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An age-related spatial learning deficit: Choline uptake distinguishes “impaired” and “unimpaired” rats
Neurobiologyof Aging, Vol. 9, pp. 363--369.©PergamonPress plc, 1988.Printed in the U.S.A.
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An Age-Related Spatial Learning Deficit: Choline Uptake Distinguishes "Impaired" and "Unimpaired" Rats MICHELA GALLAGHER'
AND MARY ANN PELLEYMOUNTER
Department o f Psychology, University o f North Carolina at Chapel Hill, Chapel Hill, N C 27514 R e c e i v e d 23 S e p t e m b e r 1987 GALLAGHER, M. AND M. A. PELLEYMOUNTER. An age-related spatial learning deficit: Choline uptake distinguishes "impaired" and "unimpaired" rats. NEUROBIOL AGING 9(4) 363-369, 1988.--A functional decline in the hippocampal formation may underlie the emergence of spatial learning deficits in aged rodents. In this study, sodiumdependent high-affinity choline uptake (HACU) was used to monitor hippocampal function in response to training on a spatial task. The subjects were male Long-Evans rats at either 4 months or 22-24 months of age. Animals were trained to locate a camouflaged escape platform in the Morris water maze. Each animal that received place training had a yoked counterpart that was exposed to swimming in the maze but was not required to learn the task. Animals, both young and aged, were sacrificed after attaining a criterion performance. Relative to animals in the yoked condition, place training significantly reduced HACU in both the young rats and in a subpopulation of the aged animals that learned the task rapidly. In contrast, for aged rats that had an impaired rate of acquisition, no effect of place training on HACU was observed. These results provide evidence for a relationship between the behavioral capacities of aged rats and changes in the status of hippocampal function. Spatial learning
Cholinergic
Hippocampus
High-affinity choline uptake
AGED rodents are commonly impaired on spatial learning tasks ([2,6] for recent reviews). There is also substantial agreement that the spatial learning capacity of young adult rats depends upon the integrity of the hippocampal formation. Damage to the hippocampal formation or treatments that alter hippocampal function disrupt performance on a wide variety of spatial tasks including those most often used to assess age-related deficits [11-14, 19]. Thus, changes in the aged hippocampal formation could conceivably provide a neurobiological substrate for age-related spatial learning deficits. Previous research in our laboratory was conducted to monitor hippocampal function during spatial learning in order to evaluate the relationship between the behavioral capacities of aged rats and changes in the status of this circuitry. This work demonstrated that a selective marker for the function of hippocampal cholinergic neurons, i.e., sodium-dependent high-affinity choline uptake (HACU), is altered in young rats as a specific consequence of spatial learning in the Morris water maze: when young adult rats are trained to locate a camouflaged escape platform in the water maze by using a configuration of spatial information, a change in hippocampal HACU is observed [4]. Specifically, young rats were trained until proficient behavioral performance on the task was demonstrated and were then sacrificed 15 min after the final training session. The velocity (Vma~) of
hippocampal HACU was significantly reduced in the animals that learned the spatial task relative to either experimentally naive animals or rats that were yoked to the trained animals for the amount of time spent swimming in the maze but were not required to learn the spatial task. A decrease in HACU is typically associated with treatments that reduce cholinergic activity [17]. Thus, this result indicated that the activity of the septo-hippocampal cholinergic system was decreased in placed-trained animals at the time of sacrifice as a consequence of training, but did not necessarily imply that cholinergic activity was reduced during the training session itself. Further work indicated that the appearance of a training-induced change in hippocampal HACU in young rats depends on the phase of acquisition as it is not observed in animals that are overtrained and does not occur in animals that are sacrificed early in training prior to acquisition of the task [4]. Furthermore, the response of the cholinergic system to training as reflected in HACU was diminished in aged rats [4]. In an initial experiment, groups of young and aged rats were sacrificed after a comparable amount of training: at the time of sacrifice a deficit in performance was evident in the aged group. The experiment showed that training in the water maze task failed to alter hippocampal H A C U in the aged subjects. This result indicated that a change in the regulation of septo-hippocampal cholinergic activity during train-
~Requests for reprints should be addressed to Michela GaUagher, Ph.D., Department of Pyschology, Davie Hall 013A, University of North Carolina, Chapel Hill, NC 27514.
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ing on a spatial task coincides with the behavioral impairment of aged rats. The distinctive effect of place training on hippocampal H A C U in young and aged rats is especially noteworthy because in experimentally naive male LongEvans rats, kinetic parameters for H A C U in hippocampus and cortex are not significantly altered in aged subjects when compared to young adult rats [4,22]. To the extent that measurement of HACU reflects the complement of ACh innervation and the activity of ACh neurons in their basal state, these results indicate no apparent change in the status of the basal forebrain ACh system during aging. An agerelated difference in the dynamic function of this system was, however, revealed by the results obtained when animals are trained on a spatial learning task. The present experiment was designed to further examine this phenomenon by addressing several features of the performance of aged animals in the water maze task. First, aged animals have a relatively mild/moderate deficit on this task. For example, a previous study reported that with extended training the performance of aged animals (23-28 months) will eventually match that of younger rats (6 months and 12 months) [16]. Thus, the study was designed to examine whether a training-induced change in hippocampal H A C U will be characteristic of aged animals when acquisition of the task, albeit delayed, is ultimately achieved. In order to address this question the performance of young and aged rats was matched by training to a criterion performance prior to sacrifice and determination of HACU. In addition, when place training is conducted using aged animals at 22-24 months of age, a substantial subpopulation of these subjects learn the task within the range of training trials required by younger rats (unpublished observations). A second major goal of the present study, therefore, was to determine whether the H A C U response to training will distinguish between impaired and unimpaired aged rats at the same chronological age. This was accomplished by using aged subjects that were 22-24 months at the time of the experiment. METHOD
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FIG. I. A schematic representation of the maze that illustrates the analysis of the interpolated probe trials that were used to assess criterion performance. Starting positions around the perimeter of the maze are designated as (N)orth, (S)outh, (E)ast, and (W)est. The amount of time spent in each quadrant of the maze was recorded. The four possible locations of the escape platform are shown positioned in the center of each quadrant. Annulus crossing were determined by the number of times an animal traversed each of those locations. merged 1 cm below the water surface in the center of one of the four quadrants in the maze. The maze was surrounded by white muslin curtains with black felt patterns affixed to provide a configuration of spatial cues. A TV camera with a wide angle lens was suspended above the center of the maze. Probe trials were videotaped with a JVC VHS videocassette recorder that was located immediately outside the door to the maze room. An RCA 12" monitor was located outside the curtained area to aid the experimenter in locating the animal's position in the maze. Data obtained on an initial habituation trial was analyzed with a video tracking system (HVS Image Analyzing VP-112) and IBM PC computer using software developed for the water maze by San Diego Instruments, Inc.
Subjects
Procedures
Young (4-5 months) and aged (22-24 months) male Long-Evans rats were used. The aged animals (N=28) were obtained as pathogen-free retired breeders at 8-9 months of age from Charles River Laboratories. They were maintained in a separate room within the Psychology Department vivarium. Survival of these animals from the time of purchase to 23-24 months in this facility is approximately 80%. The 16 naive young animals used in this experiment were obtained as.pathogen-free rats (Charles River Laboratories) and were resident in the Psychology Department vivarium for one month prior to the experiment. Each animal was single housed with food and water available ad lib. The vivarium is maintained on a 12 hr light/dark cycle (lights on at 7 a.m.) and climate controlled at 25°C.
