The influences of rearing environment and neonatal choline dietary supplementation on spatial learning and memory in adult rats

Behavioural Brain Research 105 (1999) 173 – 188 www.elsevier.com/locate/bbr

Research report

The influences of rearing environment and neonatal choline dietary supplementation on spatial learning and memory in adult rats Richard C. Tees * Department of Psychology, Uni6ersity of British Columbia, 2136 West Mall, Vancou6er, BC V6T 1Z4, Canada Received 7 January 1999; received in revised form 10 May 1999; accepted 11 May 1999

Abstract The facilitative effects of early environmental enrichment and perinatal choline chloride dietary supplementation on adult rat spatial learning and memory were examined using delayed match-to-place (DMTP) and delayed spatial win-shift (DSWSh) discrimination tasks. Animals were either maintained in a standard lighted colony (LR) or were given supplementary exposure to a complex environment (CR) for 2-h daily from 20 to 90 days of age. In each case, half the animals were exposed to the choline supplementation both prenatally (through the diet of pregnant rats) and postnatally (subcutaneous injection) for 24 days. In the first experiment, all 90-day-old rats were given trials in which they first found a hidden platform in a Morris water maze (MWM) in a particular location (acquisition trial), and then were required to remember that position 10 min later (test trial). Both environmental enrichment and early diet had significant impacts on performance. CR animals, given neonatal choline pretreatment, found the platform on test trials significantly faster than any of the other groups. CR animals exposed to the control saline diet showed better retention than did the LR animals given the early choline diet, which in turn, were superior to animals given neither environmental enrichment nor choline. All animals were subsequently tested in the same paradigm immediately following atropine sulfate injections. The atropine eliminated the difference between the four groups of animals on test trials. In a second experiment, both CR, and neonatal choline treatment facilitated performance on a DSWSh radial arm maze (RAM) task previously found to be sensitive to hippocampal and/or medial prefrontal lesions. Performance differences between groups were facilitated by the anticholinesterase drug, tacrine and attenuated by the cholinergic antagonist, Atropine. The present study extends the descriptions of long-term functional enhancements produced by perinatal choline supplementation and environmental enrichment and to relate these effects to common modifications to targets of cholinergic basal forebrain systems. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Environment; Complex rearing; Diet; Neonatal choline; Spatial memory; Learning

1. Introduction Numerous proposals have been made concerning memory systems and their dependence on cholinergic pathways (e.g. [16,49,81]). Much of the focus of these proposals and the related research efforts has been on spatial learning and memory. Temporary pharmacological manipulation of these cholinergic systems using muscarinic and nicotinic receptor antagonists and agonists have consistently shown to predictably impair or * Tel.: +1-604-822-3245; fax: + 1-604-822-6923. E-mail address: [email protected] (R.C. Tees)

enhance performance on place learning tasks (reviewed by [36,42]). Evidence that cholinergic activation [as measured by high-affinity choline uptake (HACU)] is induced in frontal cortex and hippocampus during learning and retention testing in a radial arm maze has also been reported [12]. Lesions of cholinergic cell groups, or maintenance on a choline-deficient diet, have been shown to impair performance on a wide-range of spatial learning and memory tasks in experimental research animals (e.g. [42,82]). These deficits are thought to occur as a result of interference with these same specific cholinergic systems originating in the basal forebrain and projecting to extensive neocortical and hippocampal terminal fields (e.g. [14,76]).

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While there are several reports of improved memory function in adult animals and humans following dietary supplementation with various forms of choline (e.g. [47], there is evidence to the contrary. For instance, Bartus and his coworkers [4] were unable to find any significant improvement in memory task performance in aged humans or other animals. However, there are several plausible reasons why this would have been so. An age-related diminishment in the blood – brain barrier transport of choline [48], and/or in the ability to incorporate extra amounts of choline into ACh [4] both would lead to a lack of improvement resulting from dietary supplementation in aged subjects. Evidence also does suggest that the activity of certain cholinergic pathways progressively decrease with age (e.g. [10]). Some time ago, researchers have become interested in the effects of choline dietary exposure during the early stages of development. Choline is a critical dietary component and the developing brain may have a high demand for choline as it functions both as a precursor for major phospholipid components of cellular membranes and as a precursor of ACh (see [82] for a review). The blood– brain barrier has been shown to more readily transport choline into the brain in neonates [55]. Moreover, dietary intake of choline by pregnant rats or newborn animals directly, does contribute to high concentration of choline in the blood of such young rats, and elevated concentrations of choline and its metabolites, such as phosphorylcholine and betaine, in the neonatal brain itself [21,83]. Krnjevic and Reinhardt [34], and others (e.g. [81]), have reported that these high concentrations of choline may function directly as a cholinergic antagonist and modify neural organization as well as directly increasing ACh concentrations in the brain. Evidence has been reported for an increase in muscarinic receptor density as indexed by (3H) quinuclidinyl benzilate binding and alterations in ChAT levels in the hippocampus and frontal cortex in adult rats [44,45] with neonatal dietary choline supplementation. Such a diet has also been found to increase nicotinic cholinergic receptor density in frontal cortex and hippocampus [8,9], increase glutamate-stimulated hippocampal phospholipase D activity [6,27], and produce long-term effects on morphology and distribution of cholinergic basal forebrain hippocampal and frontal, and medial septum/diagonal band neurons that are immunoreactive to nerve growth factor and the P75 neurotrophin [37,80]. Recently, the neonatal availability of dietary choline has been shown to affect growth-related proteins (e.g. MAP1) and the differentiation of specific regions (CA1-CA3) of the developing rat hippocampus [2]. Neonatal administration of choline in rats not only cause these permanent changes in the biochemical and neuroanatomical markers of basal forebrain cholinergic neurons and their target sites in hippocampus and frontal cortex, it produces long-term

facilitative effects on both the working and reference memory of treated animals as reflected in their performance as adults on 12- and 18- arm radial arm maze (RAM) tasks [43,46]. The neurochemical mechanisms underlying this memory facilitation, while correlated with the aforementioned changes in the basal forebrain during development, are not known. However, they could include long-term alterations of ACh synthesis and release resulting from changes in the number of cholinergic nerve terminals (e.g. [81]) or from changes in the activities of enzymes and macromolecules (e.g. phospholipids) within those terminals that are important in cell signaling (e.g. [6,82]). We have argued that the ability to learn and remember distal-cue-based place information is also an example of a competence whose initial development involves the establishment of an organizational framework dependent on a variety of early experiences with signals from different modalities (e.g. [68,69]). Numerous studies have reported that early rearing environments do affect spatial maze performance (e.g. [30]). While there has been controversy regarding the relative influence of various aspects of the environmental enrichment or complex-rearing (CR) experiences, animals exposed to a complex environment exhibit superior performance in learning and remembering place(s) in a variety of landand water-based mazes compared with light-reared (LR) rats raised in standard laboratory cages [15,24,67,69]. Less than ‘normal’ early experience has also been shown to have deleterious effect on competence. For example, a lack of an early visual stimulation history has also been shown to adversely affect adult spatial working and reference memory in later tested (both blind and sighted) rats [70]. Persistent anatomical and biochemical effects in response to exposure to a complex environment have also been the subject of numerous reports since the 1960s (e.g. [62]). The extent of dendritic branching, the density of synapses per neuron, altered synaptic morphology of cortical well as the hippocampal cells have been shown to be influenced by early CR (reviewed by [23,30]). There also has been evidence on neurochemical effects of CR, particularly with respect to cholinergic neurons. For example, Park and his coworkers [56] found that CR animals not only learned a Morris water maze (MWM) task more quickly than their LR controls, they also showed higher ChAT levels in various cortical brain regions, as well as the caudate nucleus. Of particular interest was the fact that CR, animals, but not their LR controls, showed increased hippocampal and anterior cortical ChAT after maze training. They argued extended maze training and complex rearing cause similar effects on ACh neurons in the basal forebrain, priming them to respond to a subsequent learning experience with an increase in ACh.

