Performance more than working memory disrupted by acute systemic inflammation in rats in appetitive tasks

Physiology & Behavior 73 (2001) 201 ± 210

Performance more than working memory disrupted by acute systemic inflammation in rats in appetitive tasks Ethan Gahtana,*, J. Bruce Overmierb a

Department of Biology, Mugar Life Sciences, Room 414, Northeastern University, Boston, MA 02115, USA b Department of Psychology, University of Minnesota, Minneapolis, MN 55455-0344, USA Received 18 October 2000; received in revised form 8 February 2001; accepted 21 February 2001

Abstract Evidence from molecular biology, epidemiology, behavioral pharmacology, and clinical science support the conclusion that brain inflammation contributes to the pathogenesis of cognitive symptoms in Alzheimer's disease (AD) and other neuropsychological disorders. Three different tests were conducted to determine whether the acute inflammatory response induced by systemic lipopolysaccharide (LPS) treatment is accompanied by a selective disruption of working memory functioning in rats. Doses of LPS sufficient to induce a thermoregulatory response were administered intraperitoneally and their effects on behavioral measures of symbolic working memory, spatial learning, and spatial memory consolidation, were assessed. LPS-induced immune activation was found not to significantly affect memory processes in any of the behavioral tests used. However, LPS-induced immune activation caused performance deficits consistent with a disruptive effect of LPS on motivation and arousal. These results suggest that sickness behavior induced by immune stimulation is not necessarily accompanied by selective impairment in memory processes. The importance of distinguishing cognitive disruption from performance impairment in interpreting the behavioral effects of inflammatory mediators is discussed. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Lipopolysaccharide; Delayed-matching; Neuroinflammation; Rat; Y-maze

1. Introduction Proinflammatory mediators released by activated glial cells during brain inflammation have been proposed to contribute to neuropathology underlying cognitive deficits in sickness and in Alzheimer's disease (AD) [1]. Immuneactivated glial cells synthesize and secrete a host of neuroactive and neurotoxic molecules, including the cytokines interleukin (IL)-1, IL-2, IL-6, TNFa, and IFNg [2,3], as well as complement proteins [4,5], nitrogen and oxygen free radicals [6,7], proteases and protease inhibitors [8,9], and glutamate [10]. Secretory products of immune-activated glial cells have been shown to selectively disrupt neurochemical systems and brain structures involved in learning and memory processes, including serotonin [11,12], acetylcholine [13], and nitric oxide [10] neurotransmission substances, and hippocampal [14] and basal forebrain [15] cell * Corresponding author. Tel.: +1-617-373-4034; fax: +1-617-3733724. E-mail address: [email protected] (E. Gahtan).

function and survival. The hippocampus, a cortical structure involved in working memory, shows high levels of induced cytokine and cytokine receptor expression relative to other brain areas [2,13], and hippocampal neuronal function, including the long-term potentiation electrophysiological model of working memory, is selectively disrupted by inflammatory mediators [14,16]. These findings establish a compelling theoretical link between the processes of brain inflammation and cognitive function. Evidence linking brain inflammation to cognitive dysfunction has come from several types investigative and experimental studies in humans and animals. At least four retrospective investigations have reported a decreased incidence of AD and a decreased rate of progression of Alzheimer's pathology in elderly patients with histories of anti-inflammatory drug use [17 ± 20] (for recent reviews, see Refs. [21,22]). A 6-month prospective study comparing Alzheimer's patients treated with the anti-inflammatory drug, indomethacin, with placebo-treated control subjects, reported similar findings [23]. Additionally, human subjects undergoing cytokine therapy for cancer and hepatitis have