Prior to training each animal was habituated to handling and to the maze itself. Animals were handled 3 rain/day for one week. On the day immediately preceeding training, each animal was placed into the maze without the escape platform and allowed to swim for 90 sec. The animal was then returned to its home cage. During subsequent training all animals received three trials/day using a 60 sec intertrial interval. On each trial the animal was introduced into the maze from one of the four equally spaced positions around the perimeter of the tank. The starting location was varied with the provision that each starting location was used in a series a four trials. Animals in each age group were randomly assigned to either the place training or yoked conditions. Place-trained animals learned to locate the escape platform that was camoflaged and submerged below the surface of the water. For these animals the platform was located in the same position in the maze from trial to trial: its localization depended on the use of spatial information surrounding the maze. If an animal did not locate the escape platform within 120 sec on any training trial, the experimenter placed the animal onto the platform where it remained for 60 sec. F o r animals in the yoked training condition, the escape platform was not immediately available, but instead was placed in the maze after a period of time corresponding to the es-
Apparatus Behavioral testing was conducted using a water maze that consists of a circular galvanized steel tank measuring 1.83 m in diameter and 0.58 m in height. The interior surface of the tank is painted white. During testing the tank was filled to a depth of 35.5 cm tepid water ( 2 7+- I°C) that was clouded by the addition of 0.9 kg powdered milk. A white escape platform (10.2 cm in diameter and 34.5 cm in height) was used for place training. During place training this platform was sub-
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FIG. 2. The seconds (mean+SEM) spent in each quadrant of the maze during the first probe trial are shown in a. The number of annulus crossings (mean-+SEM) in each quadrant on the first probe trial are shown in b. Training=quadrant or annulus where the escape platform was located during training. Adj-R=quadrant or annulus located immediately to the right of the training quadrant. Adj-L=quadrant or annulus located immediately to the left of the traimng quadrant. Opp=quadrant or annulus located 180 degrees from the training quadrant. cape latency of a place-trained counterpart had elapsed. After the designated time swimming in the maze, the escape platform was introduced at a location that varied from trial to trial in a quasi-random fashion such that each of the four possible locations was sampled within a block of four training trials. When the escape platform was introduced, the experimenter place the yoked subject onto the platform where it remained for a 60 sec interval. In this way the amount of time that yoked subjects spent swimming in the maze over the entire course of training was matched to that of the placetrained animals. In contrast to the place-trained subjects, however, the spatial location of the platform for the animals in the yoked condition was not relevant to task performance. Every sixth trial throughout training consisted of a probe trial. During these probe trials each animal was placed in the maze for a 30 sec interval with no escalJe platform present. Each probe trial was videotaped and analyzed to assess the search pattern. Quadrant time was determined as the number of seconds that an animal spent in each of the four quadrants of the maze. A second measure, annulus crossings, refers to the number of times an animal traversed each of the four points where a platform could be positioned (see Fig. 1). A criterion performance was attained when a place-trained animal spent at least 10 seconds in the training quadrant and
crossed the annulus in the training quadrant at least twice during a probe trial (see schematic represented in Fig. 1). Each placed-trained animal along with its yoked control was sacrificed on the day after criterion was achieved. On the sacrifice day each animal received 3 additional trials and was sacrificed 15 min after the completion of this final session. Animals were sacrificed by decapitation and brains were rapidly removed and dissected over ice. The left hippocampus was weighed and homogenized in 20 vol. of ice-cold 0.32 M sucrose using a hand held teflon on glass homogenizer (8--10 strokes). After centrifugation of the homogenate at 100xg for 10 min at 2 degrees C, the supernatant was centrifuged at 18000xg for 20 rain to yield a crude synaptosomal/mitochodrial pellet (P2 fraction). The pellet was resuspended in a 24 vol. of cold 0.32 M sucrose, and 50/zl of this suspension (approximately 75/xg protein) was added to tubes containing 400/zl of ice-cold incubation buffer (final concentration: 126 mM NaC1, 4.75 mM KC1, 1.27 mM CaC12, 15.8 mM Na2HPO4, 1.42 mM MgCI2, and 2 mg/ml dextrose; pH=7.4) to which 50/zl of all-choline (final specific activity: 4.2 Ci/mM, New England Nuclear) was added at a final concentration of 0.4/~M. This concentration approximates the Km for H A C U in hippocampus as determined in previous work [4]. Sodium-dependent uptake was measured in paral-
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lel using incubation buffer in which NaCI had been replaced with LiCI and Na2HPO4 had been replaced with Tris phosphate. Tubes were then placed in a shaking water bath for 2.5 min at 37°C followed by cooling in an ice water bath. After the addition of 2 ml of ice cold sodium-free buffer, samples were f'dtered over glass fiber f'dters (Gelman, A/E). The filters were then rinsed twice with 2 ml of ice-cold buffer and counted in I0 ml of Scintiverse E (Fisher Scientific). HACU in all samples was measured in triplicate, and HACU was determined by subtracting sodium-independent uptake from total uptake. Protein was determined by the method of Lowry [10]. Rapid measurement of HACU in vitro with these methods can be used to determine the relative in vivo activity of cholinergic neurons prior to sacrifice. Typically, interventions which increase ACh neuronal activity are found to increase HACU, and, conversely HACU is decreased by treatments that inhibit ACh neurons [18]. RESULTS
Analysis of the behavioral data revealed an age difference in the acquisition of place learning. The young animals that received place training rapidly learned to locate the escape platform: at the first probe trial these subjects had a significant spatial bias for the appropriate training quadrant and for the precise former location of the escape platform. In contrast, at this relatively early point in training a spatial bias was not evident in the aged group (Fig. 2). Overall analysis of the quadrant time data on the first probe trial was conducted using a three factor (Age x Quadrant × Training Condition) mixed design analysis of variance. The analysis revealed a significant main effect for Quadrant, F(3,120) =5.86, p<0.001, and significant interactions for Age x Quadrant and for Age x Quadrant x Training Condition, F(3,120) =3.76, p<0.01 and F(3,120)=4.13, p<0.01, respectively. Further analysis of the Age x Quadrant interaction with F tests for simple effects indicated that the spatial bias for the training quadrant was limited to the young subjects (p<0.001). In contrast, no significant spatial bias was evident for the aged subjects. Further analysis indicated that the quadrant effect was significant only for animals that were place trained (,o<0.02) with no significant difference in the amount of time distributed across quadrants for the yoked subjects. Comparisons between the young and aged groups revealed that the young rats spent significantly more time in the training quadrant and significantly less time in the quadrant opposite to the location of the escape platform during training (p's<0.02). Analysis of the data on annulus crossings during the first probe trial yielded similar results: in the overall analysis significant main effects were obtained for Training Condition, F(1,41)=4.22, p<0.05, and Annulus Crossings, F(3,123)= 2.64, p<0.05, and for the Age x Annulus Crossing interaction, F(3,123) = 5.97, p < 0.001. Again the distribution of swimming across annuli was significantly different for the place training condition (p<0.002), but not for the yoked condition. The young subjects traversed the training annulus more frequently than the aged subjects (/7<0.005). On the first probe trial, 3 of the 8 subjects in the young group that received place training attained a criterion performance, the criterion was not met by any of the 14 aged subjects. It is important to note that the impaired performance of the aged rats early in place training does not appear to be due to an age difference in swimming ability upon initial exposure to the maze. A general estimate of swimming speed and proficiency is provided by the total distance traversed during