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Certainly the induction of (hippocampal) long-term potentiation (LTP) is a well-established manifestation of synaptic plasticity that positively correlates with spatial competence (e.g. [40]), and both perinatal dietary choline supplementation and environmental CR has been shown to enhance LTP induction in CA1 hippocampal slices from adult animals [19,57]. The deleterious effects of aging, deficient diets, and brain damage, including septohippocampal lesion, have been found to be attenuated by exposure to complex environmental housing (reviewed by [24,61]). The expression of positive behavioural effects of cholinergic cell grafts into frontal cortex or hippocampus after lesion have also been found to be augmented by subsequent CR (e.g. [32]). If perinatal choline dietary supplementation initiates organizational changes to the developing basal forebrain systems, one might expect that the impact of the diet on the treated animal, who subsequently grows up in a more demanding environmental circumstances, with the additional opportunities to learn and remember, would be potentiated. This highly experience-modifiable system (e.g. [81]), putatively thought to be engaged by (and altered by) such cumulative effects of spatial experiences, would enhanced by the combination of early exposure to perinatal choline and subsequent environmental stimulation.

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subjects [46,43,72]. For example, Meck and his coworkers [46] found that memory enhancement was only manifest in RAM tasks that involved at least 12 arms/ places. The facilitative effects of environmental CR on spatial and other perceptual competencies are also most evident on more difficult problems [68,69]. In our version of a demanding water maze-based working memory test, a series of one-trial, delayedmatch-to-place (DMTP) sessions are utilized in which the animal swims to a novel, quasi-randomly selected platform location on an (acquisition) trial and then, after a 10–12 min delay, is required to return to that same location on a test (retention) trial. This DMTP task has proved to be sensitive to the effects of early complex rearing [72] and, it has been argued, can be more sensitive to subtle deficits and enhancements of place learning in other circumstances [38,42]. Similar DMTP tasks have been used effectively to assess the effects of aging, cholinergic antagonists, entorhinal– perirhinal, prefrontal cortical and hippocampal lesions (e.g. [5,50]). If the support, which is largely based on superior performance in the radial arm maze, for altered spatial competence in neonatally choline-treated and CR animals is robust, we would expect to find evidence of it using different (albeit demanding) tasks, involving different motivational and response demands.

2.1. Method 2. Experiment 1 In the first study, we set out to examine the hypothesis that neonatal choline administration and early CR would both facilitate later performance on a demanding working spatial memory version of a MWM task we have used in other circumstances [72]. The enhancements in spatial abilities reported for choline-treated rats have largely been evident on RAM tasks (e.g. [43]). Schenk and Brander [63] did report ‘indirect’ facilitative effects of perinatal choline supplementation in watermaze-based place-learning tasks. In fact, their cholinetreated rats were not superior to controls in learning to swim to a standard, simple, single invisible platform location over four days of testing. An easier version of the task, in which an addition ‘landmark’ or cue, i.e. a black cylinder above the platform location, yielded some evidence of superior performance by cholinetreated animals. The simple water maze task Schenk and Brandner employed is one which yields no impairment in animals with medial septal lesions in spite of the significantly reduced cholinergic innervation to their hippocampal formations [11]. Earlier work by Meck, Williams and their co-workers, as well as preliminary work of ours, strongly suggests that the spatial tasks that are likely to prove sensitive to the facilitative effects of the choline diet are those that place significant demands on the spatial memory capacity of the test

2.1.1. Subjects and rearing conditions The subjects were 24 normally LR and 24 CR male Long–Evans (Rattus nor6egicus) rats from 16 litters, born and reared in the Biopsychology colonies at the University of British Columbia. The general rearing conditions have been described previously [69,70]. The rats were raised in 25× 47× 20 cm plastic maternity bins until 24 days of age at which time they were weaned and placed in groups of four to six in ‘group’ hanging wire mesh cages (66× 25×18 cm). Food (Purina Rat Chow) and water were available ad lib throughout the experiment and the colony rooms were on a 12-h-light:12h-dark schedule. The environmental ‘supplementation’ for the CR subjects began at 20 days of age. The animals from the eight litters assigned to the enriched or complex rearing condition were given 2 h daily access in groups of four to six to a large closed field or maze. This daily period of enriched environmental exposure has been reported to have an effect equivalent to continuous exposure in terms of many brain and behavioral measures (e.g. [24,62]). The open field or maze consisted of a tall, wire mesh cage (180× 92× 62 cm). It’s floor and two bridges were covered with Corncob Granules, and the two bridges were located 40 and 100 cm above the floor. Wire mesh racks connected the bridges with one another and with the floor of the chamber. The floor of the chamber was

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filled with an assortment of toys, many of which were changed daily. This daily exposure continued until behavioral testing began. At the time of testing, all the rats were separated and single-housed in hanging wire mesh cases (20×25× 18 cm) and had been handled for a short time once a day for the previous week. All the animals were 90 to 100 days of age and weighed between 350 and 500 g at the start of behavioral testing which took place during the light cycle.