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shown dramatic alterations in learning, memory, affective state, attention, and perceptual and motor functions resulting from peripheral cytokine administration [24 ± 28]. Preliminary evidence that brain-derived inflammatory mediators contribute to the pathogenesis of cognitive dysfunction in AD has stimulated efforts to establish an animal model of immune-mediated cognitive impairment. In an early study, rats injected with IL-1b 60 min prior to training in the Morris water maze spatial navigation learning and memory task were shown to be significantly impaired relative to saline-treated controls [29]. Subsequent studies have also reported impaired learning and memory performance after acute or chronic immune stimulation in rodents, and proinflammatory agents have been reported to induce cytokine production in the brain and disrupt cognitive functioning after central [14] or peripheral [30,31] administration (for a recent review, see Ref. [32]). The demonstration in animals that brain immune activation reliably and selectively disrupts cognitive functioning would further support a pathogenic role of immune mediators in AD and, importantly, facilitate the investigation of the molecular mechanisms mediating the interaction between immune mediators and cognitive processes. An impediment to assessing the effects of immune mediators on cognition is ``sickness behavior,'' a motivational state that increases the probability of recuperative behaviors [33]. Specifically, soporific and anorectic components of sickness behavior may diminish the vigor of performance of behavioral tasks in animals in a manner falsely suggesting cognitive impairment. The present study addressed the question of whether acute inflammation selectively affects learning or memory in rats. Rats were trained to perform in behavioral tasks that were designed to measure, in separate tests: (a) symbolic working memory in a matching-tosample task, (b) spatial learning in a Y-maze task, and (c) spatial memory consolidation in a Y-maze task. A transient systemic inflammatory response was induced by intraperitoneal injection of endotoxin (lipopolysaccharide or LPS), and the effect on performance of behavioral tasks was assessed. In each test, separate behavioral indexes of cognitive functioning and motivation were obtained with the goal of disambiguating the endotoxin treatment effects on motivation vs. those on cognitive functioning. 2. Methods 2.1. Animals Forty-two male Wistar rats (weight 395± 500 g) served as subjects in this study, with each rat included in one of three separate tests. All rats were housed individually under a 12:12-h light ± dark cycle (lights on at 7:00 a.m.), and water was available ad libitum in the home cages. In the delayed matching-to-sample (DMTS) and Y-maze learning tests, food was restricted to maintain weights at 85% of free-

feeding weight, as determined for each animal after at least 4 weeks of free feeding prior to the start of the training. In the Y-maze memory consolidation test, food and water were available ad libitum in the home cages except for a 20-h period before pretraining and retraining sessions during which rats were food-deprived. 2.2. Treatments The LPS used was a phenol extract from Escherichia coli (serotype 0128:B12; Sigma, St. Louis, MO). LPS was dissolved in pyrogen-free saline for use in intraperitoneal injection, with solutions prepared on the day of use. LPS and saline injection volumes were 1.0 ml/kg for all doses. The LPS dose was either 125 or 250 mg/kg. The lower dose (125 mg/kg) was chosen based on its ability to induce a thermoregulatory response in a separate group of Wistar rats (data not shown) and because this dose has been used previously with the same strain of rat to induce transient immune activation [31]. The high dose (250 mg/kg) was designated as twice the concentration of the low dose. 2.3. Apparatus Rats in the DMTS test were trained in Plexiglas and stainless steel operant chambers with dimensions: height = 26.5 cm; length = 30 cm; width = 24 cm. Affixed to the right wall (interface panel) of each chamber was a houselight, two stimulus lights located 8 cm to the left or right of the vertical midline 5 cm from the ceiling of the chamber, a `center' stimulus light, tone generator, and food trough connected to the pellet dispenser (located on the vertical midline at 2, 6, 10, and 25 cm, respectively, from the top of the chamber), two choice lights located 8 cm to the left or right of the vertical midline and each 17cm below the top of the chamber, and two response levers, one located 5 cm directly below each choice light. A PC computer running the Med-PC software and hardware interface system (Med Associates, St. Albans, VT) was used to control all experimental events within operant chambers and to record lever press responses. The Y-maze was constructed of a wood exterior and stainless steel and plastic interior. Three arms, 62 cm in length and 10.25 cm in width at the floor, were connected at 120° angles. Guillotine doors were located 20 cm from the end of each arm and controlled remotely with a manual pulley. Turn direction and trial duration were recorded by the experimenter on each Y-maze trial. 2.4. Procedure 2.4.1. Delayed matching-to-sample Seven rats were trained in a DMTS conditional discrimination task, used to assess nonspatial, ``symbolic'' working memory. In the DMTS procedure, the sample stimulus to be remembered was a light or a tone, generated by devices