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a standard 90 sec swim in the maze on the day prior to the commencement of training. Statistical analysis (one-way ANOVA) showed that there was no significant difference between the age groups on this measure. The total path lengths for the young and aged groups that subsequently received place training were 26.4+--1.15 and 28.1 +_-0.73 meters, respectively. Compared to the young rats, the aged animals required more training to reach criterion. The mean blocks of five training trials to reach criterion for the young and aged groups were 1.9__-0.3 and 3.7---0.3, respectively, a difference that was statistically significant, F(I,20) = 15.85, p <0.001. As shown in Fig. 3, with extended training every aged animal attained criterion even though the majority of aged subjects fell outside the entire range of training required by the younger rats. When criterion was met, the performance of animals in the young and aged groups was well matched. The spatial bias for each age group based on performance during the criterion probe trial is shown in Fig. 4. Analysis of the data for quadrant time and annulus crossings revealed no significant main effects of Age and no significant interactions involving age as a factor. In contrast to the data from the first probe trial, at criterion both young and aged place-trained groups exhibited a comparable spatial bias for the training quadrant and training annulus. The overall analysis for quadrant time yielded a significant effect for Quadrant, F(3,120)=20.50, p<0.0001, and a significant Quadrant x Training Condition interaction, F(3,120)=20.96, p<0.0001. Subsequent analysis revealed a significant spatial bias in the place-trained subjects (/9<0.001) but not in the yoked control groups. In comparison with the yoked subjects, placetrained animals spent significantly more time in the training quadrant and significantly less time in the quadrant opposite to the one in which the escape platform was located during training 09's <0.0001). The overall analysis for annulus crossings revealed significant main effects for both Training Condition and Annulus Crossings, F(1,39)= 16.15, p<0.0005 and F(3,117)=31.63, p<0.0001, respectively, and a significant Training Condition x Annulus Crossing interaction, F(3,117)=21.13, p<0.0001. Analysis for simple effects indicated that significant spatial bias for annulus crossings was present in the place-trained animals (p<0.001) but not in the yoked subjects. Significantly more annulus crossings in the
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training quadrant occurred in the place-trained relative to the yoked subjects (p<0.001). In order to independently confirm that the young and aged subjects performed with equal proficiency prior to sacrifice, escape latencies for the three training trials that occurred on the day following criterion performance were compared for the young and aged place-trained groups. A two-way analysis of variance (Age x Trial) conducted on these data revealed no significant main effects and no significant interaction. The mean escape latencies on this final block of trials for the young and aged subjects were 17.7---5.0 and 14.2___2.0 see, respectively. The effects of training on H A C U were assessed when the aged animals were considered as two distinct subpopulations. A number of the aged subjects (n=6) attained criterion within the range of training trials required by the younger animals (see Fig. 3). A second subpopulation of aged subjects (n=8) required more training to reach criterion than was required by any animal in the younger group. The aged animals in these two subpopulations were designated as "unimpaired" and " i m p a i r e d . " A two-way analysis of variance for a mixed design was conducted on the H A C U data for the resulting three groups of place-trained animals and their
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FIG. 5. High-affinity choline uptake determined at a 0.04 tzM concentration of [aH]-choline in hippocampus. See text for description groups. *Differs from yoked control, p<0.02. **Differs from yoked control, p <0.001. yoked controls (see Fig. 5 for data). This analysis revealed a significant main effect for Training Condition, F(1,38)= 15.20, p < 0 . 0 0 0 5 , and a significant Group x Training Condition interaction, F(2,38)=6.40, p < 0 . 0 0 5 . Subsequent analyses indicated that H A C U was significantly lower in
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place-trained animals relative to the yoked controls for both the young subjects (p<0.02) and the unimpaired aged subjects (p<0.001) but no difference in H A C U as a function of training condition was found in the impaired aged animals. DISCUSSION In aggreement with other reports [13], the results of this experiment demonstrate that young rats exhibit rapid learning during place training. For these animals escape latencies declined early in training, and by the first probe trial the young rats had a spatial bias for the vicinity of the maze where the escape platform was located during training. In contrast, animals in the yoked condition did not develop a spatial bias in the maze but distributed their swimming more evenly among the quadrants and annuli. When young rats were sacrificed on the day following attainment of criterion performance, hippocampal H A C U was significantly reduced in the place-trained animals relative to their yoked controls. This result confirms our earlier observation that hippocampal H A C U is decreased as a consequence of place training, an effect that can be distinguished from the physical stress of swimming in the maze. In our previous research we found that H A C U determined in yoked control subjects did not differ from that of experimentally naive animals [4]. Thus, the amount of swimming in tepid water to which animals are subjected during place training was not sufficient to alter H A C U in hippocampus. The relation of learning to ACh function as reflected in H A C U is presently not well understood. Consistent with our results, a decrease in hippocampal HACU has been previously observed in young rats that were sacrificed after training on a brightness discrimination task [9]. Other studies, however, have indicated that training experiences can increase H A C U in young animals. For example, a recent study reported that training on any of a wide range of tasks (the radial arm maze, a T-maze, and cued nonmatch-to-sample task, or active avoidance training in a shuttle-box) elevated hippocampal H A C U relative to control subjects [21]. In another series of experiments, Raaijmakers [15] found that exposure to a more circumscribed set of training procedures elevated hippocampal HACU. In this study no effect of avoidance training in a shuttle box was found on hippocampal HACU. The discrepancy between this result and the results of Wenk et al. [21] could conceivably reflect strain differences in the effects of exposure to footshock on hippocampal H A C U [17]. Raaijmakers did observe elevated hippocampal H A C U after go/no-go avoidance discrimination training. However, the author notes that rats which performed well on no-go trials tended to exhibit less, or even no increase in hippocampal HACU compared to rats that performed poorly. Some greater uniformity of results is found across several studies in which training-induced elevations in hippocampal HACU have been observed following training on appetitive spatial learning tasks [15,21]. Although the decrease in H A C U that we observe after place-training in the water maze is in contrast with these reports, the consistent observation is that training on spatial tasks that require the integrity of hippocampal function modifies choline uptake in this system. The direction of change in cholinergic function may be better understood by further experiments explicitly designed to assess a number of variables that differ across studies, including motivational conditions, extent of training, the sacrifice interval in relation to the training sessions, the strain of animals used.