2.1.2. Dietary manipulation Initially, 16 Long– Evans female rats were exposed to either 0 or 5 ml/l solution of 70% choline chloride which was delivered in 0.02 M saccharin solution in tap water. The solution was freely given to the rats as their only source of drinking water. The presence or absence of choline in the water has been demonstrated not to have significant effects on the amount of water ingested by rats [46]. Choline supplementation started approximately 2 days before conception and was maintained throughout pregnancy. Prior to giving birth, the female rats were singly housed in plastic maternity cages. At birth, pups from each of the experimental conditions were cross-fostered among the eight mothers in that condition. During the next 24 days, the prenatally choline treated pups received 70% choline chloride solution in 25.0 ml/l of physiological saline once per day sub-cutaneously (SQ) at a volume of 0.05 ml for the first 5 days and 0.1 ml thereafter. The ‘0% choline’ treated animals received the same volume of saline solution without choline chloride. Half of animals from the choline-treated litters were randomly assigned to be exposed to the CR experience and half the normal LR conditions. Similarly the saline-treated animals were assigned to either CR or LR conditions. Thus, there were four groups, each consisting of 12 animals: A choline-treated CR, a choline-treated LR, a salinetreated CR, and a saline-treated LR group. 2.1.3. Test apparatus The animals were tested in a large circular white swimming pool, 180 cm in diameter and 54 cm in height. The pool was filled to a height of 37 cm with approximately 18°C water that was rendered opaque with the addition of approximately 1.5 containers of white Tempura Powder paint (454 g each). The invisible escape platform was 34 cm in height and 14 cm in diameter. On the top of the platform a circular metal mesh was attached on which the animal rested when it reached the platform. The data were recorded by a tracking system obtained from HVS Image (18 Ormond Crescent, Hampton TW12 2TH, UK). The system included a camera which was mounted vertically on the ceiling directly over the center of the pool so that the entire surface of the pool was inside a viewpoint. The camera was

attached to a video monitor and a video recorder. The video recorder was connected to a microcomputer. The tracking system recorded samples of the animal’s position in the pool at a rate of ten samples per second. The system kept a record of the latency to reach the platform, and the route taken. The computer, the video monitor, and the video recorder were on a table separated from the rest of the room by a room-divider and were not visible to the animals. The animals were kept in holding cages between trials. The walls on the north, west, and east sides of the room with the pool were light beige. On each wall there was a distant visual distal cue attached. The cues were the black letters, XX, OO and II on sheets of white paper (30× 40 cm). The size of the visual angle subtended by each of the lines was 3°. Overall, both letters subtended visual angles of approximately 15°. The distal cues remained on the walls throughout the experiment. The illumination was provided by two 100 W lamps placed below the level of the pool at the northwest and south-east corners and was constant throughout the experiment.

2.1.4. Procedure Our ‘working memory’ version of the water maze task (a delayed memory for location), involved 2 days of pretraining and 5 days of testing. On day 1, the animals were habituated to the swimming pool environment. They were placed in the pool individually for a period of 70 s and allowed to swim. They were then returned to the holding cages and after approximately 10 min the 70 s habituation was repeated. Day 2 consists of ten trials with the invisible platform in the pool. During the first six trials the invisible platform was located at the center of quadrant 3. The animals were released randomly from either the north or west point of the pool and allowed to swim for a maximum of 70 s. During this time if they were unable to find the platform they were led to it by the experimenter placing a hand on top of the platform and the animals moved towards the hand as though to be picked up. When on the platform, regardless of whether finding it independently or being guided, the animals were allowed to stay for 20 s and observe their surroundings. The animals were then returned to the holding cages. On trial 7 of day 2 the platform was moved to the center of quadrant 1, and a small gray colored ball with black stripes (approximately 4 cm in diameter) was attached to the platform as a proximal cue. Each animal was placed in the pool and allowed to search for the platform. If unable to find the platform the animal was led to it. Again when on the platform the animal was left there for 20 s and was then removed from the pool. On trial 8 the cue was removed from the platform which remained in quadrant 1. The animal was released and allowed to search for the invisible platform, and if

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unsuccessful it was guided to the platform. Trials 9 and 10 were similar to trials 7 and 8, respectively, except the platform was the center of quadrant 4. The procedure for days 3 – 7 was divided into two sessions: morning and afternoon. Each session consisted of two trials: an acquisition trial and a test trial. During the acquisition trial an invisible platform was placed at the center of one of the four quadrants without any proximal cue. Each animal was released from one of the two farthest corners with respect to platform position, and allowed to swim and search for the platform. After 20 s on the platform, the animal was removed and returned to a holding cage, there it remained for 10–12 min. After 10–12 min retention interval, the animal was released from the other farthest point (the test trial), with the platform remaining in the same position as it was in the acquisition trial. The animal was removed from the pool after it found the platform (or 70 s had passed). On days 6 and 7, 30 min prior to each session, the animals were injected intraperitoneally (i.p.) with 50 mg/kg atropine sulfate dissolved in saline solution. This dosage has been shown to be effective in altering forebrain ACh activity [13], motor behavior and forebrain EEG activity [78]. Albanus et al. [1] among other research groups, have found that such a dose is necessary to produce virtually complete occupation of muscarinic binding sites in rats. In addition to latency to reach the invisible platform after release, each of the search paths followed by the rats in reaching there, was later categorized separately by two research assistants. Agreement between the two was high (0.93). Three somewhat distinct search strategies were observed for the swimming animals throughout the experiment and, thus all search paths were categorized as either direct, quadrant-by-quadrant, or random (see Fig. 1 for representative examples). This was done both for the acquisition trials and the subse-

Fig. 1. Representative examples of three types (‘direct’, ‘quadrant-byquadrant’, and ‘random’ of swim/search patterns exhibited by the rats in the Morris water maze during the experiment. Swim paths on each trial were categorized as to which of these three it most resembled.

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quent test trials for each of the (initial, intermediate and atropine) phases, covering days 3–7.

2.2. Results A repeated measures analysis of variance (ANOVA) was used to assess differences in escape latency on the 10 trials of the preliminary day (day 2) of testing for the four groups of animals (CR/choline, CR/saline, LR/ choline, and LR/saline). Not surprisingly, the trial order itself had a significant impact on overall performance [F(9,396)=141.2, PB0.001]. Overall performance improved significantly from the first trial (M= 55.0 s) through to trial 6 (M= 12.1 s), then increased slightly, on trial 7 (and 8), when the location of the invisible platform was moved to a new location (M=19.4 and 21.1 s), and again when to moved to another location (M= 26.8 and 18.6 s) in trials 9 and 10. However no significant difference in performance was found due to rearing (CR versus LR), dietary treatment (choline versus saline), nor any interaction between rearing, diet, and trial order. Data for the pairs of (acquisition and test) trials on days 3 and 7 were divided into three phases. The initial phase constituted the data from the first three pairs of trials (on days 3 and 4), and that for the next three pairs of trials (on days 4 and 5) was designed as the intermediate phase. During the next four pairs of trials (on days 6 and 7) the animals received atropine sulfate prior to each session and the final three pairs of trials were designated as the atropine phase. (The first of these four pairs was planned to be a ‘habituation to-the-effects-of-injection’ trial and was not part of this analysis.) A repeated measures ANOVA was used to assess differences in performance of the four groups of animals (CR/choline-, CR/saline-, LR/choline-, and LR/saline-treated groups during these different phases of the experiment. An analysis of variance revealed general improvement across the three phases of the experiment for both the acquisition [F(2,132)=48.2, PB 0.001, and test trials [F(2,132)= 9.4, PB 0.001]. Not surprisingly (see Fig. 2), there was clear evidence that, after one acquisition trial, the animals retained the information about location during the 10–12 min. delay, performing significantly better on the test trials (M for three phases= 22.6, 13.2, and 16.5 s) than they had on the related acquisition (M= 44.9, 35.9, and 30.7 s) trials [F(2,131)=542.5, PB 0.0001]. Further comparative analysis (Tukey HSD, PB 0.05) of performance during the three phases revealed significant evidence of significant retention (acquisition versus test) during all phases, including the atropine phase. However, while analysis revealed no significant differences between the performance of the four groups of animals on acquisition trials in any phase of the experiment, rearing (CR

