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Individual Differences in Spatial Memory and Striatal ChAT Activity among Young and Aged Rats
NEUROBIOLOGY OF LEARNING AND MEMORY ARTICLE NO.
70, 314 –327 (1998)
NL983857
Individual Differences in Spatial Memory and Striatal ChAT Activity among Young and Aged Rats Paul J. Colombo and Michela Gallagher Department of Psychology, Johns Hopkins University, Baltimore, Maryland 21218
Individual differences in spatial memory among young and aged rats were assessed using memory tasks related to integrity of the hippocampus and the neostriatum. Relationships were then examined between measures of spatial memory and regional choline acetyltransferase (ChAT) activity, a marker for cholinergic integrity. Twentyfour-month-old Long-Evans rats were impaired in comparisons with 6-month-old rats on measures of place learning, working memory, reference memory, and perseveration in water-maze tasks. Aged rats that were impaired on one measure of memory, however, were not necessarily impaired on other measures. ChAT activity in the ventromedial and dorsolateral neostriatum of aged rats was significantly reduced in comparisons with young rats whereas no difference was found in the hippocampus. Aged rats with the most ChAT activity in the anterior ventromedial neostriatum performed best on the place-learning and reference memory tasks but also made the most perseverative errors on the working memory task. In addition, young and aged rats with the most ChAT activity in the anterior dorsolateral neostriatum were those with the least accurate working memory. No relationships were found between ChAT activity in the hippocampus and spatial memory. Thus age-related memory impairment has components that can be segregated by measuring relationships between cholinergic integrity in subregions of the anterior neostriatum and memory tasks with different strategic requirements. © 1998 Academic Press Key Words: aging; spatial learning; memory; reference memory; working memory; hippocampus; neostriatum; ChAT activity; cholinergic system.
Evidence linking the central cholinergic systems to age-related memory impairment began with pharmacological studies of the effects produced by cholinergic antagonists. In humans, scopolamine produced memory impairments in young subjects that were comparable to those observed in aged subjects (Drachman & Leavitt, 1974). More recently, relationships were reported between memory impairment and the integrity of cholinergic systems in young and aged rats. Among several examples, spatial memory was correlated with the size of acetylcholinesterase (AChE)-positive neurons in the medial septum and the number of AChE-positive neurons in the medial septum, diagonal band of Broca, and striatum (Fischer, Gage & Bjorklund, 1989). Relationships between choline acetyltransferase (ChAT) activity and age-related memory impairment were reported in the basal forebrain (Gallagher, This research was supported by F32-MH11337 to P.J.C. and by K05-MH01149 and PO-AG00973 to M.G. Correspondence and reprint requests should be addressed to Paul J. Colombo at the above address. Fax: (410) 516-4478. E-mail: [email protected]. 1074-7427/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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Burwell, Kodsi, McKinney, Southerland, Vella-Roundtree & Lewis, 1990), medial septum (Lee, Ross, Gower, Paris, Martensson & Lorens, 1994), vertical limb of the diagonal band of Broca (Luine & Hearns, 1990), striatum (Gallagher et al., 1990; Ishimaru, Ogawa, Fuji, Fukuta, Kameyama & Nabeshima, 1991), and hippocampus (Dunbar, Rylett, Schmidt, Sinclair & Williams, 1993). Taken together, these studies indicate that spatial memory formation, and age-related memory impairment, are associated with the basal forebrain cholinergic system that projects to the hippocampus and with the cholinergic interneurons of the neostriatum both. Studies of the effects of damage to the hippocampus and to the neostriatum indicate that their relative contributions during memory formation can be dissociated. Complete damage to the hippocampus impaired both trial-dependent and trial-independent spatial learning in young rats tested on appetitively motivated radial-arm maze tasks (Jarrard, 1983); these two types of memory are sometimes termed working memory and reference memory, respectively. Hippocampal damage caused by global cerebral ischemia, and restricted primarily to the CA1 region, impaired working memory but had no effect on reference memory (Volpe, Davis & Colombo, 1989). In contrast, lesions restricted to the dorsal neostriatum impaired acquisition of reference memory, but had no effect on working memory (Colombo, Davis & Volpe, 1989; Packard & White, 1989). Other evidence suggests that the hippocampus is involved primarily during tasks requiring win-shift and spatial strategies whereas the neostriatum is necessary for normal performance of tasks requiring win-stay and cue-related strategies (McDonald & White, 1993, 1994; Packard & Teather, 1997). In addition to dissociations between the hippocampus and the neostriatum, other reports indicate that regions within the neostriatum make differential contributions to performance of some memory tasks. In general, regional functional variations in the neostriatum are related to variations in connectivity across the anterior–posterior and dorsal–ventral planes. For example, anterodorsal, but not posteroventral, caudate lesions impaired working memory on the radial-arm maze when extramaze cues were minimized (Masuda & Iwasaki, 1984). Anterior–posterior caudate dissociations were reported in monkeys in relation to acquisition and performance of motor sequences (Miyachi, Hikosaka, Miyashita, Karadi & Rand, 1997) as well as performance of delayed spatial and object alternation tasks (Levy, Friedman, Davachi & Goldman-Rakic, 1997). Most reports indicate that aged rats, as a group, are impaired in comparisons with young rats on both the working and the reference memory components of spatial tasks (Ando & Ohashi, 1991; Jucker, Oettinger & Battig, 1988; Pitsikas & Algeri, 1992; van der Staay & de Jonge, 1993). One of the hallmarks of research on age-related spatial memory impairment, however, is that groups of aged rats show a broader range of variability than groups of young rats on measures of spatial memory and on measures of relevant neurobiological markers (Barnes, 1979; Gage, Chen, Buzsaki & Armstrong, 1988; Gallagher & Burwell, 1989; Gallagher & Nicolle, 1993). Thus some aged rats are impaired on spatial memory tasks whereas other aged rats perform as well as young rats. Individual differences among young and aged rats were examined in the current study first on measures of spatial memory and then on measures of cholinergic integrity. In specific, place learning in the water maze was exam-
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ined in young and aged rats. Two weeks later, acquisition of spatial working and reference memory was determined in a radial-arm maze adapted for use in a water tank (Buresˇova´, Buresˇ, Oitzl & Zaha´lka, 1985). Aged rats were reported to show spatial memory impairments in comparisons with young rats on both the ‘‘dry’’ (Gallagher, Bostock & King, 1985) and the ‘‘wet’’ (Pitsikas & Algeri, 1992) versions of the radial-arm maze task. We tested whether place learning is predominantly a measure of spatial reference memory (Barnes, 1988; van der Staay & de Jonge, 1993) and whether the two measures of performance, taken at different times, reliably detected individual differences among young and aged subjects. We then examined whether or not aged rats that showed working memory or reference memory impairment also showed signs of cholinergic neurodegeneration in brain regions implicated in working and reference memory; these included the hippocampus, the anterior dorsolateral neostriatum (DLS), which receives projections primarily from sensorimotor cortex, and the anterior ventromedial neostriatum (VMS), which receives meso- and allocortical projections including those from entorhinal and piriform cortex (McGeorge & Faull, 1989). METHODS Male Long-Evans rats (Charles River Laboratories, Raleigh, NC) were obtained either at 4 months of age and tested at 6 months of age (young group, N 5 10) or obtained as retired breeders at 9 months of age and tested at 24 months of age (aged group, N 5 19). All rats were provided water and food ad libitum and maintained in a pathogen-free vivarium at 25°C on a 12-h light– dark cycle with lights on at 0700 h. Two white circular tanks (1.8 3 0.6 m) were situated in different rooms and filled to 35 cm with 27°C water and made opaque with white tempera paint (150 ml). One tank was used for the place-learning task and was surrounded by a white curtain on which black cloth extramaze cues were