In this experiment, aged rats were impaired in place learning. Upon initial exposure to the maze, however, there was no evidence that the aged rats were deficient in their swimming ability. Nonetheless, the aged subjects were delayed in their aquisition of place learning as reflected by an age difference in performance on the initial probe trial and in the amount of training required to reach criterion by the young and aged groups. With extended training, however, all aged animals developed a significant spatial bias in the maze, and analysis of the escape latency data for the final block of training trials indicated that the young and aged subjects performed with equal proficiency. Thus, learning in the water maze does not exceed the behavioral capacity of these aged rats. Despite the fact that every aged animal in the placetraining condition achieved criterion, the majority of the aged subjects required more training than any of the subjects in the young group. An important finding in the present study was that the effect of training on hippocampal H A C U depended on the degree of behavioral impairment in the aged animals. An effect of place training on hippocampal H A C U that was similar to that found in younger animals was exhibited by a subpopulation of aged rats that achieved criterion within the range of training trials required by the young group. In contrast, no effect of training on HACU was observed in the hippocampus of " i m p a i r e d " aged rats. Thus the response of the septo-hippocampai cholinergic system to training as reflected in HACU provides a neurobiological index that distinguishes aged rats with preserved learning capacity from cohorts at the same chronological age that are clearly impaired relative to the performance of young adults. These results provide further support for the concept that agerelated changes in the function of hippocampal circuitry may underly the emergence of spatial learning deficits toward the end of the lifespan. It is noteworthy that the "'impaired" aged subjects did not exhibit an effect of training on hippocampal H A C U even though their behavioral performance indicated that they learned the task. This result might be taken to indicate that the effect of training on hippocampal H A C U is not necessary to support place learning. Indeed other research has indicated when young animals are place-trained in the water maze after muscarinic antagonist treatment, acquisition of the task is impaired but with sufficient training learning does eventually occur [20,23]. In one of these investigations it was sugeested, however, that different learning strategies may be used by normal young rats and rats treated with atropine [23]. Whereas young adult rats normally learn to swim rather directly to the escape platform from any starting location around the perimeter, atropine-treated animals appeared to use a variety a taxon strategies to locate the platform. For example, animals may swim an appropriate fixed distance from the wall and thereby traverse the platform's location. Alternatively, the animals may locate the platform in relation to some fixed cue surrounding the maze and thereby approach the platform on the same trajectory from trial to trial. Interestingly, recent analysis of the spatial learning behavior of rats during early development has indicated that young pups use rather similar "immature" taxon strategies prior to approximately 45 days of age when the more flexible strategy of directly locating a goal from any starting position emerges [3]. It is our impression that " i m p a i r e d " aged rats are also more likely to use taxon strategies. This issue was not directly addressed in the present experiment. Because animals received only 3 training trials after criterion was achieved, an
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insufficient s a m p l e o f d a t a w a s available to a n a l y z e the search p a t t e r n from different starting locations. This question, h o w e v e r , is the s u b j e c t o f ongoing investigation in o u r laboratory. In c o n c l u s i o n , a n i n c r e a s i n g b o d y o f d a t a suggests that age-related c h a n g e s in the m o r p h o l o g y / f u n c t i o n o f the hipp o c a m p a l f o r m a t i o n m a y s e r v e as a s u b s t r a t e for a declining p e r f o r m a n c e of rats o n spatial learning tasks. A particularly interesting feature of s o m e of this r e s e a r c h is the o b s e r v a t i o n that n e u r o b i o l o g i c a l m a r k e r s in this circuitry c a n distinguish b e t w e e n aged a n i m a l s that are i m p a i r e d a n d a n i m a l s at a c o m p a r a b l e age that h a v e a relatively p r e s e r v e d learning ability [I, 5-7]. T h e d i s t i n c t i v e feature o f the m e a s u r e used in this r e s e a r c h , i.e., H A C U , is that it is specifically r e s p o n s i v e
to the training p r o c e d u r e that is used to identify s u b p o p u l a tions o f " i m p a i r e d " a n d " u n i m p a i r e d " aged animals. T h u s , the results r e p o r t e d here particularly s e r v e to s u p p o r t the c o n c e p t t h a t h i p p o c a m p a l f u n c t i o n is engaged during spatial l e a r n i n g a n d to direct f u r t h e r s t u d y o f t h e c o n t r i b u t i o n of s e p t o - h i p p o c a m p a l c h o l i n e r g i c f u n c t i o n to this t y p e o f learning in b o t h y o u n g and aged p o p u l a t i o n s .
ACKNOWLEDGEMENTS This work was supported by NIMH grant MH 39180, a Research Science Development Award NIMH KO2 MH00406 to M.G. and a NIA National Research Service Award AG 05407 to M.A.P.
REFERENCES
1. Barnes, C. A. Memory deficits associated with senescence: A neurophysiological behavioral study in the rat. J Comp Physio/ 93: 74-104, 1979. 2. Barnes, C. A. Aging and the physiology of spatial memory. Neurobiol Aging, in press; 1988. 3. Chevalley, A-F. and F. Schenk. Immature processes of spatial learning in hooded rats. Soc Neurosci Abstr 13: 655, 1987. 4. Decker, M. W., M. A. Pelleymounter and M. Gallagher. The effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats. J Neurosci 8: 90-99, 1988. 5. Gage, F. H., P. A. T. Kelly and A. Bjorklund. Regional changes in brain glucose metabolism reflect cognitive impairments in aged rats. J Neurosci 4: 2856-2865, 1984. 6. Gallagher, M. and M. A. Pelleymounter. Spatial learning deficits in old rats: A model for memory decline in the aged. Neurobiol Aging, in press; 1988. 7. Geinisman, Y., L. de Toledo-Morrell and F. Morrell. Loss of perforated synapses in the dentate gyrus: Morphological substrate of memory deficit in aged rats. Proc Natl Acad Sci USA 83: 3027-3031, 1986. 8. Geinisman, Y., L. de Toledo-Morrell and F. Morrell. Aged rats need a preserved complement of perforated axospinous synapses per hippocampal neuron to maintain good spatial memory. Brain Res 398: 266-275, 1986. 9. Kammerer, E., Ch. Rauca and H. Matthies. Cholinergic activity of the hippocampus and permanent memory storage. In: Neuronal Plasticity and Memory Formation, edited by C. A. Marsan and H. Matthies. New York: Raven Press, pp. 387-395. 10. Lowry, O., N. Rosebraugh, A. Farr and R. Randal. Protein measurement with the Folin reagent. J Bio! Chem 193: 265-275, 1951. 11. McNaughton, B. L., C. A. Barnes, G. Rao, J. Baldwin and M. Rasmussen. Long-term enhancement of hippocampal synaptic transmission and the acquisition of spatial information. J Neurosci 6: 563-571, 1986. 12. Mitchell, S. J., J. N. P. Rawlins, O. Steward and D. S. Olton. Medial septal area lesions disrupt theta rhythm and alter cholinergic staining in medial entorhinal cortex and produce impaired radial arm maze behavior in rats. J Neurosci 2: 292-302, 1982.
13. Morris, R. G. M., P. Garrard, J. N. P. Rawlins and J. O'Keefe. Place navigation impaired in rats with hippocampal lesions. Nature 297: 681-683, 1982. 14. Olton, D. S., J. Walker and F. Gage. Hippocampal connections and spatial discrimination. Brain Res 139: 295-308, 1978. 15. Raaijmakers, W. G. M. High-affinity choline uptake in hippocampal synaptosomes and learning in the rat. In: Neuronal Plasticity and Memory Formation. edited by C. A. Marsan and H. Matthies. New York: Raven Press, pp. 373-385. 16. Rapp, P. R., R. A. Rosenberg and M. Gallagher. An evaluation of spatial information processing in aged rats. Behav Nettrosci 101: 3-12, 1987. 17. Schmidt, D. E., D. O. Cooper and R. J. Barrett. Strain specific alterations in hippocampal cholinergic function following acute footshock. Pharmacol Bioc'hem Behav 12: 227-280, 1980. 18. Simon, J. R., S. Atweh and M. J. Kuhar. Sodium dependent high affinity choline uptake: a regulatory,rtep in the synthesis of acetylcholine, J Neurochem 26: 90%922, 1976. 19. Sutherland, R. J., I. Q. Whishaw and Bob Kolb. A behavioral analysis of spatial localization following electrolytic, kainate- or cholchicine-induced damage to the hippocampal formation in the rat. Behav Brain Res 7: 133-152, 1983. 20. Sutherland, R. J., I. Q. Whishaw and J. C. Regeher. Cholinergic receptor blockade impairs spatial localization by distal cues in the rat. J Comp Physiol Psychol 96: 563-573, 1982. 21. Wenk, G., D. Hepler and D. Olton. Behavior alters the uptake of all-choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area. Behav Brain Res 13: 12%138, 1984. 22. Wheeler, D. D. Aging of membrane transport mechanisms in central nervous system--High affinity choline transport in rat cortical synaptosomes. Exp Gerontol 20: 73-80, 1985. 23. Whishaw, I. Q. Cholinergic receptor blockade in the rat impairs locale but not taxon strategies for place navigation in a swimming pool. Behav Neurosci 99: 97%1005, 1985.