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Fig. 2. The mean (and SE) latency to find the platform for the 12 CR/choline-, 12 CR/saline-, 12 LR/choline-, and 12 LR/saline-treated animals during their nine acquisition and nine test trials during the three phases (initial, intermediate, atropine) of the delayed spatial working memory water maze task. The asterisks (*) mark only those phases/trials in which there were significant (Tukey HSD, PB 0.05) differences due to group. The CR/choline treated group did perform significantly better than did any other group in both the initial and intermediate phases of the test trials (see text for other significant findings).

versus LR), and early dietary treatment (choline versus saline) both had an influence on test trials [F(1,132)= 167.5 and 397.4, PB 0.001] as did phase (initial, intermediate, atropine), [F(91,132) =70.2, P B 0.001], and the interaction between the three factors [F(12,132)= 11.2, P B0.001]. Further comparative analysis (Tukey HSD, P B 0.01) revealed the CR/choline treated animals performed significantly better than did an other group of animals during the test trials of both the initial and intermediate phases of the experiment. The CR/saline group, in turn, did significantly better than did the LR/choline group that did significantly better than did the LR/saline group. Following atropine sulfate injection, during the third phase, however, the significant

differences in performance due to rearing and diet disappeared. A summary of the results are presented in Fig. 2. The difference in latency between test and acquisition trials was also reflected in different search strategies used by the animals. Not surprisingly random and quadrant-by-quadrant searches (illustrated in Fig. 1) represented virtually all of the searches during acquisition trials and there were no differences due to early dietary treatment (choline versus saline) or rearing (CR versus LR) during any of the three phases for these acquisition trials. The nonparametric analysis did show significant differences between the search patterns exhibited by the animals in the four groups during test trials for the different phases of the experiment. During the initial phase, diet, but not rearing was a significant factor in swim patterns displayed. Saline animals showed more ‘random’ searches than did choline animals [x 2(2)= 4.36, PB 0.05)]. Saline animals also displayed more ‘quad’ and fewer ‘direct’ searches in the intermediate phase [x 2(2)= 14.1, PB 0.01)]. During the intermediate phase, complex rearing itself did have a significant impact as well [x 2(2)= 4.5, PB 0.05], with the animals which were CR making more direct and fewer quad searches than the LR animals. However, there were no significant differences between the searches of the two groups during the atropine phase and (see Fig. 3), the overall distribution of swim search patterns during this phase was similar to those observed in the initial stage. The proportion of three searches employed by four groups of animals during test trials are displayed in Fig. 3. Another factor that was looked at in this experiment was swimming speed itself. The average speed was calculated in terms of the distance traveled divided by the latency and that analysis revealed no significant difference in swimming speed due to rearing (CR vsersus LR) or diet (choline versus saline) or any interaction. However, there was a significant effect due to atropine injection [F(2,132)= 7.9, PB0.01]; the average speed for the animals in all groups was about approximately 8% faster during the atropine phase of the experiment.

3. Experiment 2 Damage to the hippocampal formation and to the septohippocampal cholinergic system adversely affects performance on a variety of (spatial) learning and memory tasks, while leaving performance unaffected on many others [49]. Proposals concerning the nature of the specific processes that are especially compromised after such damage are numerous, and include proposals about working memory (e.g. [52]), spatial mapping [51], and configural learning and memory [35,66]. Similarly a

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variety of ideas have been offered about the role(s) played by subfields of the prefrontal cortex (e.g. [22,33,65]), as well as early stimulation history (e.g. [68]). In virtually all of these accounts, there is an acceptance of the existence of multiple encoding and memory systems (e.g. [16,49]), and an attempt to specify the neural substrates for specific memory systems and the ‘signature’ task-specific deficits produced by damage to it. Indeed, one key to help understand the impact of CR and choline diet is to employ tasks that have been reported to be selectively influenced by lesions or neurochemical manipulation of cells in specific brain regions. One candidate is the spatial win-shift (DSWSh) radial arm maze task originally developed by Olton [53] which requires the animal to use previously acquired trial-unique information to perform a ‘prospective’ sequence of responses. The animal is allowed to visit four randomly selected arms of an eightarm radial maze, and, after a delay, is returned for a retention test in which the four arms not visited initially, now contain food. Such a test has been used to measure memory dysfunction in aging rats [7] and is a task which draws on many of the functions attributed to the hippocampus (working memory, spatial memory and behavioral flexibility) and to medial prefrontal cortex (prospective foraging, planning motor response strategies, flexibility in the use of recently acquired information). An important finding is that it is disrupted by hippocampal or prefrontal lesions but not by other lesions to, for example, the striatum or amydala (e.g. [41,64]). A recent elegant investigation [17], utilizing unilateral, multiple site, lidocaine-induced transient inactivations demonstrated that performance on this

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task is dependent on interconnected network involving the ventral CA1/subiculum region of the hippocampus and the prelimbic region of the rat’s medial prefrontal cortex. Furthermore, this is a task on which performance is likely to influence with the administration of cholinergic drugs. For example, AF-DX-116, a M2 receptor antagonist has been shown to yield a time-dependent improvement in retention [54]. In general, pharmacological blockage of cholinergic (muscarinic) receptors by drugs such as atropine sulfate, scopolamine, and pirenzepine have been shown to impair performance on several maze and delayed response tasks (reviewed by [42]). While the enhancement of cholinergic activity by reducing enzymatic breakdown with ACh inhibitors such as physostigmine have been shown to attenuate spatial learning and memory deficits produced by lesions or receptor blockade, procholinergic drugs including anticholinesterases have not consistently been shown to improve working (spatial) in normal rodents (e.g. [16,29]) in other circumstances. However there are reports that the cholinesterase inhibitor tacrine (THA) as well as physostigmine can improve retention in both middle-aged and aged rodents in a win-shift task [60]. The pharmacology of THA has been widely studied since it was first synthesized in 1945, and it has been shown to rehabilitate memory in AD patients as well to have a variety of beneficial effects on learning and memory in experimental animals (reviewed, e.g. [20]). In the present experiment, the strategy was to utilize THA as a pharmacological probe potentially further enhancing any cholinergically related spatial memory system ‘improvements’ produced by the early choline diet and/or

Fig. 3. The average proportion of ‘direct’, ‘quadrant-by-quadrant’, and ‘random’ swim patterns employed during the nine test trials by the four groups of animals during the three phases of the experiment.. The asterisks (*) mark only those swim patterns/phases for which there were significant (Tukey HSD, PB 0.05) differences due to group (see text for the nature of group differences).

