Exposure to Kynurenic Acid During Adolescence Produces Memory Deficits in Adulthood

Schizophrenia Bulletin vol. 38 no. 4 pp. 769–778, 2012 doi:10.1093/schbul/sbq151 Advance Access publication on December 20, 2010 Exposure to Kynurenic Acid During Adolescence Produces Memory Deficits in Adulthood 1 Department of Psychological and Brain Sciences, Dartmouth College, Hanover, NH; 2Department of Psychiatry, Dartmouth Medical School, Lebanon, NH; 3New York State Psychiatric Institute, New York, NY *To whom correspondence should be addressed; tel: 603-646-3439, fax: 603-646-1419, e-mail: david.j.bucci@dartmouth.edu by astrocytes.1,2 KYNA acts as an antagonist of both a7 nicotinic acetylcholine receptors (nAChRs) and the glycineB site of n-methyl-d-aspartate glutamate receptors (n-methyl-d-aspartateglutamate [NMDA]-Rs),3 both of which have critical roles in neural plasticity as well as learning and memory.4–7 Interestingly, KYNA levels are elevated in the brains and cerebral spinal fluid of persons with schizophrenia,8–11 leading to the notion that changes in KYNA concentration might contribute to cognitive dysfunction associated with schizophrenia.12 Indeed, recent studies have shown that increasing endogenous KYNA concentration by administering l-kynurenine (L-KYN), the precursor of KYNA, results in spatial and contextual learning deficits in rats13,14 as well as impaired sensory gating, prepulse inhibition, and attention.15–17 Conversely, knockout mice with abnormally low levels of KYNA exhibit improved spatial learning abilities.18 These findings are consistent with evidence that persons with schizophrenia experience deficits in spatial and contextual learning and memory.19,20 Most of these prior studies of the effects of elevated KYNA concentration on cognitive function in mice or rats have examined adult subjects and the effects of acute treatment with L-KYN to temporarily increase KYNA levels. However, increased concentration of KYNA in the brains of persons with schizophrenia may occur over a much longer period of time. Indeed, recent studies indicate that genotypic alterations may underlie changes in KYNA concentration associated with schizophrenia.21–24 One implication of these findings is that exposure to high levels of KYNA may begin early in life. It is well established that both acetylcholine and glutamate play critical roles in normal brain development25–27 and that NMDA-Rs and a7 nAChRs are involved in synaptic plasticity and neural development.4,5 Thus, increases in KYNA concentration and the resulting inhibition of NMDA-Rs and/or a7 nAChRs during critical stages of The glia-derived molecule kynurenic acid (KYNA) is an antagonist of a7 nicotinic acetylcholine receptors and the glycineB binding site on n-methyl-d-aspartateglutamate receptors, both of which have critical roles in neural plasticity as well as learning and memory. KYNA levels are increased in the brains and cerebral spinal fluid of persons with schizophrenia, leading to the notion that changes in KYNA concentration might contribute to cognitive dysfunction associated with this disorder. Indeed, recent studies indicate that increasing endogenous KYNA concentration by administering l-kynurenine (L-KYN, the precursor of KYNA) impairs spatial as well as contextual learning and memory in adult rats. In the present study, rats were treated with L-KYN (100 mg/ kg) throughout adolescence to increase endogenous KYNA concentration during this critical time in brain development. Rats were then tested drug-free as adults to test the hypothesis that exposure to elevated levels of KYNA during development may contribute to cognitive dysfunction later in life. Consistent with prior studies in which adult rats were treated acutely with L-KYN, juvenile rats exposed to increased KYNA concentration during adolescence exhibited deficits in contextual fear memory, but cue-specific fear memory was not impaired. In addition, rats treated with L-KYN as adolescents were impaired on a novel object recognition memory task when tested as adults. The memory deficits could not be explained by drug-induced changes in locomotor activity or shock sensitivity. Together, these findings add to the growing literature supporting the notion that exposure to increased concentration of KYNA may contribute to cognitive deficits typically observed in schizophrenia. Key words: schizophrenia/cholinergic/fear conditioning/ object recognition Introduction Kynurenic acid (KYNA) is a product of tryptophan metabolism that is synthesized and released in the brain © The Author 2012. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com 769 Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 Cynthia O. Akagbosu1, Gretchen C. Evans1, Danielle Gulick2, Raymond F. Suckow3, and David J. Bucci*,1 J. K. Forsyth et al. Methods Subjects Forty-six male Long Evans rats (21 days old) were obtained from Harlan Laboratories. Rats were housed in groups of 4 and allowed 6 days to acclimate to the vivarium before the experiment began. Throughout the study, rats were maintained on a 12:12 light-dark cycle (lights on at 7 AM, off at 7 PM) with food available ad libitum (Purina standard rat chow; Nestle Purina). Rats were monitored and cared for in compliance with Association for Assessment and Accreditation of Laboratory Animal Care guidelines and the Dartmouth College Institutional Animal Care and Use Committee. 