019"/-4580/88$3.00 + .00
An Age-Related Spatial Learning Deficit: Choline Uptake Distinguishes "Impaired" and "Unimpaired" Rats MICHELA GALLAGHER'
AND MARY ANN PELLEYMOUNTER
Department o f Psychology, University o f North Carolina at Chapel Hill, Chapel Hill, N C 27514 R e c e i v e d 23 S e p t e m b e r 1987 GALLAGHER, M. AND M. A. PELLEYMOUNTER. An age-related spatial learning deficit: Choline uptake distinguishes "impaired" and "unimpaired" rats. NEUROBIOL AGING 9(4) 363-369, 1988.--A functional decline in the hippocampal formation may underlie the emergence of spatial learning deficits in aged rodents. In this study, sodiumdependent high-affinity choline uptake (HACU) was used to monitor hippocampal function in response to training on a spatial task. The subjects were male Long-Evans rats at either 4 months or 22-24 months of age. Animals were trained to locate a camouflaged escape platform in the Morris water maze. Each animal that received place training had a yoked counterpart that was exposed to swimming in the maze but was not required to learn the task. Animals, both young and aged, were sacrificed after attaining a criterion performance. Relative to animals in the yoked condition, place training significantly reduced HACU in both the young rats and in a subpopulation of the aged animals that learned the task rapidly. In contrast, for aged rats that had an impaired rate of acquisition, no effect of place training on HACU was observed. These results provide evidence for a relationship between the behavioral capacities of aged rats and changes in the status of hippocampal function. Spatial learning
Cholinergic
Hippocampus
High-affinity choline uptake
AGED rodents are commonly impaired on spatial learning tasks ([2,6] for recent reviews). There is also substantial agreement that the spatial learning capacity of young adult rats depends upon the integrity of the hippocampal formation. Damage to the hippocampal formation or treatments that alter hippocampal function disrupt performance on a wide variety of spatial tasks including those most often used to assess age-related deficits [11-14, 19]. Thus, changes in the aged hippocampal formation could conceivably provide a neurobiological substrate for age-related spatial learning deficits. Previous research in our laboratory was conducted to monitor hippocampal function during spatial learning in order to evaluate the relationship between the behavioral capacities of aged rats and changes in the status of this circuitry. This work demonstrated that a selective marker for the function of hippocampal cholinergic neurons, i.e., sodium-dependent high-affinity choline uptake (HACU), is altered in young rats as a specific consequence of spatial learning in the Morris water maze: when young adult rats are trained to locate a camouflaged escape platform in the water maze by using a configuration of spatial information, a change in hippocampal HACU is observed [4]. Specifically, young rats were trained until proficient behavioral performance on the task was demonstrated and were then sacrificed 15 min after the final training session. The velocity (Vma~) of
hippocampal HACU was significantly reduced in the animals that learned the spatial task relative to either experimentally naive animals or rats that were yoked to the trained animals for the amount of time spent swimming in the maze but were not required to learn the spatial task. A decrease in HACU is typically associated with treatments that reduce cholinergic activity [17]. Thus, this result indicated that the activity of the septo-hippocampal cholinergic system was decreased in placed-trained animals at the time of sacrifice as a consequence of training, but did not necessarily imply that cholinergic activity was reduced during the training session itself. Further work indicated that the appearance of a training-induced change in hippocampal HACU in young rats depends on the phase of acquisition as it is not observed in animals that are overtrained and does not occur in animals that are sacrificed early in training prior to acquisition of the task [4]. Furthermore, the response of the cholinergic system to training as reflected in HACU was diminished in aged rats [4]. In an initial experiment, groups of young and aged rats were sacrificed after a comparable amount of training: at the time of sacrifice a deficit in performance was evident in the aged group. The experiment showed that training in the water maze task failed to alter hippocampal H A C U in the aged subjects. This result indicated that a change in the regulation of septo-hippocampal cholinergic activity during train-
~Requests for reprints should be addressed to Michela GaUagher, Ph.D., Department of Pyschology, Davie Hall 013A, University of North Carolina, Chapel Hill, NC 27514.
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ing on a spatial task coincides with the behavioral impairment of aged rats. The distinctive effect of place training on hippocampal H A C U in young and aged rats is especially noteworthy because in experimentally naive male LongEvans rats, kinetic parameters for H A C U in hippocampus and cortex are not significantly altered in aged subjects when compared to young adult rats [4,22]. To the extent that measurement of HACU reflects the complement of ACh innervation and the activity of ACh neurons in their basal state, these results indicate no apparent change in the status of the basal forebrain ACh system during aging. An agerelated difference in the dynamic function of this system was, however, revealed by the results obtained when animals are trained on a spatial learning task. The present experiment was designed to further examine this phenomenon by addressing several features of the performance of aged animals in the water maze task. First, aged animals have a relatively mild/moderate deficit on this task. For example, a previous study reported that with extended training the performance of aged animals (23-28 months) will eventually match that of younger rats (6 months and 12 months) [16]. Thus, the study was designed to examine whether a training-induced change in hippocampal H A C U will be characteristic of aged animals when acquisition of the task, albeit delayed, is ultimately achieved. In order to address this question the performance of young and aged rats was matched by training to a criterion performance prior to sacrifice and determination of HACU. In addition, when place training is conducted using aged animals at 22-24 months of age, a substantial subpopulation of these subjects learn the task within the range of training trials required by younger rats (unpublished observations). A second major goal of the present study, therefore, was to determine whether the H A C U response to training will distinguish between impaired and unimpaired aged rats at the same chronological age. This was accomplished by using aged subjects that were 22-24 months at the time of the experiment. METHOD
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FIG. I. A schematic representation of the maze that illustrates the analysis of the interpolated probe trials that were used to assess criterion performance. Starting positions around the perimeter of the maze are designated as (N)orth, (S)outh, (E)ast, and (W)est. The amount of time spent in each quadrant of the maze was recorded. The four possible locations of the escape platform are shown positioned in the center of each quadrant. Annulus crossing were determined by the number of times an animal traversed each of those locations. merged 1 cm below the water surface in the center of one of the four quadrants in the maze. The maze was surrounded by white muslin curtains with black felt patterns affixed to provide a configuration of spatial cues. A TV camera with a wide angle lens was suspended above the center of the maze. Probe trials were videotaped with a JVC VHS videocassette recorder that was located immediately outside the door to the maze room. An RCA 12" monitor was located outside the curtained area to aid the experimenter in locating the animal's position in the maze. Data obtained on an initial habituation trial was analyzed with a video tracking system (HVS Image Analyzing VP-112) and IBM PC computer using software developed for the water maze by San Diego Instruments, Inc.
Subjects
Procedures
Young (4-5 months) and aged (22-24 months) male Long-Evans rats were used. The aged animals (N=28) were obtained as pathogen-free retired breeders at 8-9 months of age from Charles River Laboratories. They were maintained in a separate room within the Psychology Department vivarium. Survival of these animals from the time of purchase to 23-24 months in this facility is approximately 80%. The 16 naive young animals used in this experiment were obtained as.pathogen-free rats (Charles River Laboratories) and were resident in the Psychology Department vivarium for one month prior to the experiment. Each animal was single housed with food and water available ad lib. The vivarium is maintained on a 12 hr light/dark cycle (lights on at 7 a.m.) and climate controlled at 25°C.