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the postnatal CR. A plausible (albeit simple) argument could be made that the drug should have more (or less) impact on choline-treated and CR animals than their controls. Moreover, as was in the case in Experiment 1, the complementary strategy of attempting to attenuate any cholinergic related improvements produced by our experimental conditions with atropine was also employed.

3.1. Method 3.1.1. Subjects The subjects were 48 naı¨ve male Long – Evans rats (Rattus nor6egicus) from 18 litters, bred at the University of British Columbia. The general and the specific (CR versus LR) rearing, and neurochemical dietary manipulation conditions were the same as those used in Experiment 1. Twenty-four of the male rats had received the early choline treatment while the remaining 24 male rats had been exposed to the control (saline) solution during early rearing. Half of the saline- and half the choline-treated animals were given 2 h daily exposure to the complex rearing environment until behavioral testing began when the rats reached approximately 100 days of age and, as was the case in Experiment 1, the rats in the four conditions (CR/choline, CR/saline, LR/choline, and LR/saline) were grouphoused and given free access to food and water until 1 week before behavioral testing started. At that time, all were placed on a food-restricted diet and were maintained at 85% of their unrestricted weight throughout the testing. 3.1.2. Test apparatus A wooden eight-arm radial maze, painted gray, was used in this experiment. The maze had eight individual (12× 57 cm) arms radiating out from an octagonal center platform which measured 36 cm in diameter. The sides of the arms were ‘edged’ (2.8 cm high) and, each had a small (3 cm deep) cylindrical food cup located 2.5 cm from the end. Access to each of the arms could be blocked by 15 cm high removable metal doors. The maze was elevated 48 cm off the floor in a room containing various extra maze cues (i.e. a shelf unit, table, sink area, and black-on-white wall patterns identical to those used in Experiment 1). 3.1.3. Procedure On the first of 2 days of pretraining the rats were individually placed on the radial maze and allowed to explore for 10 min. On the second day, pieces of the breakfast cereal, Froot Loops, were scattered on the center and at the ends of the arms and the animals were allowed to explore and eat for 10 min. On both days, (and subsequent test days), the animals returned after maze exposure to their home cages and received a

measured number of Bioserv (Holland Industries) food pellets (such that their weights would be maintained at 85% of their prediet body weights). The third day marked the beginning of training trials which were given once a day. These trials consisted of a training phase and a test phase, separated by a delay. Prior to each trial, four quasi-randomly selected arms each were baited with a piece of Froot Loops cereal, placed in the food dish at the end of four arms. Access to the remaining four arms was blocked by the removable metal doors and the animal was required to retrieve the Froot Loops from the four open arms within 10 min. (In fact, there were 17 different ‘random’ patterns utilized throughout the experiment which had been selected such that none contained three consecutive baited arms). Following the training phase (either 10 min passing or successful retrieval of the four Froot Loops), the animals were removed from the maze and maintained in a closed housing chamber for the delay period (which initially was 5 min, and then was extended to 30 min). During the test phase of each daily trial in this delayed spatial win-shift (DSWSh) paradigm, all the arms were open, but only the four arms that previously were blocked, contained food. Animals were allowed 10 min to retrieve the four Froot Loops during the test phase. Animals were required to reach a criterion of four correct choices (out of their first five arm entries during the postdelay test) at the 5 min delay, and on reaching that mark, the delay was extended to 30 min and, to reach their final criterion performance level, the animals had to make the four correct choices (food retrievals) in five or fewer choices for each of three consecutive days. The day after each of the animals achieved criterion performance at the final 30-min delay, drug trials began. For all of the animals, the first ‘drug’ trial involved a 1 cc/kg i.p. isotonic saline injection administered 40 min prior to daily training. Following that day, the animals continued to be tested with saline injections until the animals had returned to/maintained criterion performance for 3 days. The second drug trial consisted of either 1.5 mg/kg or a 3.5 mg/kg i.p. injection of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride (tacrine hydrochloride/THA), a cholinesterase inhibitor (Sigma Chemical Company). The ‘dose’ order was counterbalanced. Half the animals in each of the four groups received the 1.5 mg/kg injection first and the remaining animals the 3.5 mg/kg. Once again saline daily trials followed until the animals returned to/maintained 3 days of criterion performance. The third drug trial (THA) then occurred, followed by saline to-criterion days. The fourth drug day involved a 40 mg/kg i.p. injection of the ACh antagonist, atropine sulfate salt (Sigma Chemical Company) dissolved in isotonic saline. While this was somewhat lower a dose than the 50 mg/kg dose used in Experiment 1, 30 mg/kg i.p. (and

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lower) doses of atropine have also been shown to produce behavioral impairments on spatial problems and significantly alter forebrain EEG and ACh activity (e.g. [59,74]). A final day followed on which the animals were tested with saline injections. In addition to days/trials to criterion at the 30 min delay, the number and type of errors made by the animals in their first five choices during the test phases were recorded (as well as the number of errors during made during the training phase and the total time required to either retrieve the four Froot Loops or, if errors were made, to make the first five arm entries to the food cups). Errors were scored as re-entries into arms previously visited during either phase of a trial. During the retention/test phase the animal might make return entry(s) into arms visited during the postdelay test itself (within phase or proacti6e errors) and/or the animal could return entries to arms visited during the predelay training phase (across session or retroacti6e errors).

3.2. Results A repeated measures analysis of variance (ANOVA) was used to assess differences in the initial performance of the 48 animals in the four groups (CR/choline, CR/saline, LR/choline, and LR/saline) in reaching criterion performance levels on the DSWSh at the 30 min delay. To achieve that level of achievement, the animals had to, after the delay, made four correct entries (i.e. retrieve food) during their first no-morethan-five arm entries on three consecutive days. Both diet (F(1,44)=6.9, P B 0.02) and early rearing conditions (F(1,44)=24.5, PB 0.0001) had significant impacts on the days (or trials) needed for animals to reach criterion (i.e. to begin their three consecutive day criterion ‘run’). The interaction between the two factors was not significant. Both complex rearing and early choline diet facilitated performance with respect to achieving criterion. Further comparative analysis (Tukey HSD, P B0.05) of the performance of the animals in the four groups indicated that those animals given both the perinatal choline diet and subsequently exposed to the 2-h daily of environmental enrichment (CR/choline) took significantly fewer days (see Table 1) to reach criterion than did either of the normally reared groups, those animals (LR/choline) given the choline diet and normal rearing, or (LR/saline) animals given neither. However, their (CR/choline) performance was not significantly better than the other CR (CR/saline) group given no dietary choline supplementation. The difference in performance between the CR/saline group and that of the LR/saline group was also significant, but not the difference between the LR/choline and the LR/saline groups.