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situated along the vertical midline of the response panel. The choice stimuli were two lights, located on the right and left side of the response panel above the two response levers, though only one of the two lights were illuminated on the choice phase of a trial. The rules of the matching-to-sample task were as follows: a light sample must be followed, after the delay period, by a choice response on the lever above which a choice light was illuminated, and a tone sample must be followed by a choice response on the lever above which the choice light was dark. Three lever presses were required on a single lever for a choice response to be registered and produce an outcome (FR 3), a condition designed to minimize the effect of accidental lever presses on accuracy measures. Correct matching choices (presses on lighted lever after light cue; presses on unlighted lever after tone cue) were followed by a food pellet delivery and a 30-s intertrial interval during which the houselight remained illuminated. Incorrect choices were followed by a 70-s time-out period during which all chamber lights were extinguished, followed by illumination of the houselight and 5 s later, the beginning of the next trial. A correction procedure was in place throughout the session according to which every trial ending in an incorrect choice response was repeated until a correct, reinforced response was made. Correction trials were not factored into calculations of session matching accuracy assessment of memory (that is, only initial correct or incorrect choices were counted), but correction trials were factored into calculations of response latency and total session response number. Sessions lasted until 112 reinforcers were obtained or for 3 h, whichever occurred sooner. Four delay intervals (0.1, referred to as ``no-delay,'' 2, 4, or 8 s) occurred within each session on individual trials following each of the two sample stimuli (light or tone). In addition, correct responses were programmed to occur equally often on the left and right levers. Each combination of the two-sample stimuli, two correct response locations, and four delay intervals was programmed to occur equally often within each session according to a pseudo-random schedule, resulting in a total of 16 discrete trial configurations within sessions (pseudo-random means random sampling without replacement, so that all trial types occur with the same relative frequency). As sessions were run until 112 reinforced trials, each of the 16 trial configurations was associated with seven reinforced trials in every completed session. LPS sessions began after rats had reached a criterion of at least 75% correct matching on no-delay trials. LPS was injected intraperitoneally 45 min prior to behavioral sessions. The 125- and 250-mg/kg doses of LPS were tested in DMTS sessions, with dose order counterbalanced among subjects. The purpose of the counterbalancing procedure was to ensure that any effect of LPS exposure order was distributed equally between the two dose conditions, and that an effect of dose would be interpretable as such. In an effort to diminish the possibility

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of habituation to LPS and to allow the rat to recover from the effects of the drug, all LPS sessions (days) were preceded by a saline session and followed by at least three sessions with no injections given. This experiment used a repeated measures design with four dependent measures of matching-to-sample performance: (1) matching accuracy, (2) response latency, (3) total session response number, and (4) number of reinforcers obtained. Matching accuracy, calculated as the percent correct matching, was analyzed as a function of two within-subjects factors, drug treatment (saline, 125, and 250 mg/kg LPS) and delay interval (0.1, 2, 4, and 8 s). Response latency was analyzed as a function of drug treatment and the correct or incorrect response outcome of the trial, both as within-subjects variables. Total session response number and total reinforcers obtained were analyzed as a function of drug treatment only, because these data were cumulated across delay intervals. Planned contrasts using one-tailed t tests (a=.05) were performed to test for differences in dependent measures at specific levels of the independent variables [e.g., LPS treatment (125 or 250 mg/kg) vs. saline treatment at specific delay intervals]. Onetailed t tests were used on the basis of the experimental hypothesis that LPS-induced immune activation would impair memory and/or performance factors supporting DMTS performance and that saline treatment would not affect performance. 2.4.2. Y-maze learning Twelve rats maintained at 85% free-feeding weight were divided into two groups: a saline treatment control group (n = 6) and an LPS treatment group (n = 6). Rats were injected intraperitoneally in the home cage room with saline (1.0 ml/kg) or LPS (125mg/kg) and were transported to the behavioral testing room within 10 min postinjection, before a thermoregulatory response to LPS is evident [34]. Rats were placed in a start box and after 15 s, introduced into the maze by raising a guillotine door which opened into the end of one arm facing the Y junction. On each trial, a 45-mg food pellet was placed in a hidden recess on the terminal wall of the arm either to the left or to the right of the Y-maze junction relative to the start box. The left/right placement of the reward was constant across trials for each rat but counterbalanced across rats within saline and LPS groups. All trials started from the same arm in the distal 20 cm enclosed by the lowered guillotine door (the start box). At the end of 15 s, the guillotine door was raised, and the rat was allowed to enter the maze. There were three possible `choice responses' on each trial: (1) reentry error, in which the rat traverses the length of the start arm and then reverses to reenter the start box; (2) turn error, in which the rat turns into and traverses to the end of the unbaited arm; and (3) correct turn, in which the rat turns into the baited arm and obtains the food reward. The rat was restrained for 20 s in the first goal box it entered after traversing the length of the start arm. Trial latency was measured from the raising of the