attached. The other tank was used for the radial-arm maze task and was surrounded by a black curtain on which white cloth extramaze cues were attached. Both sets of curtains and cues remained in a fixed position throughout training. For the place-learning task, a white retractable platform (12 cm diameter) was situated near the center of one of four maze quadrants and 1 cm below the water surface. A black platform extending 2 cm above the surface was used during cue training. The eight-arm radial maze used in this study is similar to one designed by other researchers for use in a water maze (Buresˇova´ et al., 1985) and tested with aged rats (Pitsikas & Algeri, 1992). The maze is made from eight, 3-mm-thick clear Plexiglas channels closed at one end and open at the other which when joined at the open ends form a central area 45 cm in diameter. Each channel/arm is 50 cm long, 18 cm wide, and 35 cm high and fitted at the closed outer end for removable platforms (12 3 18 cm) which were located at the ends of arms 2, 4, 6, and 7. The maze apparatus was placed inside the water tank on adjustable legs such that the walls of the arms extended 10 cm below, and 25 cm above, the water surface; platforms were attached 1 cm below the surface. All rats were trained first on the place-learning task and then trained 2 weeks later on the radial-arm maze task. The group of aged rats was selected
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to represent their typical range of spatial memory performance in which approximately half perform within the range of young rats. Young and aged rats were randomly ordered for testing within each task. For place learning, rats were placed in the pool individually at one of four randomly chosen start locations spaced equally around the perimeter and allowed 90 s to swim to an escape platform located in a constant position and camouflaged 1 cm below the opaque water surface. If rats did not find the platform within 90 s then they were led to it by the experimenter and allowed to remain there for 15 s. Rats received three trials per day for 8 consecutive days. Every sixth trial (probe trial), the platform was retracted to the bottom of the pool for 30 s to measure spatial bias to the platform position. Rats received an additional six cuetraining trials on the ninth day of the protocol, during which a visible platform was positioned in one the four pool quadrants for each trial. We assessed cumulative search error and learning index measures (Gallagher, Burwell & Burchinal, 1993) in addition to measures of pathlength and latency. Cumulative search error was computed by sampling 10 times per second the rat’s distance from the escape platform and averaging these values into 1-s blocks. The blocks were summed and the proximity score that would result from perfect performance was subtracted to correct for different start locations. The learning index is the sum of weighted proximity scores measured during probe trials; low scores reflect search near the escape platform, whereas high scores reflect search farther away from the target. Pathlength is the total distance swum from the start location to the target and latency is the total duration of the trial from when the rat was placed in the water until it located the escape platform. For the eight-arm radial maze task, a rat was placed in the center of the maze facing north and allowed to swim freely throughout the maze until it escaped onto one of the platforms or 90 s elapsed. Rats that did not reach a platform within 90 s were led to the nearest platform by the experimenter. After 10 s on the platform, the rat and the escape platform were removed from the maze, and the rat was immediately placed back in the center of the maze, also facing north, for the next trial. Each rat received four trials per session and two training sessions per day for 9 consecutive days. Two hours elapsed between training sessions to prevent interference, and the same four arms contained escape platforms at the start of each session. Acquisition of trialindependent and trial-dependent memory components were determined. The trial-independent (reference memory) component required that rats remember which four arms contained platforms. The trial-dependent (working memory) component required that rats remember which of the four arms with platforms were visited within a session. Entries into arms that never contained escape platforms were scored as reference memory errors. Entries into arms after the escape platforms had been removed, within each session, were scored as working memory errors. Measures of working and reference memory accuracy were computed by dividing the number of working or reference memory errors per day by the total number of choices made that day. Perseverative errors were scored if a rat entered an arm from which it escaped on the previous choice. The percentages of working memory errors that were perseverative were determined by dividing the numbers of perseverative errors per day by the numbers of working memory errors per day and multiplying by 100. On all
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of the behavioral measures used, lower scores indicate more accurate performance. One week after completion of behavioral training, rats were sacrificed by decapitation, and their brains were removed rapidly and placed on an ice-cold stage. A 2.5-mm-thick coronal section was removed extending from the decussation of the anterior commissure to the posterior end of the anterior olfactory nucleus. The neostriata were dissected ventral to the corpus callosum, lateral to the ventricles, and dorsal to a line connecting the rhinal fissures. The tissue was then bisected into dorsolateral and ventromedial portions. The hippocampi were dissected as reported (Glowinski & Iverson, 1966). All tissue samples were frozen on dry ice immediately after dissection and stored at 280°C. ChAT activity was determined in homogenates using the radioenzymatic method. Frozen tissue samples were homogenized in 2 ml of 0.32 M sucrose solution and 300-ml aliquots of homogenate were added to 75 ml of 2% Triton X-100 containing 50 mM EDTA. Forty microliters of homogenate/Triton was mixed with 40 ml reaction substrate (0.4 M sodium chloride, 0.1 M sodium phosphate, pH 7.4, 10.0 mM disodium EDTA, 15.0 mM eserine hemisulfate, 12 mM choline chloride, 1.0 mg/ml bovine serum albumin, 0.18 mM acetyl-CoA, and 0.02 mM [3H]acetyl-CoA (0.2 mCi/mmol)), vortexed, and incubated at 37°C for 15 min. All samples were run in triplicate and tubes containing 40 ml sucrose/Triton and 40 ml reaction substrate served as blanks. The reaction was terminated by placing tubes in an ice-water bath and addition of 100 ml ice-cold ddH2O to each tube. One milliliter of extraction solution (85% toluene, 15% acetonitrile, 5 mg/ml sodium tetraphenylboron) was added, and the tubes were vortexed and centrifuged at 3000 rpm for 1 min. A 500-ml aliquot of the organic phase was removed and radioactivity was counted in a scintillation counter. Sample protein content was determined (Bradford, 1976). ChAT activity was calculated as the net synthesis of [3H]acetylcholine and expressed as nanomoles of ACH per milligram of protein per hour. Analyses of variance (ANOVA) were performed between groups (aged vs young) for behavioral and neurochemical measures. A repeated-measures within-subject design was used to examine acquisition of place learning, working and reference memory, the percentage of perseverative errors, and regional differences in ChAT activity. Correlations were tested between four behavioral indices of learning (learning index, working memory accuracy, reference memory accuracy, and percentage of perseverative errors) and ChAT activity in three brain regions (hippocampus, DLS, and VMS). RESULTS Learning index scores derived from the place-learning task revealed that 24-month-old rats were significantly impaired in comparison with 6-month-old rats [t(27) 5 3.24, p , .05] (see Fig. 1). In addition, aged rats differed significantly from young rats on the other measures of performance during place learning; these included cumulative search error [F(1, 27) 5 9.54, p , .05], training trial pathlength [F(1, 27) 5 8.97, p , .05], and training trial latency [F(1, 27) 5 9.86, p , .05]. During cue training, aged rats were not significantly different from young rats on measures of cumulative search error [t(20) 5 .27, p . .05], pathlength [t(27) 5 1.25, p . .05], or latency [t(20) 5 1.10, p . .05],
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FIG. 1. Acquisition of place learning by 24-month-old rats (open squares) was impaired in comparisons with 6-month-old rats (open circles). The learning index scores (inset) reflect greater heterogeneity among 24-month-old rats (open squares) than among 6-month-old rats (open circles).