Prior to training each animal was habituated to handling and to the maze itself. Animals were handled 3 rain/day for one week. On the day immediately preceeding training, each animal was placed into the maze without the escape platform and allowed to swim for 90 sec. The animal was then returned to its home cage. During subsequent training all animals received three trials/day using a 60 sec intertrial interval. On each trial the animal was introduced into the maze from one of the four equally spaced positions around the perimeter of the tank. The starting location was varied with the provision that each starting location was used in a series a four trials. Animals in each age group were randomly assigned to either the place training or yoked conditions. Place-trained animals learned to locate the escape platform that was camoflaged and submerged below the surface of the water. For these animals the platform was located in the same position in the maze from trial to trial: its localization depended on the use of spatial information surrounding the maze. If an animal did not locate the escape platform within 120 sec on any training trial, the experimenter placed the animal onto the platform where it remained for 60 sec. F o r animals in the yoked training condition, the escape platform was not immediately available, but instead was placed in the maze after a period of time corresponding to the es-
Apparatus Behavioral testing was conducted using a water maze that consists of a circular galvanized steel tank measuring 1.83 m in diameter and 0.58 m in height. The interior surface of the tank is painted white. During testing the tank was filled to a depth of 35.5 cm tepid water ( 2 7+- I°C) that was clouded by the addition of 0.9 kg powdered milk. A white escape platform (10.2 cm in diameter and 34.5 cm in height) was used for place training. During place training this platform was sub-
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FIG. 2. The seconds (mean+SEM) spent in each quadrant of the maze during the first probe trial are shown in a. The number of annulus crossings (mean-+SEM) in each quadrant on the first probe trial are shown in b. Training=quadrant or annulus where the escape platform was located during training. Adj-R=quadrant or annulus located immediately to the right of the training quadrant. Adj-L=quadrant or annulus located immediately to the left of the traimng quadrant. Opp=quadrant or annulus located 180 degrees from the training quadrant. cape latency of a place-trained counterpart had elapsed. After the designated time swimming in the maze, the escape platform was introduced at a location that varied from trial to trial in a quasi-random fashion such that each of the four possible locations was sampled within a block of four training trials. When the escape platform was introduced, the experimenter place the yoked subject onto the platform where it remained for a 60 sec interval. In this way the amount of time that yoked subjects spent swimming in the maze over the entire course of training was matched to that of the placetrained animals. In contrast to the place-trained subjects, however, the spatial location of the platform for the animals in the yoked condition was not relevant to task performance. Every sixth trial throughout training consisted of a probe trial. During these probe trials each animal was placed in the maze for a 30 sec interval with no escalJe platform present. Each probe trial was videotaped and analyzed to assess the search pattern. Quadrant time was determined as the number of seconds that an animal spent in each of the four quadrants of the maze. A second measure, annulus crossings, refers to the number of times an animal traversed each of the four points where a platform could be positioned (see Fig. 1). A criterion performance was attained when a place-trained animal spent at least 10 seconds in the training quadrant and
crossed the annulus in the training quadrant at least twice during a probe trial (see schematic represented in Fig. 1). Each placed-trained animal along with its yoked control was sacrificed on the day after criterion was achieved. On the sacrifice day each animal received 3 additional trials and was sacrificed 15 min after the completion of this final session. Animals were sacrificed by decapitation and brains were rapidly removed and dissected over ice. The left hippocampus was weighed and homogenized in 20 vol. of ice-cold 0.32 M sucrose using a hand held teflon on glass homogenizer (8--10 strokes). After centrifugation of the homogenate at 100xg for 10 min at 2 degrees C, the supernatant was centrifuged at 18000xg for 20 rain to yield a crude synaptosomal/mitochodrial pellet (P2 fraction). The pellet was resuspended in a 24 vol. of cold 0.32 M sucrose, and 50/zl of this suspension (approximately 75/xg protein) was added to tubes containing 400/zl of ice-cold incubation buffer (final concentration: 126 mM NaC1, 4.75 mM KC1, 1.27 mM CaC12, 15.8 mM Na2HPO4, 1.42 mM MgCI2, and 2 mg/ml dextrose; pH=7.4) to which 50/zl of all-choline (final specific activity: 4.2 Ci/mM, New England Nuclear) was added at a final concentration of 0.4/~M. This concentration approximates the Km for H A C U in hippocampus as determined in previous work [4]. Sodium-dependent uptake was measured in paral-
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G A L L A G H E R AND P E L L E Y M O U N T E R
lel using incubation buffer in which NaCI had been replaced with LiCI and Na2HPO4 had been replaced with Tris phosphate. Tubes were then placed in a shaking water bath for 2.5 min at 37°C followed by cooling in an ice water bath. After the addition of 2 ml of ice cold sodium-free buffer, samples were f'dtered over glass fiber f'dters (Gelman, A/E). The filters were then rinsed twice with 2 ml of ice-cold buffer and counted in I0 ml of Scintiverse E (Fisher Scientific). HACU in all samples was measured in triplicate, and HACU was determined by subtracting sodium-independent uptake from total uptake. Protein was determined by the method of Lowry [10]. Rapid measurement of HACU in vitro with these methods can be used to determine the relative in vivo activity of cholinergic neurons prior to sacrifice. Typically, interventions which increase ACh neuronal activity are found to increase HACU, and, conversely HACU is decreased by treatments that inhibit ACh neurons [18]. RESULTS
Analysis of the behavioral data revealed an age difference in the acquisition of place learning. The young animals that received place training rapidly learned to locate the escape platform: at the first probe trial these subjects had a significant spatial bias for the appropriate training quadrant and for the precise former location of the escape platform. In contrast, at this relatively early point in training a spatial bias was not evident in the aged group (Fig. 2). Overall analysis of the quadrant time data on the first probe trial was conducted using a three factor (Age x Quadrant × Training Condition) mixed design analysis of variance. The analysis revealed a significant main effect for Quadrant, F(3,120) =5.86, p<0.001, and significant interactions for Age x Quadrant and for Age x Quadrant x Training Condition, F(3,120) =3.76, p<0.01 and F(3,120)=4.13, p<0.01, respectively. Further analysis of the Age x Quadrant interaction with F tests for simple effects indicated that the spatial bias for the training quadrant was limited to the young subjects (p<0.001). In contrast, no significant spatial bias was evident for the aged subjects. Further analysis indicated that the quadrant effect was significant only for animals that were place trained (,o<0.02) with no significant difference in the amount of time distributed across quadrants for the yoked subjects. Comparisons between the young and aged groups revealed that the young rats spent significantly more time in the training quadrant and significantly less time in the quadrant opposite to the location of the escape platform during training (p's<0.02). Analysis of the data on annulus crossings during the first probe trial yielded similar results: in the overall analysis significant main effects were obtained for Training Condition, F(1,41)=4.22, p<0.05, and Annulus Crossings, F(3,123)= 2.64, p<0.05, and for the Age x Annulus Crossing interaction, F(3,123) = 5.97, p < 0.001. Again the distribution of swimming across annuli was significantly different for the place training condition (p<0.002), but not for the yoked condition. The young subjects traversed the training annulus more frequently than the aged subjects (/7<0.005). On the first probe trial, 3 of the 8 subjects in the young group that received place training attained a criterion performance, the criterion was not met by any of the 14 aged subjects. It is important to note that the impaired performance of the aged rats early in place training does not appear to be due to an age difference in swimming ability upon initial exposure to the maze. A general estimate of swimming speed and proficiency is provided by the total distance traversed during