1-piperazineethanesulfonic acid (HEPES) buffer. The solution was brought to a neutral pH by adding 1N hydrochloric acid. Rats in the L-KYN group received a 100 mg/kg dose of L-KYN, which has been shown to produce a 3–4 fold increase in brain KYNA 2 h after injection.16,30 Treatment Regimen On postnatal day (PND) 27, rats were randomly assigned to either the L-KYN group or the control group and exposed to a 3-day-on/3-day-off drug treatment regimen as follows: On postnatal days 27–29, each rat received a daily intraperitoneal injection of either L-KYN (100 mg/kg) or a comparable volume of 0.1 M HEPES buffer (vehicle). Injections were made on alternate sides of the abdomen each day to reduce discomfort at the injection site. During the subsequent 3 days (ie, postnatal days 30–32), no injections were administered. This 6-day cycle was repeated another 4 times, resulting in a total of 15 injections of L-KYN (or vehicle), the last of which occurred on postnatal day 53. This procedure reduced distress and sensitivity at the injection site compared with injecting every day from postnatal days 27–53. In addition, the cycle of 3 injection days followed by 3 no-injection days was designed to minimize the potential for metabolic adaptations following chronic systemic L-KYN treatment. Indeed, Vecsei and colleagues31 reported that KYNA concentrations peaked during the first 3 days of daily injections of L-KYN but subsequently decreased following additional daily treatment. Assessment of KYNA Concentration following L-KYN Treatment Twenty-four of the 46 rats were sacrificed at various time points during the course of the drug treatment regimen to assess the effects of L-KYN treatment on KYNA concentration. Three vehicle-treated rats and 3 L-KYN-treated rats were sacrificed using isoflurane followed by rapid decapitation at each of the following time points: (a) 2 h after the injection on PND 27 (ie, after the first very injection of either vehicle or LKYN); (b) on PND 32 (ie, 3 days after the 1st 3-day course of treatment); (c) 2 h after the injection on PND 39 (ie, the first treatment day of the 3rd treatment cycle); and (d) on PND 61 (ie, the age corresponding to the first day of behavioral testing for the remaining rats). Brains were rapidly dissected and frozen on dry ice for subsequent analysis of KYNA concentration using high-performance liquid chromatography. Brain Kynurenic Acid Measurement Drug Preparation L-kynurenine (L-KYN; Sigma) was prepared fresh daily by dissolving in 2N sodium hydroxide and bringing to a final volume (30 mg/ml) with 0.1 M 4-(2-hydroxyethyl)770 Brain concentrations of kynurenic acid (KYNA) were determined using a modified validated method applied to human serum.32 Whole rat brains were stored frozen at 20C, then thawed to room temperature, and weighed Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 development could have lasting impacts on brain morphology and/or cognitive function in adulthood. The present study was therefore designed to test cognitive function in rats that were exposed to high levels of endogenous KYNA concentration throughout adolescence, a critical and particularly vulnerable stage in brain development. Moreover, there is substantial evidence that schizophrenia often develops during adolescence,28 and it has been shown that the number of synapses lost during adolescence is 2 times higher in patients with schizophrenia compared with controls.29 Starting on postnatal day 27, rats received 3 consecutive days of treatment with 100 mg/kg of L-KYN (or vehicle) which has previously been shown to increase KYNA concentration by 3–4 fold,16,30 a level comparable with that observed in schizophrenia.8,10 This method of increasing endogenous KYNA concentration is ideal from a clinical perspective because elevations in L-KYN are thought to underlie increases in KYNA concentration in schizophrenia.21–23 During the next 3 days, no injections were administered; this cycle (3 days on drug, 3 days off drug) was subsequently repeated through postnatal day 53. Rats were then given an 8-day rest period during which no drugs were administered and behavioral training started on day 61. Thus, subjects were tested drug-free as young adults to determine how exposure to KYNA during adolescence may impact cognitive function in adulthood. All rats were first tested in a standard fear conditioning paradigm, in which contextual but not cue-specific fear has previously been shown to be sensitive to acute treatment with LKYN.14 After the fear-conditioning task was completed, rats were tested in a novel object recognition task to evaluate object memory. It was hypothesized that exposure to elevated concentration of KYNA during adolescence would impair contextual fear memory and object recognition in