indicating that place-learning impairment was not due to sensorimotor deficits among aged rats. Acquisition of reference memory on the radial-arm maze was examined by analysis of variance using a within-subjects repeated-measures design. The analysis revealed significant effects of age [F(1, 27) 5 7.53, p , .05] and day [F(8, 216) 5 54.85, p , .05], but no age by day interaction [F(8, 216) 5 .47, p . .05] (see Fig. 2). Thus both 6- and 24-month-old rats showed significant improvement during the 9 days of testing, but aged rats were impaired relative to young rats. Analysis of covariance, using the place-learning index as a covariate, was performed to determine whether there was a relationship between the measures of place learning and reference memory. There was no significant effect of age [F(1, 26) 5 2.59, p . .05] indicating that variability in the measure of reference memory is substantially accounted for by the measure of place learning. Working memory was examined by analysis of variance using a withinsubjects repeated-measures design. The analysis revealed significant effects of age [F(1, 27) 5 13.08, p , .05], day [F(8, 216) 5 5.75, p , .05] and an age by day interaction [F(8, 216) 5 3.69, p , .05] (see Fig. 3). Analysis of covariance, using the place-learning index as a covariate, was performed to determine whether or not there was a relationship between variability during place learning and variability on the measure of working memory accuracy. The effect of age remained significant [F(1, 26) 5 8.40, p , .05] suggesting that a
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FIG. 2. Reference memory accuracy was impaired in 24-month-old rats (open squares) in comparison with 6-month-old rats (open circles).
significant amount of variability in the measure of working memory accuracy is unrelated to the measure of place learning. Among the working memory errors, perseverative errors were scored if an animal entered an arm containing a platform, escaped onto the platform, and then reentered that arm on the next choice. Because platforms were removed from arms after escape, the strategy of reentering an arm never provided an opportunity to escape from the maze. The percentage of working memory errors that were perseverative was significantly greater in aged rats than in young rats [F(1, 27) 5 5.35, p , .05]. Among aged rats, 22.6 6 2.2% of working memory errors were perseverative, whereas 15.6 6 1.6% of working memory errors were perseverative among young rats. The effect of age on regional ChAT activity was examined by analysis of variance using a within-subjects repeated-measures design. The analysis revealed significant effects of age [F(1, 26) 5 15.91, p , .05], region [F(2, 52) 5 463.5, p , .05], and an age by region interaction [F(2, 52) 5 8.42, p , .05] (see Fig. 4). Aposteriori analyses revealed significant decreases in ChAT activity between 6- and 24-month-old rats in the DLS and in the VMS. No difference was detected between young and aged rats in the hippocampus. When all animals were included in the analyses, significant positive correlations were found between the place-learning index score and reference memory accuracy (r 5 .45, p , .05) and between reference and working memory accuracy (r 5 .41, p , .05). Working memory accuracy did not correlate significantly with the place-learning index and the percentage of working memory errors that were perseverative did not correlate significantly with any other behavioral measure.
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FIG. 3. Working memory accuracy was impaired in 24-month-old rats (open squares) in comparison with 6-month-old rats (open circles).
FIG. 4. Regional choline-acetyltransferase activity in the neostriatum and hippocampus of 6-month-old (open bars) and 24-month-old (filled bars) rats. In comparisons with 6-month-old rats, ChAT activity in 24-month-old rats was significantly lower in dorsolateral (DLS) and ventromedial (VMS) striatum, but not in hippocampus.
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Relationships between regional ChAT activity and the various measures of spatial memory were tested by correlational analysis. The ChAT activity values from the hippocampus, the DLS, and the VMS were each compared to the four behavioral measures that follow: spatial learning index, reference memory accuracy, working memory accuracy, and the percentage of working memory errors that were perseverative. Hippocampal ChAT activity did not correlate significantly with any of the measures of learning and memory. In contrast, ChAT activity in the VMS correlated significantly with the place-learning index and reference memory accuracy when all rats were included in the analyses (r’s 5 2.50 and 2.47, respectively, p’s , .05). These correlations were influenced, however, by age-related group differences as well as by the relationships between ChAT activity and spatial memory within the young and the aged groups. Similar relationships were found among aged, but not young, rats when the two groups were analyzed independently; high ChAT activity in the VMS tended to be related to lower (better) scores for both the place-learning index and reference memory accuracy measures (r 5 2.41, p 5 .08 and r 5 2.42, p 5 .07, respectively; see Figs. 5A and 5B). In contrast to the relationships between the VMS and place-learning or reference memory wherein higher ChAT activity was related to better spatial memory, higher levels of ChAT activity in the DLS were related to less accurate spatial working memory in young rats (r 5 .69, p , .05; see Fig. 6A) and in aged rats (r 5 .46, p , .05; see Fig. 6B). Finally, a relationship was found among aged, but not young, rats between the percentage of working memory errors that were perseverative and ChAT activity in the VMS (r 5 .62, p , .05; see Fig. 5C). DISCUSSION Aged rats were impaired in comparisons with young rats on measures of place-learning, reference memory, working memory, and perseveration. The age-related impairments were not due to sensorimotor or motivational deficits in the water maze because no effects of age were observed during nonspatial cue learning. Measures of place-learning and reference memory were related across two separate tasks and may, therefore, assess similar or overlapping elements of memory (Barnes, 1988). As the two tasks were conducted at 2-week intervals, individual differences among rats in this study population are stable over at least that length of time, a finding consistent with other behavioral assessments conducted in this laboratory (Colombo, Wetsel & Gallagher, 1997; Gallagher & Burwell, 1989). Individual aged rats that were impaired on one measure of memory were not necessarily impaired on a different measure. For example, neither working memory accuracy nor the percentage of working memory errors that were perseverative was correlated significantly with place learning, indicating that the effects of age in tasks that require variable, trial-dependent responses (working memory) are not related to performance of tasks that require constant, trial-independent responses (place learning and reference memory). This finding extends previous evidence that impairments in different behavioral domains can be uncorrelated in aged rats, as indicated by assessments of sensorimotor function and spatial cognition (Gage et al., 1988; Gallagher & Burwell, 1989). The current results are consistent also with evidence from
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FIG. 5. Twenty-four-month-old rats with higher levels of ChAT activity in the VMS tended to perform better, indicated by lower scores, than rats with lower ChAT activity in the VMS on tasks requiring invariant solution strategies. These include the place-learning task (A) and the reference memory component of the radial-arm maze task (B). Twenty-four-month-old rats with higher ChAT activity in the VMS also showed a higher percentage of working memory errors that were perseverative than rats with lower VMS ChAT activity (C).
research with elderly humans indicating that decline in different cognitive functions can occur somewhat independently (Glisky, Polster & Routhieaux, 1995). Such findings suggest that patterns of age-related degeneration or dysfunction may vary among aged individuals within neuronal systems that are required for different functions. In the context of the components of memory examined in the current research this was investigated further by examining the relationships between regional ChAT activity, which is a measure of cholinergic integrity, and measures of spatial memory among individual aged rats. There was neither an effect of age on hippocampal ChAT activity nor a relationship between hippocampal ChAT activity and spatial memory among young or aged rats; those findings are consistent with other reports (Fischer et al., 1989; Gallagher et al., 1990; but see Dunbar et al., 1993). Markers of hippocampal circuitry other than ChAT activity, however, are related to spatial memory among aged rats (Barnes, 1979; Barnes, Rao, Foster & McNaughton, 1992; de Toledo-Morrell, Geinisman & Morrell, 1988; Gallagher & Nicolle, 1993) and both age- and learning-related changes in the hippocampus were
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FIG. 6. Rats with lower levels of ChAT activity in the DLS tended to perform better, indicated by lower scores, than rats with higher ChAT activity in the DLS on the working memory component of the radial-arm maze task. This relationship was found among 6-month-old rats (A) and among 24-month-old rats (B).