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start door, and a maximal trial time of 5 min was imposed (i.e., if a response was not made within 5 min, the trial was scored incorrect and the rat was removed from the maze). At the end of each trial, rats were removed from the maze by the experimenter and returned to individual cages on a rack identical to the home cage rack but located in the Y-maze testing room. Rats were run in series in three separate, ordered groups of four (i.e., Trial 1: Rat a, b, c, d; Trial 2: Rat a, b, c . . .) to a total of 25 trials each. In groups in which a rat was dropped from the experiment, the remainder of the rats were run in series with 60 s extra intertrial interval at the order position that the discontinued rat had previously occupied. Sessions lasted on average 120 min, so the LPS-induced immune response was active during the criterial run. The learning acquisition criterion was defined as six correct out of seven consecutive trials. Specifically, trials were counted in sliding blocks of seven (i.e., Block 1 refers to Trials 1 through 7, Block 2 to Trials 2 through 8, etc.), and the acquisition score was the first block number with six correct trials. For example, a rat that acquires on Block 10 will have six correct trials among Trials 10 through 16. Rats not reaching criterion within 25 trials were given an acquisition score of 20, one more than the maximum score possible within 25 trials. This experiment used a between-groups design, with the differences between LPS- and saline-treated rats evaluated using independent samples t tests. Three such analyses were done, comparing LPS- and saline-treated rats on: (1) the number of trials taken to reach a learning criterion, (2) the average trial latency, and (3) the number of trials completed across the 25-trial training procedure. One-tailed t tests were used to test the experimental hypotheses that LPS treatment would (1) increase in the number of trials taken to reach learning acquisition criterion, (2) increase trial latency, and (3) decrease the total number of Y-maze trials completed. 2.4.3. Y-maze memory consolidation Twenty-three Wistar rats were randomly divided into three treatment groups, referred to as (1) the partial learning saline group (n = 9 for partial learning phase, n = 7 for acquisition phase), (2) nonpartial learning saline group (n = 6), and (3) partial learning LPS group (n = 8). Y-maze trials were run as in the previous Y-maze learning test, and the behavioral dependent measures were recorded in the same manner. Three groups were run on successive days in the following order: (1) a partial learning saline group, (2) a nonpartial learning saline group, and (3) a partial learning LPS group. The partial learning training condition for Groups 1 and 3 was identical. The partial learning procedure consisted of 12 consecutive training trials in the Ymaze spatial choice task. The nonpartial learning saline condition, which can be thought of as a `Y-maze exposure' condition, was included as a negative control for the partial learning saline group, because the partial learning group

would be expected to show faster acquisition if the partial learning effect was operative. Nonpartial learning consisted of 12 Y-maze trials, which were subdivided into three blocks of four trials each. Each four-trial block was designed to include one reinforced and one unreinforced goal box visit for both the left and right sides. This was accomplished in the following way: the first trial was a free-choice trial in which both right and left goal boxes were baited. The second trial was a free-choice trial in which only the goal box not visited on Trial 1 was baited. If the rat had turned in the same direction on the first two trials, a turn in the opposite direction was forced on the third and fourth trials by inserting a boundary at the entry of one arm. In this case, the third trial was reinforced and the forth trial, forced to the same direction, was not. If the rat alternated sides on the first two trials, the third trial was an unreinforced free choice, and the fourth trial was an unreinforced forced choice in the direction needed to balance the right and left turns in that four-trial block. All trial blocks were conducted in the same way. A 50% reinforcement rate was chosen because this was the average reinforcement rate attained during partial learning by the partial learning saline group, the strategy being that the nonpartial learning condition have equivalent Y-maze exposure as the partial learning group (in particular, that the same number of rewards were obtained) with the exception of the contingencies for spatial discrimination learning. All rats received a single injection (saline or LPS) in the home cage room within 5 min following the 12th trial of the initial phase, and were then returned to their home cages where they remained under routine care conditions for 10 days. Acquisition training was conducted 10 days following the pretraining conditions, with each group run on successive days in the same order as in the partial learning condition so that the interval between training sessions was 10 days for all groups. The acquisition training condition was identical for all three groups. Individual rats from the two partial learning groups were retrained to criterion under the same (left or right turn) reward contingency that they had been trained in during the previous partial learning, whereas half of the rats of the nonpartial learning group were randomly assigned to a left turn, and the other half to a right turn reward condition. Criterion for learning was six correct trials in seven consecutive trials, and performance was analyzed in sliding blocks of seven trials (as for the previous Y-maze learning test). Training was discontinued for individual rats when the rat had achieved the learning criterion or after 25 training trials, whichever occurred sooner. In this test as in the Y-maze learning test, rats that entered the baited goal box always consumed the food reward, as determined by the experimenter after each trial. This experiment used a between-groups design. Differences among three groups in the values of two dependent measures were analyzed using a one-factor analysis of variance procedure, and differences between pairs of groups