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Table 1 Performance of the four groups of animals in learning the delayed spatial win-shift radial arm maze task in Experiment 2 Group

Subjects (N)

Rearing/diet CR/choline LR/choline CR/saline LR/saline

12 12 12 12

Trials/days to reach criterion M

SD

11.9 15.7 14.0 17.3

1.5 2.9 2.1 3.2

Having reached criterion levels of performance, the animals began to receive i.p. drug injections 40 min prior to testing. The first of these was a saline ‘drug’ day, next, either a 1.5 mg/kg THA or 3.5 mg/kg THA injection was administered, the third involved the remaining THA injection and finally, the animals received a 40 mg/kg i.p. injection of atropine sulfate. Interspersed between (and following) these drug days were saline injection days. A repeated measures of analysis (ANOVA) revealed no significant differences in performance due to diet or rearing on these interspersed saline days. An further analysis was done of the performance on four specific saline days—the first saline ‘drug’ day and the three saline days that followed immediately after the two THA and atropine drug days—and there were no significant differences due to trials, diet, or rearing or any interactions between variables (see Fig. 4). In fact the animals in all four groups maintained relatively high levels of performance on all interspersed saline injection days. However, a few animals in each group did display less than criterion performance on their first saline ‘drug’ day and the saline days immediately following the THA and atropine days. Nevertheless no animal took more than 4 days to achieve the three consecutive ‘criterion’ (80– 100% correct) days between each of the four drug days. An ANOVA with repeated measures was also done on the total time spent to retrieve the four Froot Loops (or make the first four or five arm entries) on the test phase of each of the four drug days and it revealed no significant effects due to diet or rearing or any interaction between either or both of these factors and drug day. However, drug itself did produce a significant effect (F(3,132)= 43.2, PB 0.001). A further comparative analysis (Tukey HSD, PB 0.05) revealed that animals being tested under the influence of atropine took less time (M= 123.8 s) in making their arm entries during the test phase than did those same animals on any other drug day (M= 153.2–186.4 s). A repeated measures ANOVA on the total errors (see Fig. 4) made by the four groups of animals on the four drug (first saline, 1.5 THA, 3.5 THA, atropine) days revealed the order of the THA injection also had no

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significant impact on performance. More importantly, diet (F(1,44)=8.8, P B0.01), drug (F(3,132) =90.0, P B 0.001), early rearing (F(1,44) =8.7, P B 0.01), and the interaction between diet and drug (F(3,132) = 4.4, P B 0.01) all had significant effects on performance. Choline-treated animals made significantly fewer (M= 1.25) errors across all four drug days than did salinetreated animals (M= 1.58). Normally reared animals made more errors than did CR animals (M =1.57 versus 1.24). Further comparative analysis (Tukey HSD, PB0.05) the animals made significantly more errors (M =2.7, SD = 0.85) in their arm choices (maximum =5) under the influence of atropine, than they did after the first saline injection (M =1.25, SD = 0.7), and significantly fewer errors (M=0.56, SD= 0.6) under the influence of the 3.5 mg/kg dose of THA. Performance under the influence of the lower 1.5 mg/kg dose of THA (M= 1.1, SD =0.73) was not significantly different from performance under saline, but was significantly worse than under the higher dose of THA, and significantly better than that under atropine. The CR/choline animals performed significantly (Tukey HSD, PB 0.05) better (M =1.0 errors) on drug days than did the animals in any of the other three groups (M = 1.48–1.67 errors). The differences in performance between the three other groups were not significant. A comparative analysis (Tukey HSD, P B 0.05) of the significant interaction between drug and diet revealed the performance of choline-treated animals was significantly better than saline-treated animals at the lower dose of THA, and just missed significance (P B0.08) at the higher dose of THA (as that dose improved performance for all groups). No significant differences in

performance due to diet were observed on either the saline or atropine days. On the 1.5 THA day, animals in the CR/choline made significantly fewer errors (M = 0.42) than the animals in the LR/choline (M = 1.1) which, in turn made fewer errors than either the animals in the LR/saline (M= 1.7) or the CR/saline (M = 1.3) groups. Under the influence of the higher dose of THA, the performance of the animals in all of the four groups was significantly improved, and the order of the differences between the four groups matched that found for the lower dose, these lower error scores were not significantly different from one another. (M = 0.17–0.91). The comparative analysis also revealed that for the performance of CR/choline animals across the four drug days was significantly better on the 3.5 THA day (M= 0.17 errors) than their performance on the saline day (M= 1.0) which was better than that on the atropine day (M= 2.5). Their performance on the 1.5 THA day (M= 0.41) was only significantly better than the atropine day. In the case of the LR/choline animals only the poor performance on the atropine day (M = 3.0) proved to be significantly different from their other three drug days. That pattern was also true in the case of the LR/saline and CR/saline groups. Thus, while performance across all groups was significantly better under 3.5 THA drug conditions, only in the case of the CR/choline animals’ performance did those improvements reach significance (Tukey HSD, PB 0.05). A repeated measures ANOVA which looked at the within phase or proacti6e errors yielded only the fact that the animals (F(1,44)= 42.4, P B 0.001) made more such errors under the influence of atropine (M=1.45,

Fig. 4. Performance (mean and SD) on the four drug probe days (saline, 1.5 mg/kg, THA, 3.5 mg/kg and 40 mg/kg atropine sulphate) and the four interspersed Saline days for the four groups of animals (CR/choline, LR/choline, CR/saline, LR/saline). The saline days presented are the first saline test day, and those (three) saline days that followed immediately after the other drug injections. The asterisks (*) mark only those drug days on which there were significant (Tukey HSD, PB 0.05) differences due to group (see text for the nature of group differences).

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SD =0.65) than on any other drug day (M= 0.31– 0.52, SD=0.47–0.52). There was no other significant difference between the four groups. An examination (repeated measures ANOVA, Tukey’s HSD, PB 0.05) of the across phase or retroacti6e errors (in which the animal revisits an arm originally baited during the training phase in the test phase, after the delay), yielded a pattern of results somewhat similar to that found for the total errors. In this case diet (F(1,44)=12.2, P B0.01), rearing (F(1,44) = 5.7, P B0.01), drug day (F(3,132) =28.3, P B 0.001), and the interaction between the two factors (F(3,132) = 3.3, P B 0.05) had a significant influence. Not surprisingly, animals made significantly more (M = 1.25, SD= 0.48) across phase errors under the influence of atropine and fewer (M= 0.25, SD =0.43) under the influence of the higher dose of THA. Such errors on saline (M= 0.75, SD = 0.6) and under the lower does of THA (M = 0.60, SD =0.61) were not significantly different from one another. The comparative analysis (Tukey HSD, PB 0.05) of the significant interaction between diet and drug day revealed significantly fewer across phase errors were made by adult animals given the perinatal choline diet under the influence of the lower (M = 0.29 versus 91 errors) dose of THA than animals on the neonatal saline/control diet. The difference between the two diet at the higher dose of THA (M=0.08 versus 41 errors) did not reach significance. There were also no significant differences in such error-making between groups on the saline and atropine.

