t h e t h r e e dependent measures revealed a significant difference a m o n g g r o u p s on r e s p o n s e s p e r r e i n f o r c e m e n t [F(2,27) = 3.95; P < 0.05]. S u b s e q u e n t A N O V A s r e v e a l e d t h a t this was due to a significant difference b e t w e e n t h e two lesion g r o u p s [F(1,18) = 5.92; P < 0.05] with t h e differ-

ence b e t w e e n t h e S h a m vs t h e H I - N o D r u g groups only a p p r o a c h i n g significance [F(1,18) = 3.83; P < 0.066, ns]. As e x p e c t e d , t h e r e w a s a s i g n i f i c a n t m a i n effect o f blocks on this m e a s u r e [F(9,243) = 11.68; P < 0.001], b u t t h e r e was no groups b y blocks i n t e r a c t i o n [F(18,243) = 1.30; P > 0.05, ns]. T h e g r o u p scores s h o w e d t r e n d s t o w a r d statistical significance on t h e efficiency a n d r e s p o n s e r a t e m e a s u r e s . This was most notable when the HI-Drug and HI-No D r u g g r o u p s were c o m p a r e d [F's(1,18) = 3.28 a n d 3.12; P ' s < 0.09, ns]. T h e blocks effect a g a i n was significant [F's(9,243) = 21.69 a n d 7.30; P ' s < 0.001]. DISCUSSION T h e p u r p o s e of t h e p r e s e n t s t u d y was to d e t e r m i n e w h e t h e r t h e c a l c i u m e n t r y blocker, n i m o d i p i n e , would be able to e n h a n c e b e h a v i o r a l s p a r i n g a n d / o r r e c o v e r y in r a t s s u s t a i n i n g h i p p o c a m p a l lesions. D R L schedule p e r f o r m a n c e w a s c h o s e n b e c a u s e this t a s k requires t h e subject to m a k e e s t i m a t e s of t h e p a s s a g e of time, as well as to w i t h h o l d r e s p o n s e s , a n d b e c a u s e earlier studies have shown that animals with hippocampal lesions

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40-s measures. (The scores a p p r o a c h e d statistical signifi c a n c e on t h e r e m a i n i n g two D R L 40-s m e a s u r e s . ) T h e s e d a t a show t h a t n i m o d i p i n e can e n h a n c e perform a n c e after h i p p o c a m p a l lesions, even on t a s k s as challenging as high D R L schedules of reinforcement. T h e r e is no published work on new learning in animals with acute, focal b r a i n lesions t r e a t e d with nimodipine with w h i c h t h e s e findings c a n be c o m p a r e d directly. T h e closest is a recent s t u d y by LeVere e t al. (18) in w h i c h r a t s w i t h large p o s t e r i o r c o r t e x lesions rel e a r n e d a b r i g h t n e s s discrimination with fewer errors w h e n t r e a t e d with at least 1.0-3.0 m g / k g oral nimodipine t h a n w h e n given lower doses of the drug or given vehicle. In t h a t study, however, drug a d m i n i s t r a t i o n was n o t s t a r t e d until 2 weeks after ablation, a n d the animals were t e s t e d for reacquisition of the t a s k l e a r n e d prior to cortical damage.

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There have been related experiments demonstrating positive effects of nimodipine on performance of aged animals where hippocampal integrity might be questioned. These studies have reported beneficial effects of nimodipine on classically conditioned eye-blink responses in aged rabbits (6), motor abilities in rats (11, 24, 25), and water maze and passive avoidance learning in rats (25). In addition, a few experiments have been conducted on animals with widespread damage produced by ischemia (14, 29, 30). Although these studies have been concerned with survival and general neurological status and not with learning, for the most part, the results found with nimodipine also have been encouraging. Two actions of nimodipine might account for its effectiveness in this and related studies. First, nimodipine can dilate remaining cerebral blood vessels and prevent vasospasm after brain injury (9, 14, 32). And second, nimodipine, which can pass the blood-brain barrier (33) and bind with specific dihydropyridine receptors in the brain (2, 14), has the ability to prevent excessive influx of calcium by affected neurons in directly or indirectly damaged areas (14, 27, 28). These actions, which are not mutually exclusive, could reduce the secondary loss of cells in regions outside the area of primary damage. Data showing that hippocampal neurons are very sensitive to changes in calcium homeostasis (15-17), and the observation that nimodipine can protect CA1 hippocampal neurons after arterial occlusion (20), are especially notable in the context of the present findings on recovery from subtotal hippocampal lesions. Attention should also be given to the possibility that nimodipine administered soon after neural insult might improve the functional capabilities of neurons spared by the lesions. Transient secondary effects, such as neural shock, might suppress spared hippocampus and/ or neighboring or related areas that may mediate recovery or permit behavioral compensation. The observation that the HI-No Drug rats showed improvement with continued testing/time, and that cell counts made in a sample of the hilus of the dentate fascia showed no apparent differences between the two lesion groups, would be consistent with this explanation. Our results extend previous findings concerning positive effects of nimodipine (e.g., 6, 10, 18, 24, 29, 30) by demonstrating facilitation of new learning in animals with acute, focal brain lesions. While much remains to be learned about dihydropyridines, the present results suggest that in addition to its ability to meliorate some of the symptoms of advanced age and diffuse damage to the nervous system (6, 10, 18, 24), nimodipine also has the potential to attenuate higher-order behavioral and cognitive deficits following focal brain damage.

ACKNOWLEDGMENT

This study was supported in part by a stipend from the Pharmaceutical Division of Miles Laboratories, Inc., a subsidiary of Bayer AG.

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