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Fig. 1. DMTS performance summary charts. (A) Matching accuracy as a function of trial delay interval and LPS (low = 125 mg/kg, high = 250 mg/kg) or saline treatment. There was no statistical effect of treatment on response accuracy. (B) Choice response latency as a function of choice accuracy and LPS or saline treatment, depicting the significant effect of response accuracy on latency under the 250-mg/kg treatment condition. (C) Total session response number as a function of LPS treatment. (D) Number of correct trials completed (or reinforcers obtained) as a function of treatment, showing a significant reduction in completed correct trials under the high-dose LPS condition relative to saline. * P < .05.

were evaluated using independent samples t tests. The dependent measures were: (1) the ordinal value of the trial block upon which a rat achieved learning criterion in the acquisition training phase, and (2) the average trial latency during acquisition training. 3. Results 3.1. Delayed matching-to-sample The primary question addressed in the data analysis was whether LPS treatment disrupted memory-mediated choice accuracy. Fig. 1A depicts the group average matching accuracy as a function of delay interval and drug treatment. The repeated measures ANOVA analyzing percent correct matching revealed a significant effect of trial delay interval, reflecting the decreasing trend in matching accuracy with increasing delay interval [ F(3,18) = 9.66, P < .01]. No effect of LPS treatment on matching accuracy was found [ F(2,12) = 0.51], and there was no statistically significant interaction between the effects of delay interval and drug treatment [ F(3,36) = 1.12]. The ANOVA analyzing choice response latency as a function of drug treatment and matching accuracy (whether the choice response was correct or incorrect on the trial for which a latency value was recorded) revealed a significant effect of matching accuracy, with incorrect trials associated with longer choice latencies than correct trials [ F(1,6) = 5.93, P =.04] (see Fig. 1B). Separate repeated measures ANOVAs comparing average latency values between correct and incorrect responses within individual

treatment conditions showed that response latencies on incorrect trials were significantly higher than latencies on correct trials only in the 250-mg/kg LPS treatment condition [ F(1,6) = 8.01, P < .05]. Although total session responding was numerically lower under both LPS treatment conditions relative to saline, a repeated measures ANOVA testing the effect of drug treatment on total session lever responding failed to detect that LPS significantly affected responding [ F(2,12) = 2.07, P =.16] (Fig. 1C). Importantly, LPS treatment at both doses resulted in a subset of rats (four under the 125-mg/kg dose condition and three under 250 mg/kg) not obtaining the maximum 112 session rewards, an event that never occurred under saline injection (see Fig. 1D). The failure of rats to obtain all 112 session rewards on LPS sessions was a result of a cessation of responding, as is apparent by the absence of a significant difference in matching accuracy between sessions in which all rewards were (saline sessions) or were not (LPS sessions) obtained. The average number of session rewards obtained under LPS treatment was significantly lower than the average number of rewards obtained under the saline condition for both the 125-mg/kg LPS dose [t(6) = 2.14, P < .05] and the 250-mg/kg dose [t(6) = 2.09, P < .05]. 3.2. Y-maze spatial learning Two of the rats (one from the LPS group, one from the saline group) were eliminated from the experiment because they consistently failed to exit the start box within 5 min of the start of the trial. A third rat, from the LPS group, was eliminated from the experiment because an injury (broken

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Fig. 2. (A) Effect of presession LPS or saline on the rate of acquisition of Y-maze learning criterion. Learning criterion was defined as six correct out of seven consecutive trials. (B) Shows group means for (left) trial block of criterion attainment, (middle) number of trials completed (uncompleted trials resulted from exceeding the 5-min maximum trial duration), and (right) average trial latency (units are seconds). * P < .05 for indicated pair.