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a standard 90 sec swim in the maze on the day prior to the commencement of training. Statistical analysis (one-way ANOVA) showed that there was no significant difference between the age groups on this measure. The total path lengths for the young and aged groups that subsequently received place training were 26.4+--1.15 and 28.1 +_-0.73 meters, respectively. Compared to the young rats, the aged animals required more training to reach criterion. The mean blocks of five training trials to reach criterion for the young and aged groups were 1.9__-0.3 and 3.7---0.3, respectively, a difference that was statistically significant, F(I,20) = 15.85, p <0.001. As shown in Fig. 3, with extended training every aged animal attained criterion even though the majority of aged subjects fell outside the entire range of training required by the younger rats. When criterion was met, the performance of animals in the young and aged groups was well matched. The spatial bias for each age group based on performance during the criterion probe trial is shown in Fig. 4. Analysis of the data for quadrant time and annulus crossings revealed no significant main effects of Age and no significant interactions involving age as a factor. In contrast to the data from the first probe trial, at criterion both young and aged place-trained groups exhibited a comparable spatial bias for the training quadrant and training annulus. The overall analysis for quadrant time yielded a significant effect for Quadrant, F(3,120)=20.50, p<0.0001, and a significant Quadrant x Training Condition interaction, F(3,120)=20.96, p<0.0001. Subsequent analysis revealed a significant spatial bias in the place-trained subjects (/9<0.001) but not in the yoked control groups. In comparison with the yoked subjects, placetrained animals spent significantly more time in the training quadrant and significantly less time in the quadrant opposite to the one in which the escape platform was located during training 09's <0.0001). The overall analysis for annulus crossings revealed significant main effects for both Training Condition and Annulus Crossings, F(1,39)= 16.15, p<0.0005 and F(3,117)=31.63, p<0.0001, respectively, and a significant Training Condition x Annulus Crossing interaction, F(3,117)=21.13, p<0.0001. Analysis for simple effects indicated that significant spatial bias for annulus crossings was present in the place-trained animals (p<0.001) but not in the yoked subjects. Significantly more annulus crossings in the
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training quadrant occurred in the place-trained relative to the yoked subjects (p<0.001). In order to independently confirm that the young and aged subjects performed with equal proficiency prior to sacrifice, escape latencies for the three training trials that occurred on the day following criterion performance were compared for the young and aged place-trained groups. A two-way analysis of variance (Age x Trial) conducted on these data revealed no significant main effects and no significant interaction. The mean escape latencies on this final block of trials for the young and aged subjects were 17.7---5.0 and 14.2___2.0 see, respectively. The effects of training on H A C U were assessed when the aged animals were considered as two distinct subpopulations. A number of the aged subjects (n=6) attained criterion within the range of training trials required by the younger animals (see Fig. 3). A second subpopulation of aged subjects (n=8) required more training to reach criterion than was required by any animal in the younger group. The aged animals in these two subpopulations were designated as "unimpaired" and " i m p a i r e d . " A two-way analysis of variance for a mixed design was conducted on the H A C U data for the resulting three groups of place-trained animals and their
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FIG. 5. High-affinity choline uptake determined at a 0.04 tzM concentration of [aH]-choline in hippocampus. See text for description groups. *Differs from yoked control, p<0.02. **Differs from yoked control, p <0.001. yoked controls (see Fig. 5 for data). This analysis revealed a significant main effect for Training Condition, F(1,38)= 15.20, p < 0 . 0 0 0 5 , and a significant Group x Training Condition interaction, F(2,38)=6.40, p < 0 . 0 0 5 . Subsequent analyses indicated that H A C U was significantly lower in
368
GALLAGHER AND PELLEYMOUNTER
place-trained animals relative to the yoked controls for both the young subjects (p<0.02) and the unimpaired aged subjects (p<0.001) but no difference in H A C U as a function of training condition was found in the impaired aged animals. DISCUSSION In aggreement with other reports [13], the results of this experiment demonstrate that young rats exhibit rapid learning during place training. For these animals escape latencies declined early in training, and by the first probe trial the young rats had a spatial bias for the vicinity of the maze where the escape platform was located during training. In contrast, animals in the yoked condition did not develop a spatial bias in the maze but distributed their swimming more evenly among the quadrants and annuli. When young rats were sacrificed on the day following attainment of criterion performance, hippocampal H A C U was significantly reduced in the place-trained animals relative to their yoked controls. This result confirms our earlier observation that hippocampal H A C U is decreased as a consequence of place training, an effect that can be distinguished from the physical stress of swimming in the maze. In our previous research we found that H A C U determined in yoked control subjects did not differ from that of experimentally naive animals [4]. Thus, the amount of swimming in tepid water to which animals are subjected during place training was not sufficient to alter H A C U in hippocampus. The relation of learning to ACh function as reflected in H A C U is presently not well understood. Consistent with our results, a decrease in hippocampal HACU has been previously observed in young rats that were sacrificed after training on a brightness discrimination task [9]. Other studies, however, have indicated that training experiences can increase H A C U in young animals. For example, a recent study reported that training on any of a wide range of tasks (the radial arm maze, a T-maze, and cued nonmatch-to-sample task, or active avoidance training in a shuttle-box) elevated hippocampal H A C U relative to control subjects [21]. In another series of experiments, Raaijmakers [15] found that exposure to a more circumscribed set of training procedures elevated hippocampal HACU. In this study no effect of avoidance training in a shuttle box was found on hippocampal HACU. The discrepancy between this result and the results of Wenk et al. [21] could conceivably reflect strain differences in the effects of exposure to footshock on hippocampal H A C U [17]. Raaijmakers did observe elevated hippocampal H A C U after go/no-go avoidance discrimination training. However, the author notes that rats which performed well on no-go trials tended to exhibit less, or even no increase in hippocampal HACU compared to rats that performed poorly. Some greater uniformity of results is found across several studies in which training-induced elevations in hippocampal HACU have been observed following training on appetitive spatial learning tasks [15,21]. Although the decrease in H A C U that we observe after place-training in the water maze is in contrast with these reports, the consistent observation is that training on spatial tasks that require the integrity of hippocampal function modifies choline uptake in this system. The direction of change in cholinergic function may be better understood by further experiments explicitly designed to assess a number of variables that differ across studies, including motivational conditions, extent of training, the sacrifice interval in relation to the training sessions, the strain of animals used.