reported within the study population and place-learning model used for the current research (Chouinard, Gallagher, Yasuda, Wolfe & McKinney, 1995; Gallagher & Burwell, 1989; Jiang, Owyang, Hong & Gallagher, 1989; Nicolle, Bizon & Gallagher, 1996; Stenvers, Lund & Gallagher, 1996; Sugaya, Chouinard, Greene, Robbins, Personett, Kent, Gallagher & McKinney, 1996; Tanila, Shapiro, Gallagher & Eichenbaum, 1997). These studies include evidence that postsynaptic signal transduction mediated by cholinergic activation in the hippocampus is reduced in aged rats with place-learning impairments compared to their aged cohorts with relatively preserved performance. It is likely, however, that this effect of age alone is insufficient to account for deficits in spatial learning because selective immunolesions that remove cholinergic input to the hippocampus do not impair the performance of young rats in place learning (for review, see Gallagher & Colombo, 1995). In addition to the likely contribution of other hippocampal alterations to age-related cognitive decline, the current results indicate that alterations contributing to this impairment may not be limited to the hippocampus. The current study revealed that aged rats with more ChAT activity in the VMS performed better on the place-learning and reference memory tasks than did aged rats with less ChAT activity in this region. At the same time, aged rats with more ChAT activity in the VMS had a higher percentage of working memory errors that were perseverative than aged rats with less ChAT activity. These findings suggest that aged rats with the greatest ChAT activity in this subregion of the striatum may rely on strategies that facilitate learning invariant contingencies (e.g., place learning), but also render those rats more prone to perseverative errors. It is important to note that ChAT activity in the VMS of young rats was not correlated with any of the behavioral measures. Relationships between ChAT activity in the VMS and behavioral status were restricted to aged rats, whereas relationships between ChAT activity in the DLS and behavior were observed among young and aged rats both. When the young and aged groups were analyzed separately, rats within each age group that had the highest ChAT activity in the DLS also had the least
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accurate working memory. This finding resembles a result described in an earlier report in which high levels of NMDA receptor binding in the striatum were associated with poor spatial learning, a relationship that was evident among young rats and aged rats alike (Nicolle et al., 1996). Research indicating that the striatum and hippocampus may operate in parallel during performance of some memory tasks may help to account for such findings (McDonald & White, 1994). There is evidence, for example, that under some circumstances the functions for which striatal processing is specialized may compete with hippocampal functions, a phenomenon that was invoked recently to account for the behavioral performance of young rats with damage to either the dorsal striatum or the hippocampal system in a version of the water-maze task (McDonald & White, 1994). By this view, aged rats with the highest ChAT activity in the dorsal striatum, a neural system implicated in learning invariant stimulus–response contingencies, may be less likely to adopt flexible, hippocampal-dependent strategies that are optimal for solution of the working memory component of the task. Taken together, the current results show that variability in spatial memory among individual aged rats is related to the integrity of more than one neuronal system and can be assessed using memory tasks with different strategic requirements. REFERENCES Ando, S., & Ohashi, Y. (1991). Longitudinal study on age-related changes of working and reference memory in the rat. Neuroscience Letters, 128, 17–20. Barnes, C. A. (1979). Memory deficits associated with senescence: A behavioral and neurophysiological study in the rat. Journal of Comparative Physiological Psychology, 93, 74 –104. Barnes, C. A. (1988). Aging and the physiology of spatial memory. Neurobiology of Aging, 9, 563–568. Barnes, C. A., Rao, G., Foster, T. C., & McNaughton, B. L. (1992). Region-specific age effects on AMPA sensitivity: Electrophysiological evidence for loss of synaptic contacts in hippocampal field CA1. Hippocampus, 2, 457– 468. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Analytical Biochemistry, 72, 248 –254. Buresˇova´, O., Buresˇ, J., Oitzl, M. S., & Zaha´lka, A. (1985). Radial maze in the water tank: An aversively motivated spatial working memory task. Physiology and Behavior, 34, 1003–1005. Chouinard, M., Gallagher, M., Yasuda, R. P., Wolfe, B. B., & McKinney, M. (1995). Hippocampal muscarinic receptor function in spatial learning-impaired aged rats. Neurobiology of Aging, 6, 955–963. Colombo, P. J., Davis, H. P., & Volpe, B. T. (1989). Allocentric spatial and tactile memory impairments in rats with dorsal caudate lesions are affected by preoperative behavioral training. Behavioral Neuroscience, 103, 1242–1250. Colombo, P. J., Wetsel, W. C., & Gallagher, M. (1997). Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase-C isoforms in young and aged rats. Proceedings of the National Academy of Sciences of the USA, 94, 14195–14199. de Toledo-Morrell, L., Geinisman, Y., & Morrell, F. (1988). Individual differences in hippocampal synaptic plasticity as a function of aging: Behavioral, electrophysiological and morphological evidence. In T. L. Petit & G. O. Ivy (Eds.), Neural plasticity: A lifespan approach (pp. 283–328). New York: A. R. Liss. Drachman, D. A., & Leavitt, J. L. (1974). Human memory and the cholinergic system: A relationship to aging? Archives of Neurology, 30, 113–121. Dunbar, G. L., Rylett, R. J., Schmidt, B. M., Sinclair, R. C., & Williams, L. R. (1993). Hippocampal choline acetyltransferase activity correlates with spatial learning in aged rats. Brain Research, 604, 266 –272.
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Fischer, W., Gage, F. H., & Bjorklund, A. (1989). Degenerative changes in forebrain cholinergic nuclei correlate with cognitive impairments in aged rats. European Journal of Neuroscience, 1, 34 – 45. Gage, F. H., Chen, K. S., Buzsaki, G., & Armstrong, D. (1988). Experimental approaches to age-related cognitive impairments. Neurobiology of Aging, 9, 645– 655. Gallagher, M., Bostock, E., & King, R. (1985). Effects of opiate antagonists on spatial memory in young and aged rats. Behavioral and Neural Biology, 44, 374 –385. Gallagher, M., & Burwell, R. D. (1989). Relationship of age-related decline across several behavioral domains. Neurobiology of Aging, 10, 691–708. Gallagher, M., Burwell, R., & Burchinal, M. (1993). Severity of spatial learning impairment in aging: Development of a learning index for performance in the Morris water maze. Behavioral Neuroscience, 107, 618 – 626. Gallagher, M., Burwell, R. D., Kodsi, M. H., McKinney, M., Southerland, S., Vella-Roundtree, L., & Lewis, M. H. (1990). Markers for biogenic amines in the aged rat brain: Relationship to decline in spatial learning ability. Neurobiology of Aging, 11, 507–514. Gallagher, M., & Colombo, P. J. (1995). Ageing and cognitive function. Current Opinion in Neurobiology, 5, 161–168. Gallagher, M., & Nicolle, M. M. (1993). Animal models of normal aging: relationship between cognitive decline and markers of hippocampal circuitry. Behavioral Brain Research, 57, 155–162. Glisky, E. L., Polster, M. R., & Routhieaux, B. C. (1995). Double dissociation between item and source memory. Neuropsychology, 9, 229 –235. Glowinski, J., & Iverson, L. L. (1966). Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]-norepinephrine, [3H]-dopamine and [3H]-DOPA in various regions of the brain. Journal of Neurochemistry, 13, 655– 669. Ishimaru, H., Ogawa, S., Fuji, K., Fukuta, T., Kameyama, T., & Nabeshima, T. (1991). Age-related changes in learning and memory, choline acetyltransferase activity and number of neuronal cells in rats. Journal of Pharmacobiological Dynamics, 14, 321–325. Jarrard, L. E. (1983). Selective hippocampal lesions and behavior: Effects of kainic acid lesions on performance of place and cue tasks. Behavioral Neuroscience, 97, 873– 889. Jiang, H. K., Owyang, V., Hong, J. S., & Gallagher, M. (1989). Elevated dynorphin in the hippocampal formation of aged rats: relation to cognitive impairment on a spatial learning task. Proceedings of the National Academy of Sciences of the USA, 86, 2948 –2951. Jucker, M., Oettinger, R., & Battig, K. (1988). Age-related changes in working and reference memory performance and locomotor activity in the wistar rat. Behavioral and Neural Biology, 50, 24 –36. Lee, J. M., Ross, E. R., Gower, A., Paris, J. M., Martensson, R., & Lorens, S. A. (1994). Spatial learning deficits in the aged rat: neuroanatomical and neurochemical correlates. Brain Research Bulletin, 33, 489 –500. Levy, R., Friedman, H. R., Davachi, L., & Goldman-Rakic, P. S. (1997). Differential activation of the caudate nucleus in primates performing spatial and nonspatial working memory tasks. Journal of Neuroscience, 17, 3870 –3882. Luine, V., & Hearns, M. (1990). Spatial memory deficits in aged rats: Contributions of the cholinergic system assessed by ChAT. Brain Research, 523, 321–324. Masuda, Y., & Iwasaki, T. (1984). Effects of caudate lesions on radial arm maze behavior in rats. Japanese Psychological Research, 26, 42– 49. McDonald, R. J., & White, N. M. (1993). A triple dissociation of memory systems: Hippocampus, amygdala, and dorsal striatum. Behavioral Neuroscience, 107, 3–22. McDonald, R. J., & White, N. M. (1994). Parallel information processing in the water maze: Evidence for independent memory systems involving dorsal striatum and hippocampus. Behavioral and Neural Biology, 61, 260 –270. McGeorge, A. J., & Faull, R. L. M. (1989). The organization of the projection from the cerebral cortex to the striatum in the rat. Neuroscience, 29, 503–537. Miyachi, S., Hikosaka, O., Miyashita, K., Karadi, Z., & Rand, M. K. (1997). Differential roles of monkey striatum in learning of sequential hand movement. Experimental Brain Research, 115, 1–5.