toenail) prevented it from completing the training procedure. Fig. 2A depicts the rate of learning for the LPS- and salinetreated rats in the Y-maze task. A t test comparing the salineand LPS-treated groups on acquisition trial block showed no statistical effect of LPS treatment on the rate of learning [mean acquisition block = 13.5 ‹ 4.34 and 10.80 ‹ 1.46 for LPS and saline groups, respectively; t(7) = 0.64, P =.53; see Fig. 2B]. A secondary test of the effects of LPS treatment on learning acquisition was based on the latency values for trials comprising the acquisition block. Acquisition block trial latencies were analyzed using a repeated measures ANOVA with trial as a within-subjects repeated measure and treatment group as a between-subjects factor. This analysis was performed because it was expected that once a rat had learned the task, trial latency values would decrease, providing another index of the strength of learning in the two groups. The mean trial latency across all seven trials of the acquisition block was 9.32 ‹ 1.98 and 7.07 ‹ 0.70 for the LPS and saline group, respectively. The ANOVA showed no significant effect of trials within-subjects [ F(6,36) = 1.62, P =.15], no betweengroups LPS treatment effect [ F(1,6) = 0.69, P =.43] and no Trials  Treatment interaction [ F=(6,36) = 0.74, P =.62].

Therefore, latency did not decrease significantly across the seven trials of the acquisition block in either the saline or the LPS treatment group, and overall latency on the acquisition block was not significantly different between groups. Trial latency for completed trials was not statistically different between the LPS- and saline-treated groups [t(7) = 1.31, P =.22], although the average latency value was somewhat higher in the LPS group (mean latency = 12.43 ‹ 3.09 vs. 7.78 ‹ 1.99 s for LPS and saline groups, respectively; Fig. 2B). However, the LPS treatment group did differ significantly from the saline group on the total number of training trials completed (Fig. 2B). The LPS group completed fewer trials on average than the saline treatment group [t(7) = 2.89, P < .05], with all rats in the saline group completing all 25 trials while only one rat in the LPS group completed all 25 trials (uncompleted trials were trials in which the 5-min maximum trial duration was exceeded). 3.3. Y-maze partial learning consolidation Fig. 3 depicts the spatial choice accuracy performance of rats in the nonpartial learning saline group, partial learning

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saline group, and partial learning LPS group during the partial learning phase (recall that the LPS or saline was administered after Phase 1 partial training). The proportion of reinforced trials among the 12 partial learning trials was exactly 0.50 ( ‹ 0.14) for the partial learning saline group and, with the nonpartial learning condition designed to be equivalent in proportion correct during partial learning as the partial learning saline group, this value was also 0.50 in the nonpartial learning saline group. The partial learning LPS group had on average a slightly higher choice accuracy than did the saline groups, but this difference was not statistically significant [means = 0.50 ‹ 0.14 and 0.60 ‹ 0.14 for saline and LPS partial learning groups, respectively; t(15) = 1.75, P =.10]. Similarly, the average trial latency did not differ among the three groups [ F(2,20) = 0.89, P =.42; see Fig. 3B]. As anticipated, the partial learning saline group attained the learning criterion significantly faster than did the nonpartial learning saline group, as determined by a t test comparing acquisition block [average acquisition block was 7.71 and 12.16 for saline partial learning and saline nonpartial learning group, respectively; t(11) = 1.87, P < .05]. The LPS partial learning group, while having a mean acquisition block slightly higher than that of the saline partial learning group (9.75 vs. 7.71 for the LPS and saline partial learning groups, respectively), did not differ significantly from the saline partial learning group on acquisition rate [t(13) = 0.59, P =.56; see Fig. 4A]. Acquisition rate also did not differ significantly between the LPS partial learning group and saline nonpartial learning group [t(12) = 0.66, P =.52].

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Fig. 4. Y-maze spatial discrimination performance during the learning criterion acquisition phase. (A) Average acquisition block for each group. (B) Group average trial latencies. * One-tailed P < .05.

The saline partial learning group did not differ significantly on average trial latency during the final acquisition phase from the LPS partial learning group [t(13) = 2.00, P > .06] or the saline nonpartial learning group [t(11) = 1.09, P =.29; see Fig. 4B] Ð nor were they expected to, because all were performing while in a normal physiological state.

4. Discussion

Fig. 3. Y-maze spatial choice performance during the partial training phase. (A) Percent correct discrimination. (B) Average trial latency.