In this experiment, aged rats were impaired in place learning. Upon initial exposure to the maze, however, there was no evidence that the aged rats were deficient in their swimming ability. Nonetheless, the aged subjects were delayed in their aquisition of place learning as reflected by an age difference in performance on the initial probe trial and in the amount of training required to reach criterion by the young and aged groups. With extended training, however, all aged animals developed a significant spatial bias in the maze, and analysis of the escape latency data for the final block of training trials indicated that the young and aged subjects performed with equal proficiency. Thus, learning in the water maze does not exceed the behavioral capacity of these aged rats. Despite the fact that every aged animal in the placetraining condition achieved criterion, the majority of the aged subjects required more training than any of the subjects in the young group. An important finding in the present study was that the effect of training on hippocampal H A C U depended on the degree of behavioral impairment in the aged animals. An effect of place training on hippocampal H A C U that was similar to that found in younger animals was exhibited by a subpopulation of aged rats that achieved criterion within the range of training trials required by the young group. In contrast, no effect of training on HACU was observed in the hippocampus of " i m p a i r e d " aged rats. Thus the response of the septo-hippocampai cholinergic system to training as reflected in HACU provides a neurobiological index that distinguishes aged rats with preserved learning capacity from cohorts at the same chronological age that are clearly impaired relative to the performance of young adults. These results provide further support for the concept that agerelated changes in the function of hippocampal circuitry may underly the emergence of spatial learning deficits toward the end of the lifespan. It is noteworthy that the "'impaired" aged subjects did not exhibit an effect of training on hippocampal H A C U even though their behavioral performance indicated that they learned the task. This result might be taken to indicate that the effect of training on hippocampal H A C U is not necessary to support place learning. Indeed other research has indicated when young animals are place-trained in the water maze after muscarinic antagonist treatment, acquisition of the task is impaired but with sufficient training learning does eventually occur [20,23]. In one of these investigations it was sugeested, however, that different learning strategies may be used by normal young rats and rats treated with atropine [23]. Whereas young adult rats normally learn to swim rather directly to the escape platform from any starting location around the perimeter, atropine-treated animals appeared to use a variety a taxon strategies to locate the platform. For example, animals may swim an appropriate fixed distance from the wall and thereby traverse the platform's location. Alternatively, the animals may locate the platform in relation to some fixed cue surrounding the maze and thereby approach the platform on the same trajectory from trial to trial. Interestingly, recent analysis of the spatial learning behavior of rats during early development has indicated that young pups use rather similar "immature" taxon strategies prior to approximately 45 days of age when the more flexible strategy of directly locating a goal from any starting position emerges [3]. It is our impression that " i m p a i r e d " aged rats are also more likely to use taxon strategies. This issue was not directly addressed in the present experiment. Because animals received only 3 training trials after criterion was achieved, an
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insufficient s a m p l e o f d a t a w a s available to a n a l y z e the search p a t t e r n from different starting locations. This question, h o w e v e r , is the s u b j e c t o f ongoing investigation in o u r laboratory. In c o n c l u s i o n , a n i n c r e a s i n g b o d y o f d a t a suggests that age-related c h a n g e s in the m o r p h o l o g y / f u n c t i o n o f the hipp o c a m p a l f o r m a t i o n m a y s e r v e as a s u b s t r a t e for a declining p e r f o r m a n c e of rats o n spatial learning tasks. A particularly interesting feature of s o m e of this r e s e a r c h is the o b s e r v a t i o n that n e u r o b i o l o g i c a l m a r k e r s in this circuitry c a n distinguish b e t w e e n aged a n i m a l s that are i m p a i r e d a n d a n i m a l s at a c o m p a r a b l e age that h a v e a relatively p r e s e r v e d learning ability [I, 5-7]. T h e d i s t i n c t i v e feature o f the m e a s u r e used in this r e s e a r c h , i.e., H A C U , is that it is specifically r e s p o n s i v e
to the training p r o c e d u r e that is used to identify s u b p o p u l a tions o f " i m p a i r e d " a n d " u n i m p a i r e d " aged animals. T h u s , the results r e p o r t e d here particularly s e r v e to s u p p o r t the c o n c e p t t h a t h i p p o c a m p a l f u n c t i o n is engaged during spatial l e a r n i n g a n d to direct f u r t h e r s t u d y o f t h e c o n t r i b u t i o n of s e p t o - h i p p o c a m p a l c h o l i n e r g i c f u n c t i o n to this t y p e o f learning in b o t h y o u n g and aged p o p u l a t i o n s .
ACKNOWLEDGEMENTS This work was supported by NIMH grant MH 39180, a Research Science Development Award NIMH KO2 MH00406 to M.G. and a NIA National Research Service Award AG 05407 to M.A.P.
REFERENCES
1. Barnes, C. A. Memory deficits associated with senescence: A neurophysiological behavioral study in the rat. J Comp Physio/ 93: 74-104, 1979. 2. Barnes, C. A. Aging and the physiology of spatial memory. Neurobiol Aging, in press; 1988. 3. Chevalley, A-F. and F. Schenk. Immature processes of spatial learning in hooded rats. Soc Neurosci Abstr 13: 655, 1987. 4. Decker, M. W., M. A. Pelleymounter and M. Gallagher. The effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats. J Neurosci 8: 90-99, 1988. 5. Gage, F. H., P. A. T. Kelly and A. Bjorklund. Regional changes in brain glucose metabolism reflect cognitive impairments in aged rats. J Neurosci 4: 2856-2865, 1984. 6. Gallagher, M. and M. A. Pelleymounter. Spatial learning deficits in old rats: A model for memory decline in the aged. Neurobiol Aging, in press; 1988. 7. Geinisman, Y., L. de Toledo-Morrell and F. Morrell. Loss of perforated synapses in the dentate gyrus: Morphological substrate of memory deficit in aged rats. Proc Natl Acad Sci USA 83: 3027-3031, 1986. 8. Geinisman, Y., L. de Toledo-Morrell and F. Morrell. Aged rats need a preserved complement of perforated axospinous synapses per hippocampal neuron to maintain good spatial memory. Brain Res 398: 266-275, 1986. 9. Kammerer, E., Ch. Rauca and H. Matthies. Cholinergic activity of the hippocampus and permanent memory storage. In: Neuronal Plasticity and Memory Formation, edited by C. A. Marsan and H. Matthies. New York: Raven Press, pp. 387-395. 10. Lowry, O., N. Rosebraugh, A. Farr and R. Randal. Protein measurement with the Folin reagent. J Bio! Chem 193: 265-275, 1951. 11. McNaughton, B. L., C. A. Barnes, G. Rao, J. Baldwin and M. Rasmussen. Long-term enhancement of hippocampal synaptic transmission and the acquisition of spatial information. J Neurosci 6: 563-571, 1986. 12. Mitchell, S. J., J. N. P. Rawlins, O. Steward and D. S. Olton. Medial septal area lesions disrupt theta rhythm and alter cholinergic staining in medial entorhinal cortex and produce impaired radial arm maze behavior in rats. J Neurosci 2: 292-302, 1982.
13. Morris, R. G. M., P. Garrard, J. N. P. Rawlins and J. O'Keefe. Place navigation impaired in rats with hippocampal lesions. Nature 297: 681-683, 1982. 14. Olton, D. S., J. Walker and F. Gage. Hippocampal connections and spatial discrimination. Brain Res 139: 295-308, 1978. 15. Raaijmakers, W. G. M. High-affinity choline uptake in hippocampal synaptosomes and learning in the rat. In: Neuronal Plasticity and Memory Formation. edited by C. A. Marsan and H. Matthies. New York: Raven Press, pp. 373-385. 16. Rapp, P. R., R. A. Rosenberg and M. Gallagher. An evaluation of spatial information processing in aged rats. Behav Nettrosci 101: 3-12, 1987. 17. Schmidt, D. E., D. O. Cooper and R. J. Barrett. Strain specific alterations in hippocampal cholinergic function following acute footshock. Pharmacol Bioc'hem Behav 12: 227-280, 1980. 18. Simon, J. R., S. Atweh and M. J. Kuhar. Sodium dependent high affinity choline uptake: a regulatory,rtep in the synthesis of acetylcholine, J Neurochem 26: 90%922, 1976. 19. Sutherland, R. J., I. Q. Whishaw and Bob Kolb. A behavioral analysis of spatial localization following electrolytic, kainate- or cholchicine-induced damage to the hippocampal formation in the rat. Behav Brain Res 7: 133-152, 1983. 20. Sutherland, R. J., I. Q. Whishaw and J. C. Regeher. Cholinergic receptor blockade impairs spatial localization by distal cues in the rat. J Comp Physiol Psychol 96: 563-573, 1982. 21. Wenk, G., D. Hepler and D. Olton. Behavior alters the uptake of all-choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area. Behav Brain Res 13: 12%138, 1984. 22. Wheeler, D. D. Aging of membrane transport mechanisms in central nervous system--High affinity choline transport in rat cortical synaptosomes. Exp Gerontol 20: 73-80, 1985. 23. Whishaw, I. Q. Cholinergic receptor blockade in the rat impairs locale but not taxon strategies for place navigation in a swimming pool. Behav Neurosci 99: 97%1005, 1985.