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Nicolle, M. M., Bizon, J. L., & Gallagher, M. (1996). In vitro autoradiography of ionotropic glutamate receptors in hippocampus and striatum of aged Long-Evans rats: Relationship to spatial learning. Neuroscience, 74, 741–756. Packard, M. G., & Teather, L. A. (1997). Double dissociation of hippocampal and dorsal-striatal memory systems by posttraining intracerebral injections of 2-amino-5-phosphonopentanoic acid. Behavioral Neuroscience, 111, 543–551. Packard, M. G., & White, N. M. (1989). Lesions of the caudate nucleus selectively impair ‘‘reference memory’’ acquisition in the radial maze. Behavioral and Neural Biology, 53, 369 –373. Pitsikas, N., & Algeri, S. (1992). Deterioration of spatial and nonspatial reference and working memory in aged rats: Protective effect of life-long calorie restriction. Neurobiology of Aging, 13, 369 –373. Stenvers, K. L., Lund, P. K., & Gallagher, M. (1996). Increased expression of type 1 insulin-like growth factor receptor messenger RNA in rat hippocampal formation is associated with aging and behavioral impairment. Neuroscience, 72, 505–518. Sugaya, K., Chouinard, M., Greene, R., Robbins, M., Personett, D., Kent, C., Gallagher, M., & McKinney, M. (1996). Molecular indices of neuronal and glial plasticity in the hippocampal formation in a rodent model of age-induced spatial learning impairment. Journal of Neuroscience, 16, 3427–3443. Tanila, H., Shapiro, M., Gallagher, M., & Eichenbaum, H. (1997). Brain aging: Changes in the nature of information coding by the hippocampus. Journal of Neuroscience, 17, 5155–5166. van der Staay, F. J., & de Jonge, M. (1993). Effects of age on water escape behavior and on repeated acquisition in rats. Behavioral and Neural Biology, 60, 33– 41. Volpe, B. T., Davis, H. P., & Colombo, P. J. (1989). Preoperative training modifies radial maze performance in rats with ischemic hippocampal injury. Stroke, 20, 1700 –1706.
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Individual Differences in Spatial Memory and Striatal ChAT Activity among Young and Aged Rats Paul J. Colombo and Michela Gallagher Department of Psychology, Johns Hopkins University, Baltimore, Maryland 21218
Individual differences in spatial memory among young and aged rats were assessed using memory tasks related to integrity of the hippocampus and the neostriatum. Relationships were then examined between measures of spatial memory and regional choline acetyltransferase (ChAT) activity, a marker for cholinergic integrity. Twentyfour-month-old Long-Evans rats were impaired in comparisons with 6-month-old rats on measures of place learning, working memory, reference memory, and perseveration in water-maze tasks. Aged rats that were impaired on one measure of memory, however, were not necessarily impaired on other measures. ChAT activity in the ventromedial and dorsolateral neostriatum of aged rats was significantly reduced in comparisons with young rats whereas no difference was found in the hippocampus. Aged rats with the most ChAT activity in the anterior ventromedial neostriatum performed best on the place-learning and reference memory tasks but also made the most perseverative errors on the working memory task. In addition, young and aged rats with the most ChAT activity in the anterior dorsolateral neostriatum were those with the least accurate working memory. No relationships were found between ChAT activity in the hippocampus and spatial memory. Thus age-related memory impairment has components that can be segregated by measuring relationships between cholinergic integrity in subregions of the anterior neostriatum and memory tasks with different strategic requirements. © 1998 Academic Press Key Words: aging; spatial learning; memory; reference memory; working memory; hippocampus; neostriatum; ChAT activity; cholinergic system.
Evidence linking the central cholinergic systems to age-related memory impairment began with pharmacological studies of the effects produced by cholinergic antagonists. In humans, scopolamine produced memory impairments in young subjects that were comparable to those observed in aged subjects (Drachman & Leavitt, 1974). More recently, relationships were reported between memory impairment and the integrity of cholinergic systems in young and aged rats. Among several examples, spatial memory was correlated with the size of acetylcholinesterase (AChE)-positive neurons in the medial septum and the number of AChE-positive neurons in the medial septum, diagonal band of Broca, and striatum (Fischer, Gage & Bjorklund, 1989). Relationships between choline acetyltransferase (ChAT) activity and age-related memory impairment were reported in the basal forebrain (Gallagher, This research was supported by F32-MH11337 to P.J.C. and by K05-MH01149 and PO-AG00973 to M.G. Correspondence and reprint requests should be addressed to Paul J. Colombo at the above address. Fax: (410) 516-4478. E-mail: [email protected]. 1074-7427/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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Burwell, Kodsi, McKinney, Southerland, Vella-Roundtree & Lewis, 1990), medial septum (Lee, Ross, Gower, Paris, Martensson & Lorens, 1994), vertical limb of the diagonal band of Broca (Luine & Hearns, 1990), striatum (Gallagher et al., 1990; Ishimaru, Ogawa, Fuji, Fukuta, Kameyama & Nabeshima, 1991), and hippocampus (Dunbar, Rylett, Schmidt, Sinclair & Williams, 1993). Taken together, these studies indicate that spatial memory formation, and age-related memory impairment, are associated with the basal forebrain cholinergic system that projects to the hippocampus and with the cholinergic interneurons of the neostriatum both. Studies of the effects of damage to the hippocampus and to the neostriatum indicate that their relative contributions during memory formation can be dissociated. Complete damage to the hippocampus impaired both trial-dependent and trial-independent spatial learning in young rats tested on appetitively motivated radial-arm maze tasks (Jarrard, 1983); these two types of memory are sometimes termed working memory and reference memory, respectively. Hippocampal damage caused by global cerebral ischemia, and restricted primarily to the CA1 region, impaired working memory but had no effect on reference memory (Volpe, Davis & Colombo, 1989). In contrast, lesions restricted to the dorsal neostriatum impaired acquisition of reference memory, but had no effect on working memory (Colombo, Davis & Volpe, 1989; Packard & White, 1989). Other evidence suggests that the hippocampus is involved primarily during tasks requiring win-shift and spatial strategies whereas the neostriatum is necessary for normal performance of tasks requiring win-stay and cue-related strategies (McDonald & White, 1993, 1994; Packard & Teather, 1997). In addition to dissociations between the hippocampus and the neostriatum, other reports indicate that regions within the neostriatum make differential contributions to performance of some memory tasks. In general, regional functional variations in the neostriatum are related to variations in connectivity across the anterior–posterior and dorsal–ventral planes. For example, anterodorsal, but not posteroventral, caudate lesions impaired working memory on the radial-arm maze when extramaze cues were minimized (Masuda & Iwasaki, 1984). Anterior–posterior caudate dissociations were reported in monkeys in relation to acquisition and performance of motor sequences (Miyachi, Hikosaka, Miyashita, Karadi & Rand, 1997) as well as performance of delayed spatial and object alternation tasks (Levy, Friedman, Davachi & Goldman-Rakic, 1997). Most reports indicate that aged rats, as a group, are impaired in comparisons with young rats on both the working and the reference memory components of spatial tasks (Ando & Ohashi, 1991; Jucker, Oettinger & Battig, 1988; Pitsikas & Algeri, 1992; van der Staay & de Jonge, 1993). One of the hallmarks of research on age-related spatial memory impairment, however, is that groups of aged rats show a broader range of variability than groups of young rats on measures of spatial memory and on measures of relevant neurobiological markers (Barnes, 1979; Gage, Chen, Buzsaki & Armstrong, 1988; Gallagher & Burwell, 1989; Gallagher & Nicolle, 1993). Thus some aged rats are impaired on spatial memory tasks whereas other aged rats perform as well as young rats. Individual differences among young and aged rats were examined in the current study first on measures of spatial memory and then on measures of cholinergic integrity. In specific, place learning in the water maze was exam-