This study failed to show evidence for a strong, selective effect of LPS-induced immune activation on working memory, learning, or learning consolidation in rats. The Y-maze partial learning experiment potentially contains evidence of disruption of memory consolidation by LPS. Partial learning did significantly enhance acquisition performance, as shown by the significantly faster acquisition in the saline partial learning vs. saline nonpartial learning group. As memory consolidation performance in the LPS group was intermediate between the partial learning saline group and the nonpartial learning group, though not statistically different from either, it can be said that the effect of LPS is neither null nor completely obliterative of memory consolidation. That is, the data may reflect a partial disruption of memory consolidation by LPS. LPS clearly did disrupt performance in DMTS and Ymaze learning tasks in a manner suggesting decreased motivation to respond, but not impaired mnemonic functioning. These results appear to conflict with other recent reports of learning and memory impairment in rats induced by peripheral immunostimulant treatment. Existing reports

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which have addressed whether peripherally administered LPS or cytokines have direct effects on mnemonic functioning in rats have indicated that such effects, when apparent, are labile and highly sensitive to procedural and methodological parameters both of pharmacological treatments and behavioral tasks. Additionally, the expression of sickness may compromise the specificity of a behavioral index of cognition. In a previous study of the cognitive effects of LPS treatment, male Wistar rats were injected intraperitoneally with LPS (250 mg/kg) 90 min prior to the second training session in a series of autoshaping/classical conditioning sessions [30]. Rats in this study were reported to be impaired in learning acquisition of the autoshaping task across sessions, as indicated by longer response latencies on subsequent trials relative to a saline-injected group. Because learning impairments were first apparent in this study on the session following (24 h after) LPS injection, but not on the LPS day itself, the authors interpret the effect on latency as a selective action of LPS on postsession-2 learning consolidation. However, as the authors note, LPS may have induced conditioned aversion to the relatively novel testing environment on Day 2 of training, which could account for the increased response latencies observed in LPS-treated rats. This interpretation is supported by the previous demonstration that intraperitoneal LPS is a potent inducer of conditioned taste aversion in rats [35]. An important design attribute of our Y-maze memory consolidation test reported in the current study is that it minimized the possibility of LPS conditioned aversion to the testing environment, because rats were not exposed to the testing environment under the acute effects of LPS. A disruptive effect of peripheral LPS on learning consolidation of a classically conditioned fear response in rats has also been reported [31]. In this experiment, Wistar rats given LPS (ip) immediately following contextual fear conditioning were impaired on a memory test conducted 48 h after conditioning, as demonstrated by a diminished contextevoked freezing response. Acute behavioral suppressive effects of LPS cannot account for this finding because, as in the memory consolidation test reported here, LPS was given after initial training and the subsequent test of learning occurred days after LPS injection. Moreover, the impairment in contextual fear learning among LPS-treated rats was manifest as increased, not decreased, locomotor behavior. In the study by Pugh et al. [31], IL-1 receptor antagonist administered directly into the brain ventricles immediately after conditioning trials prevented the disruption of memory consolidation caused by intraperitoneal LPS. This finding, as well as other previous studies [36,37] suggests that IL-1 is induced in the brain following intraperitoneal LPS injection, and supports the conclusion that intraperitoneal LPS treatment in the present study induced central cytokine production. Surprisingly, Pugh et al. [31] found that lower doses of LPS (0.125 and 0.25 mg/kg) impaired contextual learning, while a higher dose (0.5 mg/kg) had no effect. This