adulthood. Adolescent KYNA Exposure and Memory Behavioral Testing The remaining 22 rats were used to assess the effects of L-KYN treatment during adolescence on learning and memory. Training and testing began 8 days after the final injection, ie, when rats were 61 days old. Using this regimen, rats were thus treated with L-KYN or vehicle throughout adolescence and then behavioral tested as young adults without drug on board. Behavioral Apparatus Fear Conditioning. The fear conditioning procedure was carried out in standard conditioning chambers (Med Associates Inc.) measuring 24 3 30.5 3 29 cm. The chambers were connected to a computer and enclosed in sound-attenuating chambers (62 3 563 56 cm) outfitted with an exhaust fan to provide airflow and background noise (;68 dB). The chambers consisted of aluminum front and back walls, clear acrylic sides and top, and grid floors. A dimly illuminated food cup was recessed in the center of the front wall, and a panel light was located 5 cm above the opening to the recessed food cup, neither of which was utilized in this experiment. A 2.8-W house light providing background illumination was mounted 15 cm above the food cup. A speaker was located 15 cm above and to the right of the food cup and was used to present the auditory stimulus (1500 Hz, 78 dB). In addition, a 500-ms delivery of a 0.75 mA constant current shock through the grid floor of the operant chamber served as the unconditioned stimulus. Surveillance cameras located inside the surrounding shell were used to record the rats’ behavior. Novel Object Recognition. The novel object recognition task was conducted in a small dimly lit room. The apparatus consisted of a plastic tub (30 W 3 34 L 3 38 H cm) and objects made of plastic building blocks (Learning Resources Inc) constructed into distinct configurations with approximately the same dimensions (7 W 3 7 L 3 9 H cm). One of the objects was purple and roughly similar in shape to a dumbbell, and the other was green and shaped like a 3-dimensional axis. A video camera was mounted above the tub, and behavior was recorded on a DVD recorder. Behavioral Procedures Sixteen rats (8 previously treated with vehicle and 8 previously treated with L-KYN) were first trained in a fear conditioning procedure and then the novel object recognition memory task as follows. Fear Conditioning. Rats were trained in a widely used fear-conditioning task described previously.14,34 The training session consisted of two 10-s presentations of the tone followed immediately by a 500 ms, 0.75 mA foot shock (intertrial interval of 64 s). The first trial began 3 min after the rat was placed in the chamber. Twentyfour hours after the initial training session, rats were reexposed to the original training chamber for a 6 min–24 s context test session during which no tones or shocks were presented. Twenty-four hours later, the tone test session was carried out by placing the rats in a novel context and presenting the tone 20 times (10 s each, 30-s intertrial interval) beginning 30 s after the rat was placed in the chamber. Again, no shock was delivered during this test session. The novel context consisted of the original training chambers outfitted with plain white paper on the walls of the chamber to hide the recessed food cup and other stimuli present on the aluminum walls. Cardboard was also placed on top of the grid floor to provide a different tactile stimulus, and a cup containing Vicks VapoRub and vinegar was placed in each soundattenuating chamber to provide different olfactory cues. It has been shown previously that rats exhibit very little freezing behavior to the new context itself.14,34 All rats received the context test session first, followed by the cue test session because this has previously been shown to be the optimal method for obtaining the most independent assessment of both auditory and contextual fear conditioning in the same rats.35 Our laboratory has also previously examined whether the order of testing influences levels of freezing to the context and tone during the test sessions, and we have found identical results when the cue test session was conducted prior to the context test session.34 Novel Object Recognition. Twenty-four hours after the fear conditioning procedure was completed, rats were tested using a 3-day novel object recognition task.36,37 On day 1 (habituation session), each rat was exposed 771 Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 prior to analysis. Each brain was homogenized in 5.0 ml deionized water, and 500 ll of brain homogenate was treated with 3.4 M perchloric acid, vortexed, and centrifuged at 13 000 rpm. Fifteen microliters of the supernatant was injected for chromatographic analysis. The chromatographic system employed a Supelcosil octasilyl LC-8 column (75 3 3.0 mm, 3 lm particle size) with a mobile phase consisting of 92% (v/v) ammonium acetate and zinc acetate (50 mmol/l each; pH adjusted to 6.5),33 and 8% (v/v) methanol. A flow rate of 0.55 ml/min eluted KYNA at ;3.9 min and was detected using an Agilent Model 1321 Fluorescence Detector operating