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ined in young and aged rats. Two weeks later, acquisition of spatial working and reference memory was determined in a radial-arm maze adapted for use in a water tank (Buresˇova´, Buresˇ, Oitzl & Zaha´lka, 1985). Aged rats were reported to show spatial memory impairments in comparisons with young rats on both the ‘‘dry’’ (Gallagher, Bostock & King, 1985) and the ‘‘wet’’ (Pitsikas & Algeri, 1992) versions of the radial-arm maze task. We tested whether place learning is predominantly a measure of spatial reference memory (Barnes, 1988; van der Staay & de Jonge, 1993) and whether the two measures of performance, taken at different times, reliably detected individual differences among young and aged subjects. We then examined whether or not aged rats that showed working memory or reference memory impairment also showed signs of cholinergic neurodegeneration in brain regions implicated in working and reference memory; these included the hippocampus, the anterior dorsolateral neostriatum (DLS), which receives projections primarily from sensorimotor cortex, and the anterior ventromedial neostriatum (VMS), which receives meso- and allocortical projections including those from entorhinal and piriform cortex (McGeorge & Faull, 1989). METHODS Male Long-Evans rats (Charles River Laboratories, Raleigh, NC) were obtained either at 4 months of age and tested at 6 months of age (young group, N 5 10) or obtained as retired breeders at 9 months of age and tested at 24 months of age (aged group, N 5 19). All rats were provided water and food ad libitum and maintained in a pathogen-free vivarium at 25°C on a 12-h light– dark cycle with lights on at 0700 h. Two white circular tanks (1.8 3 0.6 m) were situated in different rooms and filled to 35 cm with 27°C water and made opaque with white tempera paint (150 ml). One tank was used for the place-learning task and was surrounded by a white curtain on which black cloth extramaze cues were attached. The other tank was used for the radial-arm maze task and was surrounded by a black curtain on which white cloth extramaze cues were attached. Both sets of curtains and cues remained in a fixed position throughout training. For the place-learning task, a white retractable platform (12 cm diameter) was situated near the center of one of four maze quadrants and 1 cm below the water surface. A black platform extending 2 cm above the surface was used during cue training. The eight-arm radial maze used in this study is similar to one designed by other researchers for use in a water maze (Buresˇova´ et al., 1985) and tested with aged rats (Pitsikas & Algeri, 1992). The maze is made from eight, 3-mm-thick clear Plexiglas channels closed at one end and open at the other which when joined at the open ends form a central area 45 cm in diameter. Each channel/arm is 50 cm long, 18 cm wide, and 35 cm high and fitted at the closed outer end for removable platforms (12 3 18 cm) which were located at the ends of arms 2, 4, 6, and 7. The maze apparatus was placed inside the water tank on adjustable legs such that the walls of the arms extended 10 cm below, and 25 cm above, the water surface; platforms were attached 1 cm below the surface. All rats were trained first on the place-learning task and then trained 2 weeks later on the radial-arm maze task. The group of aged rats was selected
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to represent their typical range of spatial memory performance in which approximately half perform within the range of young rats. Young and aged rats were randomly ordered for testing within each task. For place learning, rats were placed in the pool individually at one of four randomly chosen start locations spaced equally around the perimeter and allowed 90 s to swim to an escape platform located in a constant position and camouflaged 1 cm below the opaque water surface. If rats did not find the platform within 90 s then they were led to it by the experimenter and allowed to remain there for 15 s. Rats received three trials per day for 8 consecutive days. Every sixth trial (probe trial), the platform was retracted to the bottom of the pool for 30 s to measure spatial bias to the platform position. Rats received an additional six cuetraining trials on the ninth day of the protocol, during which a visible platform was positioned in one the four pool quadrants for each trial. We assessed cumulative search error and learning index measures (Gallagher, Burwell & Burchinal, 1993) in addition to measures of pathlength and latency. Cumulative search error was computed by sampling 10 times per second the rat’s distance from the escape platform and averaging these values into 1-s blocks. The blocks were summed and the proximity score that would result from perfect performance was subtracted to correct for different start locations. The learning index is the sum of weighted proximity scores measured during probe trials; low scores reflect search near the escape platform, whereas high scores reflect search farther away from the target. Pathlength is the total distance swum from the start location to the target and latency is the total duration of the trial from when the rat was placed in the water until it located the escape platform. For the eight-arm radial maze task, a rat was placed in the center of the maze facing north and allowed to swim freely throughout the maze until it escaped onto one of the platforms or 90 s elapsed. Rats that did not reach a platform within 90 s were led to the nearest platform by the experimenter. After 10 s on the platform, the rat and the escape platform were removed from the maze, and the rat was immediately placed back in the center of the maze, also facing north, for the next trial. Each rat received four trials per session and two training sessions per day for 9 consecutive days. Two hours elapsed between training sessions to prevent interference, and the same four arms contained escape platforms at the start of each session. Acquisition of trialindependent and trial-dependent memory components were determined. The trial-independent (reference memory) component required that rats remember which four arms contained platforms. The trial-dependent (working memory) component required that rats remember which of the four arms with platforms were visited within a session. Entries into arms that never contained escape platforms were scored as reference memory errors. Entries into arms after the escape platforms had been removed, within each session, were scored as working memory errors. Measures of working and reference memory accuracy were computed by dividing the number of working or reference memory errors per day by the total number of choices made that day. Perseverative errors were scored if a rat entered an arm from which it escaped on the previous choice. The percentages of working memory errors that were perseverative were determined by dividing the numbers of perseverative errors per day by the numbers of working memory errors per day and multiplying by 100. On all
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of the behavioral measures used, lower scores indicate more accurate performance. One week after completion of behavioral training, rats were sacrificed by decapitation, and their brains were removed rapidly and placed on an ice-cold stage. A 2.5-mm-thick coronal section was removed extending from the decussation of the anterior commissure to the posterior end of the anterior olfactory nucleus. The neostriata were dissected ventral to the corpus callosum, lateral to the ventricles, and dorsal to a line connecting the rhinal fissures. The tissue was then bisected into dorsolateral and ventromedial portions. The hippocampi were dissected as reported (Glowinski & Iverson, 1966). All tissue samples were frozen on dry ice immediately after dissection and stored at 280°C. ChAT activity was determined in homogenates using the radioenzymatic method. Frozen tissue samples were homogenized in 2 ml of 0.32 M sucrose solution and 300-ml aliquots of homogenate were added to 75 ml of 2% Triton X-100 containing 50 mM EDTA. Forty microliters of homogenate/Triton was mixed with 40 ml reaction substrate (0.4 M sodium chloride, 0.1 M sodium phosphate, pH 7.4, 10.0 mM disodium EDTA, 15.0 mM eserine hemisulfate, 12 mM choline chloride, 1.0 mg/ml bovine serum albumin, 0.18 mM acetyl-CoA, and 0.02 mM [3H]acetyl-CoA (0.2 mCi/mmol)), vortexed, and incubated at 37°C for 15 min. All samples were run in triplicate and tubes containing 40 ml sucrose/Triton and 40 ml reaction substrate served as blanks. The reaction was terminated by placing tubes in an ice-water bath and addition of 100 ml ice-cold ddH2O to each tube. One milliliter of extraction solution (85% toluene, 15% acetonitrile, 5 mg/ml sodium tetraphenylboron) was added, and the tubes were vortexed and centrifuged at 3000 rpm for 1 min. A 500-ml aliquot of the organic phase was removed and radioactivity was counted in a scintillation counter. Sample protein content was determined (Bradford, 1976). ChAT activity was calculated as the net synthesis of [3H]acetylcholine and expressed as nanomoles of ACH per milligram of protein per hour. Analyses of variance (ANOVA) were performed between groups (aged vs young) for behavioral and neurochemical measures. A repeated-measures within-subject design was used to examine acquisition of place learning, working and reference memory, the percentage of perseverative errors, and regional differences in ChAT activity. Correlations were tested between four behavioral indices of learning (learning index, working memory accuracy, reference memory accuracy, and percentage of perseverative errors) and ChAT activity in three brain regions (hippocampus, DLS, and VMS). RESULTS Learning index scores derived from the place-learning task revealed that 24-month-old rats were significantly impaired in comparison with 6-month-old rats [t(27) 5 3.24, p , .05] (see Fig. 1). In addition, aged rats differed significantly from young rats on the other measures of performance during place learning; these included cumulative search error [F(1, 27) 5 9.54, p , .05], training trial pathlength [F(1, 27) 5 8.97, p , .05], and training trial latency [F(1, 27) 5 9.86, p , .05]. During cue training, aged rats were not significantly different from young rats on measures of cumulative search error [t(20) 5 .27, p . .05], pathlength [t(27) 5 1.25, p . .05], or latency [t(20) 5 1.10, p . .05],