finding suggests additional complexity in the actions of LPS on neuronal mechanisms of learning. Two additional reports from the same group of investigators using identical behavioral tests showed that social isolation [38] and intracerebroventricular administration of the viral coat protein, gp120 [39], both increased brain IL1b protein levels and disrupted memory consolidation in contextual but not auditory cued fear conditioning. The differential effects of brain immune activation on contextual vs. cued fear conditioning [31,38,39], suggests that brain immune activation selectively disrupts hippocampal function, because the contextual task but not the cued task is believed to be hippocampal-mediated [31,38,39]. Moreover, because IL-1b antagonists consistently prevented the disruption of memory consolidation, this cytokine in particular is implicated in modulating hippocampal function. However, if the memory consolidation paradigm used in our study is not hippocampal-dependent, the studies by Pugh et al. [31,38,39] would predict no effect of our postpartial learning LPS treatment on subsequent acquisition. Whether our appetitive Y-maze spatial learning task is hippocampusmediated may depend on the strategy employed by the animal [40,41]. In our learning consolidation Y-maze experiment, the intramaze visual environment was nearly identical for each maze arm and the maze was wiped with ammonia solution after each trial to minimize olfactory cues (e.g., urine). These procedures were designed to minimize intramaze cues and thus the likelihood that rats would learn by simple association of cues with the reward Ð a learning strategy that is resistant to disruption by hippocampal lesion [42,43]. With substantial extramaze cues as in our study, the learning is likely spatial hippocampal-place learning [42]. The fact that Pugh et al. [31], used an aversive task and we used an appetitive task could account for the discrepancy in the effects of LPS on memory consolidation in the two studies. These motivationally different tasks may be differentially sensitive to the effects of LPS on memory consolidation. Food-motivated performance in our memory consolidation task was not effected by the acute anorectic effects of LPS, since acquisition training trials were conducted days after LPS treatment. However, hippocampal [44], as well as cerebellar [45] lesions, and behavioral manipulations [46], all can differentially affect appetitive and aversive memory performance in rats, suggesting the possibility that in the study by Pugh et al. [31], LPS selectively affected brain areas involved in memory for aversive events. Another previous study examined the effects of IL-1b (100 or 1000 ng, ip) on water maze spatial learning in mice injected 2 h prior to training in a spaced-trials or massedtrials water maze protocol using warm (24°C) or cool (18°C) water in the pool [32]. IL-1b at both doses tested was reported to reliably cause sickness behavior in mice. However, IL-1b had no effect on water maze learning when a spaced-trials procedure was used. In the massed-trials procedure, the low IL-1b dose produced learning impair-

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ments only when swimming water was warm, while IL-1btreated mice performed equivalently to vehicle controls when water was cool. Additionally, the high IL-1b dose actually facilitated water maze learning relative to controls. Thus, as in the previously cited study of LPS effects on fear conditioning in rats [31], the effects of peripheral immune stimulation were complex and are difficult to interpret as a simple disruptive effect on neuronal mechanisms of learning. Taken together, these studies illustrate that behavioral responses of rodents to peripheral immune stimulation is highly sensitive to the behavioral measures and procedures used, and to the dose of immunostimulant administered. Furthermore, in behavioral assessments of cognitive functioning, the timing of immune stimulation relative to the behavioral test is likely to be of crucial importance to the interpretation of changes in task performance; impaired performance of animals in a learning tasks conducted under the acute effects of immune stimulation may be attributed to noncognitive effects of treatment such as decreased motivation, conditioned aversion, or behavioral stupor. The effect of water temperature on the modulation of water maze learning by IL-1b in mice in the study cited above [32] illustrates even more clearly the influence of motivation on the behavioral response during immune-mediated sickness. The DMTS test of working memory and Y-maze test of learning reported here, in which LPS was administered shortly before behavioral sessions, showed a consistent disruptive effect of LPS treatment on task performance factors. Specifically, LPS administration was associated with increased response latency and/or decreased overall response number in the DMTS task, and decreased number of food rewards obtained in the DMTS and Y-maze learning tasks. In all sessions conducted after vehicle injection or under no-injection conditions, rats always obtained the maximum number of rewards, and the reduction by LPS in the number of rewards obtained may be a factor in the failure to detect an effect of LPS on learning or memory. That is, rats may have discontinued responding as the drug took effect, and, had rats been in some way induced to respond, a memory deficit may have been manifest as decreased matching accuracy. This study evaluated the effects of acute neuroinflammation on mnemonic functioning in rats. The relevance of these experiments as model of putative immune-mediated cognitive dysfunction in human dementias, such as in AD, depends upon the degree of homology of the neuroinflammatory processes and of the cognitive functions in the two systems. Variants of the DMTS working memory task and Y-maze learning tasks employed in this study have been used extensively as experimental models of human mnemonic function (reviewed in Refs. [47 ± 49]). In contrast, peripheral LPS administration is not a validated model of neuroinflammation in human disease, and this procedure fails to replicate critical components of the inflammatory condition in AD. Specifically, we recognize that systemic

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inflammation, as induced in this and previous [30 ± 32] studies by acute peripheral injection of a nonspecific immunostimulant, cannot recreate the chronic, central neuroinflammation of AD. The important goal of establishing a robust animal model for neuroinflammatory pathogenesis of Alzheimer's dementia would be advanced by the development of longer-acting, centrally localized immune stimulants, or a method for inducible autoimmunity against the Ab protein [50].

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