4. Discussion In the first experiment, the effects of being raised on a neonatal choline dietary supplement perinatally through to postnatal day 24, and of being subsequently exposed to daily periods of complex environmental stimulation until adulthood, were measured on a water maze-based DMTP task. First, however, the animals in the four groups were given a day of preliminary training in the water maze, learning to swim to an invisible platform location (trials 1 – 6), find a new location when it moved (trials 7–8), and rediscover it when it moved a second time (trials 9 – 10). There was no evidence that either early diet or rearing experience or the interaction between the two had any effect on the performance of the rats as they learned the initial location of the platform and responded to its two relocations. Clearly, as previous behavioral assessments have indicated (e.g. [46]), such a spatial task did not represent a sufficiently challenging probe of whatever differences in spatial competence there might be between the groups. The LR/saline animals were just as able as LR/choline, the CR/choline and the CR/saline animals to locate the platform, to remember its location relative to the distal

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cues available, and to adjust their sequences of swim movements relative to same cues when the platform was moved [79]. Even damage to the septohippocampal cholinergic fibers does not prevent all forms of spatial learning (e.g. [11]), and one shouldn’t expect that optimal tuning of the cholinergic system [6], would be indispensable to all spatial learning. Our failure to find any effects of diet and CR on performance during preliminary spatial training is consistent with the data cited, though not perhaps the interpretation offered, in a previous report on the effects of early choline diet on a comparable water maze spatial task [63] and with earlier work of our own on the effects of CR [69]. When the animals in the four groups subsequently were given a series of two-trial DMTP sessions over a 5-day period in which they had to remember a novel, invisible platform location, 10 min later when required to return to the same location, they were comparable in many respects in the MWM. All groups of animals improved their swim latencies (and search strategies) over the three phases of training on acquisition trials, and there were no differences in their performance on these trials due to early diet or rearing. On the test trials, the animals in all groups showed clear evidence of learning the locations specified by the acquisition trials, generally improving their swim latencies over the three phases of the experiment. However, importantly, both neonatal choline treatment and subsequent complex rearing did enhance the later performance involving the working spatial memory of these adult animals. Although the effect of the environmental enrichment was, in a sense, greater than that of the choline dietary supplementation, the performance of the group that received the combination of both perinatal choline and subsequent exposure to complex rearing (CR/choline) was superior to that of any of the other groups including the choline-treated animals raised in the normal lighted colony (LR/choline) on the test trials, both during the initial and intermediate phases. Those raised on the control diet with a complex stimulation history (CR/saline) performed better than the LR/choline animals on these key test trials that, in turn, were superior to those exposed to neither choline nor complex rearing (LR/saline). This difference in the relative performance between these aforementioned groups was evident in their finding the location of the platform significantly faster than their controls and, to some extent, in the nature of the swim strategies they used during these trials. While animals in all four groups improved as testing continued, the CR/choline animals consistently and significantly outperformed the animals in the other groups, displaying better working spatial memory when required to remember a location in the pool after a delay of 10–12 min, after a single acquisition trial. During the third (atropine) phase of the experiment, animals were given atropine sulfate 30 min prior to

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those two-trial sessions. While the atropine had some general effects on swim speed and search patterns, animals in all four groups did continue to show evidence of remembering the locations specified by the acquisition trials. Thus, performance on our DMTP task, after some pretraining, is not completely dependent on the now-occupied muscarinic ACh brain receptors [1]. However, what is also evident is that proposed differences in the functionality of these receptors due to choline treatment and CR may be related to the differences in spatial working memory observed in this experiment. While our animals did learn and remember the position of the platform after a single retention trial, the differences in performance, both due to early diet and to rearing, were no longer evident. Although no additional drug-free animals were tested on days 6 and 7 in the present experiment, we have tested such animals [72] previously and differences due to rearing and diet did persist through such testing. Although there are difficulties in evaluating the specificity of any effects of pharmacological agents including atropine sulfate (e.g. [42]) on learning and memory processes and on neural systems, the effects of atropine on different components of water maze spatial navigation tasks (though not the specific task used in the present experiment) have been carefully assessed and atropine-induced cholinergic blockade does produce temporary, relative specific, albeit difficult to characterize, deficits in spatial learning and memory [77]. For example, atropinized rats have been shown to not be impaired in a task in which the platform is moved randomly around a pool from trial to trial (nor were our animals on their acquisition trials). Such animals have been found also to benefit from previous experience in the pool, in utilizing distal cues and are able to learn position responses and nonspatial discriminations quite well after being subject to cholinergic [77]. In future work, the use of more selective muscarinic receptor subtype antagonists (e.g. [54,75]) and additional control groups might help to specific the nature of the ACh-related changes induced by early diet and complex rearing. In the second experiment, we further enlarged the behavioral assessment of our choline-treated and CR animals, using a land-based, food-motivated, delayed nonmatch-to-position task (DSWSh) which challenged the animal to use more (i.e. knowledge of four positions) previously acquired, trial-unique information, retain it over a greater delay (30 min), then perform a more extensive, prospective sequence of four spatial responses than was the case in Experiment 1. However one common feature of both tasks is that, after a single exposure to the trial-unique information the animals can predict the location of the platform in the MWM, or the food locations in the RAM at the start of the test trials. Once again, those (CR/choline) animals exposed to the perinatal dietary supplementation, and then to a

more complex stimulation history, learned the task to criterion more quickly than did animals raised under other circumstances. While both diet and rearing significantly improved performance, the impact of rearing was again greater. The normally reared (LR) group, given dietary choline, reached criterion faster than did the LR group given the control saline diet; however, their performance was significantly slower than the CR/saline group. Once the animals had reached criterion, all four groups maintained their levels of performance on the first and other (saline) i.p. injection drug day(s). Early diet and rearing did not affect performance on those days, neither in terms of the total number of errors made, nor in the nature of their error-making. Under the influence of atropine sulfate (on the fourth and final drug day) the performance of the four groups were all adversely affected and not significantly different from one another. The attenuation of the performance of all groups on this DSWSh task by somewhat lower (40 versus 50 mg/kg) dose of atropine was much greater than that observed on the DMTP task. Importantly, on the two drug days in which the animals receive tacrine (THA), dose-dependent differences between four groups were evident. THA did improve performance (i.e. reduce errors made) and, once again the improvement was significantly greater for the CR/choline animals than it was for the animals in the other three groups, including the LR/choline group. At the lower level of THA (1.5 mg/kg) both choline groups were superior to both saline groups. Thus, in this instance, while both influenced performances under the influence of THA, diet proved to have a greater impact than did rearing. That was the case whether one focused on the total errors made on retention trials, or on the across phase or retroactive errors. Although the attenuation of the group differences in performance under the influence of the anticholinergic drug atropine observed in Experiments 1 and 2 is open to a wide variety of interpretations, the dose-related improvements in performance under the influence of cholinesterase inhibitor THA provided very preliminary evidence that this pharmacological intervention impacted most on the ‘optimally tuned’ cholinergic spatial memory system of the CR/choline animals. At the lower dose of THA, both choline-treated groups made significantly fewer errors than did the two saline groups and the influence of the perinatal diet per se on the spatial memory system was exposed more clearly. Thus, in addition, the evidence provided for longlasting enhancement of spatial working memory involving trial-unique spatial information in a water maze (Experiment 1) we have evidence to suggest that the ability to learn and remember more trial unique information for a longer time (Experiment 2) is also enhanced in the case of our choline-treated and CR