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FIG. 1. Acquisition of place learning by 24-month-old rats (open squares) was impaired in comparisons with 6-month-old rats (open circles). The learning index scores (inset) reflect greater heterogeneity among 24-month-old rats (open squares) than among 6-month-old rats (open circles).
indicating that place-learning impairment was not due to sensorimotor deficits among aged rats. Acquisition of reference memory on the radial-arm maze was examined by analysis of variance using a within-subjects repeated-measures design. The analysis revealed significant effects of age [F(1, 27) 5 7.53, p , .05] and day [F(8, 216) 5 54.85, p , .05], but no age by day interaction [F(8, 216) 5 .47, p . .05] (see Fig. 2). Thus both 6- and 24-month-old rats showed significant improvement during the 9 days of testing, but aged rats were impaired relative to young rats. Analysis of covariance, using the place-learning index as a covariate, was performed to determine whether there was a relationship between the measures of place learning and reference memory. There was no significant effect of age [F(1, 26) 5 2.59, p . .05] indicating that variability in the measure of reference memory is substantially accounted for by the measure of place learning. Working memory was examined by analysis of variance using a withinsubjects repeated-measures design. The analysis revealed significant effects of age [F(1, 27) 5 13.08, p , .05], day [F(8, 216) 5 5.75, p , .05] and an age by day interaction [F(8, 216) 5 3.69, p , .05] (see Fig. 3). Analysis of covariance, using the place-learning index as a covariate, was performed to determine whether or not there was a relationship between variability during place learning and variability on the measure of working memory accuracy. The effect of age remained significant [F(1, 26) 5 8.40, p , .05] suggesting that a
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FIG. 2. Reference memory accuracy was impaired in 24-month-old rats (open squares) in comparison with 6-month-old rats (open circles).
significant amount of variability in the measure of working memory accuracy is unrelated to the measure of place learning. Among the working memory errors, perseverative errors were scored if an animal entered an arm containing a platform, escaped onto the platform, and then reentered that arm on the next choice. Because platforms were removed from arms after escape, the strategy of reentering an arm never provided an opportunity to escape from the maze. The percentage of working memory errors that were perseverative was significantly greater in aged rats than in young rats [F(1, 27) 5 5.35, p , .05]. Among aged rats, 22.6 6 2.2% of working memory errors were perseverative, whereas 15.6 6 1.6% of working memory errors were perseverative among young rats. The effect of age on regional ChAT activity was examined by analysis of variance using a within-subjects repeated-measures design. The analysis revealed significant effects of age [F(1, 26) 5 15.91, p , .05], region [F(2, 52) 5 463.5, p , .05], and an age by region interaction [F(2, 52) 5 8.42, p , .05] (see Fig. 4). Aposteriori analyses revealed significant decreases in ChAT activity between 6- and 24-month-old rats in the DLS and in the VMS. No difference was detected between young and aged rats in the hippocampus. When all animals were included in the analyses, significant positive correlations were found between the place-learning index score and reference memory accuracy (r 5 .45, p , .05) and between reference and working memory accuracy (r 5 .41, p , .05). Working memory accuracy did not correlate significantly with the place-learning index and the percentage of working memory errors that were perseverative did not correlate significantly with any other behavioral measure.
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FIG. 3. Working memory accuracy was impaired in 24-month-old rats (open squares) in comparison with 6-month-old rats (open circles).
FIG. 4. Regional choline-acetyltransferase activity in the neostriatum and hippocampus of 6-month-old (open bars) and 24-month-old (filled bars) rats. In comparisons with 6-month-old rats, ChAT activity in 24-month-old rats was significantly lower in dorsolateral (DLS) and ventromedial (VMS) striatum, but not in hippocampus.
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Relationships between regional ChAT activity and the various measures of spatial memory were tested by correlational analysis. The ChAT activity values from the hippocampus, the DLS, and the VMS were each compared to the four behavioral measures that follow: spatial learning index, reference memory accuracy, working memory accuracy, and the percentage of working memory errors that were perseverative. Hippocampal ChAT activity did not correlate significantly with any of the measures of learning and memory. In contrast, ChAT activity in the VMS correlated significantly with the place-learning index and reference memory accuracy when all rats were included in the analyses (r’s 5 2.50 and 2.47, respectively, p’s , .05). These correlations were influenced, however, by age-related group differences as well as by the relationships between ChAT activity and spatial memory within the young and the aged groups. Similar relationships were found among aged, but not young, rats when the two groups were analyzed independently; high ChAT activity in the VMS tended to be related to lower (better) scores for both the place-learning index and reference memory accuracy measures (r 5 2.41, p 5 .08 and r 5 2.42, p 5 .07, respectively; see Figs. 5A and 5B). In contrast to the relationships between the VMS and place-learning or reference memory wherein higher ChAT activity was related to better spatial memory, higher levels of ChAT activity in the DLS were related to less accurate spatial working memory in young rats (r 5 .69, p , .05; see Fig. 6A) and in aged rats (r 5 .46, p , .05; see Fig. 6B). Finally, a relationship was found among aged, but not young, rats between the percentage of working memory errors that were perseverative and ChAT activity in the VMS (r 5 .62, p , .05; see Fig. 5C). DISCUSSION Aged rats were impaired in comparisons with young rats on measures of place-learning, reference memory, working memory, and perseveration. The age-related impairments were not due to sensorimotor or motivational deficits in the water maze because no effects of age were observed during nonspatial cue learning. Measures of place-learning and reference memory were related across two separate tasks and may, therefore, assess similar or overlapping elements of memory (Barnes, 1988). As the two tasks were conducted at 2-week intervals, individual differences among rats in this study population are stable over at least that length of time, a finding consistent with other behavioral assessments conducted in this laboratory (Colombo, Wetsel & Gallagher, 1997; Gallagher & Burwell, 1989). Individual aged rats that were impaired on one measure of memory were not necessarily impaired on a different measure. For example, neither working memory accuracy nor the percentage of working memory errors that were perseverative was correlated significantly with place learning, indicating that the effects of age in tasks that require variable, trial-dependent responses (working memory) are not related to performance of tasks that require constant, trial-independent responses (place learning and reference memory). This finding extends previous evidence that impairments in different behavioral domains can be uncorrelated in aged rats, as indicated by assessments of sensorimotor function and spatial cognition (Gage et al., 1988; Gallagher & Burwell, 1989). The current results are consistent also with evidence from
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FIG. 5. Twenty-four-month-old rats with higher levels of ChAT activity in the VMS tended to perform better, indicated by lower scores, than rats with lower ChAT activity in the VMS on tasks requiring invariant solution strategies. These include the place-learning task (A) and the reference memory component of the radial-arm maze task (B). Twenty-four-month-old rats with higher ChAT activity in the VMS also showed a higher percentage of working memory errors that were perseverative than rats with lower VMS ChAT activity (C).
research with elderly humans indicating that decline in different cognitive functions can occur somewhat independently (Glisky, Polster & Routhieaux, 1995). Such findings suggest that patterns of age-related degeneration or dysfunction may vary among aged individuals within neuronal systems that are required for different functions. In the context of the components of memory examined in the current research this was investigated further by examining the relationships between regional ChAT activity, which is a measure of cholinergic integrity, and measures of spatial memory among individual aged rats. There was neither an effect of age on hippocampal ChAT activity nor a relationship between hippocampal ChAT activity and spatial memory among young or aged rats; those findings are consistent with other reports (Fischer et al., 1989; Gallagher et al., 1990; but see Dunbar et al., 1993). Markers of hippocampal circuitry other than ChAT activity, however, are related to spatial memory among aged rats (Barnes, 1979; Barnes, Rao, Foster & McNaughton, 1992; de Toledo-Morrell, Geinisman & Morrell, 1988; Gallagher & Nicolle, 1993) and both age- and learning-related changes in the hippocampus were
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FIG. 6. Rats with lower levels of ChAT activity in the DLS tended to perform better, indicated by lower scores, than rats with higher ChAT activity in the DLS on the working memory component of the radial-arm maze task. This relationship was found among 6-month-old rats (A) and among 24-month-old rats (B).
reported within the study population and place-learning model used for the current research (Chouinard, Gallagher, Yasuda, Wolfe & McKinney, 1995; Gallagher & Burwell, 1989; Jiang, Owyang, Hong & Gallagher, 1989; Nicolle, Bizon & Gallagher, 1996; Stenvers, Lund & Gallagher, 1996; Sugaya, Chouinard, Greene, Robbins, Personett, Kent, Gallagher & McKinney, 1996; Tanila, Shapiro, Gallagher & Eichenbaum, 1997). These studies include evidence that postsynaptic signal transduction mediated by cholinergic activation in the hippocampus is reduced in aged rats with place-learning impairments compared to their aged cohorts with relatively preserved performance. It is likely, however, that this effect of age alone is insufficient to account for deficits in spatial learning because selective immunolesions that remove cholinergic input to the hippocampus do not impair the performance of young rats in place learning (for review, see Gallagher & Colombo, 1995). In addition to the likely contribution of other hippocampal alterations to age-related cognitive decline, the current results indicate that alterations contributing to this impairment may not be limited to the hippocampus. The current study revealed that aged rats with more ChAT activity in the VMS performed better on the place-learning and reference memory tasks than did aged rats with less ChAT activity in this region. At the same time, aged rats with more ChAT activity in the VMS had a higher percentage of working memory errors that were perseverative than aged rats with less ChAT activity. These findings suggest that aged rats with the greatest ChAT activity in this subregion of the striatum may rely on strategies that facilitate learning invariant contingencies (e.g., place learning), but also render those rats more prone to perseverative errors. It is important to note that ChAT activity in the VMS of young rats was not correlated with any of the behavioral measures. Relationships between ChAT activity in the VMS and behavioral status were restricted to aged rats, whereas relationships between ChAT activity in the DLS and behavior were observed among young and aged rats both. When the young and aged groups were analyzed separately, rats within each age group that had the highest ChAT activity in the DLS also had the least
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accurate working memory. This finding resembles a result described in an earlier report in which high levels of NMDA receptor binding in the striatum were associated with poor spatial learning, a relationship that was evident among young rats and aged rats alike (Nicolle et al., 1996). Research indicating that the striatum and hippocampus may operate in parallel during performance of some memory tasks may help to account for such findings (McDonald & White, 1994). There is evidence, for example, that under some circumstances the functions for which striatal processing is specialized may compete with hippocampal functions, a phenomenon that was invoked recently to account for the behavioral performance of young rats with damage to either the dorsal striatum or the hippocampal system in a version of the water-maze task (McDonald & White, 1994). By this view, aged rats with the highest ChAT activity in the dorsal striatum, a neural system implicated in learning invariant stimulus–response contingencies, may be less likely to adopt flexible, hippocampal-dependent strategies that are optimal for solution of the working memory component of the task. Taken together, the current results show that variability in spatial memory among individual aged rats is related to the integrity of more than one neuronal system and can be assessed using memory tasks with different strategic requirements. REFERENCES Ando, S., & Ohashi, Y. (1991). Longitudinal study on age-related changes of working and reference memory in the rat. Neuroscience Letters, 128, 17–20. Barnes, C. A. (1979). Memory deficits associated with senescence: A behavioral and neurophysiological study in the rat. Journal of Comparative Physiological Psychology, 93, 74 –104. Barnes, C. A. (1988). Aging and the physiology of spatial memory. Neurobiology of Aging, 9, 563–568. Barnes, C. A., Rao, G., Foster, T. C., & McNaughton, B. L. (1992). Region-specific age effects on AMPA sensitivity: Electrophysiological evidence for loss of synaptic contacts in hippocampal field CA1. Hippocampus, 2, 457– 468. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Analytical Biochemistry, 72, 248 –254. Buresˇova´, O., Buresˇ, J., Oitzl, M. S., & Zaha´lka, A. (1985). Radial maze in the water tank: An aversively motivated spatial working memory task. Physiology and Behavior, 34, 1003–1005. Chouinard, M., Gallagher, M., Yasuda, R. P., Wolfe, B. B., & McKinney, M. (1995). Hippocampal muscarinic receptor function in spatial learning-impaired aged rats. Neurobiology of Aging, 6, 955–963. Colombo, P. J., Davis, H. P., & Volpe, B. T. (1989). Allocentric spatial and tactile memory impairments in rats with dorsal caudate lesions are affected by preoperative behavioral training. Behavioral Neuroscience, 103, 1242–1250. Colombo, P. J., Wetsel, W. C., & Gallagher, M. (1997). Spatial memory is related to hippocampal subcellular concentrations of calcium-dependent protein kinase-C isoforms in young and aged rats. Proceedings of the National Academy of Sciences of the USA, 94, 14195–14199. de Toledo-Morrell, L., Geinisman, Y., & Morrell, F. (1988). Individual differences in hippocampal synaptic plasticity as a function of aging: Behavioral, electrophysiological and morphological evidence. In T. L. Petit & G. O. Ivy (Eds.), Neural plasticity: A lifespan approach (pp. 283–328). New York: A. R. Liss. Drachman, D. A., & Leavitt, J. L. (1974). Human memory and the cholinergic system: A relationship to aging? Archives of Neurology, 30, 113–121. Dunbar, G. L., Rylett, R. J., Schmidt, B. M., Sinclair, R. C., & Williams, L. R. (1993). Hippocampal choline acetyltransferase activity correlates with spatial learning in aged rats. Brain Research, 604, 266 –272.
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