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animals. On both tasks there was (albeit not all) evidence that, while both variables had an impact on spatial working memory, and combined to produce the greatest improvement, that 3 months of CR had a greater effect than the perinatal exposure to the choline diet for 50 or so days on these measures of spatial competence. Parenthetically, all of the earlier work on the effects of neonatal choline diet utilized Sprague–Dawley strain rats and our work is the first to confirm enhanced working spatial memory in Long–Evans animals. Previous research [43 – 46] has demonstrated that increasing dietary choline during pre- and early postnatal life has profound and long lasting effects on cholinergic transmission and on spatial memory capacity. Previous behavioral assessment of the effects of neonatal choline treatment has focused on spatial memory capacity as measured in terms of choice accuracy in the radial arm maze by food deprived animals retrieving food pellets at the ends of the arms of the maze. Enhanced working and reference memory has consistently been found for choline-treated animals, but only in a relatively demanding spatial memory task in which there were 12 – 18 arm locations to remember. In an earlier report [72] we provided preliminary evidence that both neonatal choline treatment and rearing in a complex environment until adulthood improved performance on the trial-unique DMTP task, but, once again, performance differences were also only evident under more challenging 10 min delay conditions. Subsequently, Dallal et al. [9] reported that prenatal exposure to choline dietary supplementation can also have long-lasting positive effects, reducing age-related impairments in working memory retention observed at long delays on a DMTP task in older animals subsequently tested at 6 – 21 months of age. Our two present behavioral tasks do provide additional support for the proposition that the dietary treatment does produce a long lasting enhancement of the systems underlying such spatial memory processes. If spatial memory capacity in choline-treated animals is enhanced as the earlier work indicated, one would expect it to be evident in other spatial tasks that are affected by temporary or permanent manipulation of cholinergic brain systems (e.g. [5]). We [71] have also found the perinatal choline diet produces superior performance at adulthood on a trans6erse patterning problem requiring the animal to solve concurrently three visual discriminations formed by only three stimuli. This kind of task has also proved to be highly sensitive to both prefrontal and hippocampal lesions [3,79]. Win-shift and transverse patterning tasks both share the fact that animals can not solve or remember them in a simple associative manner and require a configural learning/ memory system including the hippocampus [66]. The

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results of the present experiment confirms that both early rearing and early choline diet have a facilitative impact on ACh-related spatial working memory and that, at least in this instance, the combination of neonatal exposure to dietary choline supplementation and to subsequent CR interacted to produce more impact on working spatial memory capacity than either alone. Although the present experiment doesn’t provide evidence about what aspects of the CR experience contributed to the improvements, the extra handling the CR animals received when they were placed in the enrichment chamber was not likely the critical factor. There is an extensive literature (e.g. [23– 25,30]) that indicates handling itself during the period at issue does not produce superior spatial competence. Moreover, our animals were all handled daily during their first 24 postnatal days. All our animals were also handled once a week prior to and throughout the behavioral testing. Furthermore, the animals in the four groups performed and behaved very comparably during their initial training in the MWM and the RAM when handling effects might have been expected to be evident. Complex rearing in a variety of environmental circumstances generates a cascade of neurochemical events that cause plastic synaptic changes in cortex, hippocampus, cerebellum, etc. (reviewed by [23,30,61,73]). These structural and chemical changes are highly correlated with behavioral improvements including those related to spatial competence and memory (e.g. [30,67,69]). Although the mechanisms underlying these improvements in spatial competencies are uncertain, and complex, the focus of present investigation is on the possible involvement of cholinergic systems. In vivo microdialysis monitoring of hippocampal and frontal cortical ACh during auditory, tactile and visual stimulus presentation, during spatial maze testing, during exploratory of novel objects, and environments all yields an increase in ACh activity (e.g. [28]). Even physical activity (e.g. chronic running) itself can upregulated both HACU and muscarinic receptor binding in hippocampus and improve spatial memory [18]. Although rats are capable of place learning by the third postnatal week, changes in the ability to retain place information and in dependent brain structures continue to mature after that time and exposure to an enriched environment for as little as 12 days improves the ability of young 30–40day-old rats to retain, but not acquire distal-cue, but not proximal cue, place information in the water maze [31,58]. To some extent the present work is predicated on the proposition that both perinatal choline and subsequent environmental stimulation would both modulate parts of a common neural system. Both perinatal dietary choline supplementation

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[57] and complex rearing [19] do impact on cholinergic hipocampal functioning and have each been shown to even enhance LTP induction in area CA1 hippocampal slices. Another of the many potential candidates for mediating these changes produced by choline diet and complex rearing has been neurotropins such as nerve growth factor (NGF) which itself has been shown to improve spatial performance in aged rats, to be of importance for the development and maintenance of cholinergic neurons, and to improve spatial memory in a DSWSh task similar to ours [39,73,80]. Recently Meck and Williams [44] have added to the picture by demonstrating that neonatal choline supplementation also improved the adult rat’s performance in a peak-interval temporal discrimination task. Although all these neonatal choline-induced and the complex rearing-induced improvements in spatial navigation, spatial memory capacity and structure, in working memory, in configural discrimination memory systems, and in interval timing certainly must involve different underlying neural circuitry, the generality of organizational changes caused in the cholinergic cellular networks indicate the importance of both choline intake and a history of environmental stimulation in early development for the emergence of competencies related to widespread underlying cholinergic system [81]. The delayed spatial tasks we used in the present study causes the animal to forage, or seek safety, prospectively (e.g. [26]) and impairments in such tasks with lesions to prefrontal [64] cortex or hippocampal formation [14] is consistent with the idea that the two regions interact when the intact animal seeks location/positions under these circumstances. Several have argued that the hippocampus and prefrontal cortex are functionally as well as anatomically related and that, together, in collaboration with other subcortical and cortical areas regulating spatial cognition [16,22]. It is clear from our findings that both exposure to a perinatal choline dietary supplement and a history of environmental complexity modifies this circuitry to produce more effective processing (and retention) of spatial information which is reflected in superior performance on behavioral tasks which put demand on the animal’s competence to acquire remember, plan and act on such information.

Acknowledgements This investigation was supported by the Natural Sciences and Engineering Council of Canada Grant AP-0179 to R.C. Tees. The assistance of those raising and testing the animals, maintaining and building equipment, particularly Frances Montgomery, Lucille Hoover, Judi Johnston, Shauna Perry, and Manh Tran is gratefully acknowledged.

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