at kex = 251 nm and kem = 398 nm. A 7-point calibration curve encompassing the expected concentration range of KYNA was included with each run. The low limit of quantification of KYNA was ;0.4 ng/ml of brain homogenate. A correction factor ((brain weight þ volume of water)/brain weight) was generated for each brain sample to compensate for differences in brain weight in the determination of the final brain concentration of KYNA. J. K. Forsyth et al. to the tub individually for 10 min to habituate to the testing environment. On day 2 (sample session), rats were placed in the tub and given 5 min to explore 2 identical sample objects. Exploration was defined as direct sniffing or snout contact with the object. Twenty-four hours later (test session), rats were again placed in the tub with one familiar and one novel object (counter-balanced across subjects) and given 2 min to investigate the items. An investigator, who was blind to the experimental conditions, later scored the time spent exploring each object. Novel Object Recognition. The mean time spent exploring the identical objects during the sample session was analyzed using a 2-sample t-test. For the test session data, a discrimination ratio served as the dependent variable of interest and was calculated as the time spent exploring the novel object divided by total time spent exploring both objects. This measure takes into account individual differences in total exploratory behavior. A one-sample t-test (expected value = 0.5, indicating no discrimination between the novel and familiar objects) was used to determine whether each group was able to successfully discriminate between the 2 objects. A 2-sample t-test was used to compare the discrimination ratio between the vehicle and L-KYN treatment groups. In addition, a repeated measures ANOVA was conducted to assess the time spent exploring the familiar vs novel object in each group. The between-subjects variable was Group (control, L-KYN) and the within-subjects variable was object (familiar, novel). Behavioral Observations and Data Analysis Fear Conditioning. Freezing served as the index of conditioned fear and was operationally defined as total motor immobility except for breathing.38,39 On the training day, the incidence of freezing behavior was recorded during the 64 s period prior to the first trial (baseline freezing) and during the 64 s period following each trial (postshock freezing). During each of the 64-s periods, the rat’s behavior was observed and recorded every 8 s, resulting in 8 observations per period. The frequency of freezing behavior was calculated by dividing the number of instances of freezing by 8 and multiplying by 100 to convert the data into a percentage of total observations (eg, 4 observations of freezing out of 8 observations = 50%). During the context test session, the 12 min–48 s session was divided into 64-s bins and freezing was observed every 8 s as described above. For the tone test session, freezing was recorded every 2 s during each 10-s presentation of the tone. In this case, the frequency of freezing behavior was calculated by dividing the number of freezing observations by 5 (eg, 2 observations of freezing out of 5 = 40%). A single primary observer scored all the behavioral data, while a second observer scored a subset of the data to assess objectivity. Both observers were blind to treatment condition, and their observations were highly correlated (r = .9; P < .0001). For each observation period, the percentage of freezing behavior was averaged across the rats in each group. 772 Locomotor Activity. The video recordings of the rats’ behavior during the habituation session of the novel object recognition task was reanalyzed to test for treatment effects on locomotor behavior. The image of the tub was divided into 3 equal parts by drawing 2 vertical lines on the video screen. An observer who was blind to treatment condition recorded the number of line crossings made by each rat during the 10-min session. Importantly, testing locomotor activity in the same apparatus used to test novel object recognition allowed for task-relevant assessment of potential changes in locomotion. A 2-sample ttest was used to compare the average number of line crossings exhibited by rats in the saline and L-KYN treatment groups. An alpha level of .05 was adopted for all analyses in the experiment. Results KYNA Concentration The percent change in KYNA concentration at various times during the drug treatment regimen is shown in table 1. The very first dose of L-KYN increased the concentration of KYNA ;5-fold in 27-day-old rats (table 1, column 1). The difference in concentration between vehicle-treated rats and L-KYN-treated rats was statistically significant (t4 = 9.5, P < .0007), indicating that an acute 100 mg/kg dose of L-KYN effectively increases KYNA Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 Shock Sensitivity. Six rats did not receive training or testing in the tasks described above and were used to determine if L-KYN treatment during adolescence affected shock sensitivity. On postnatal day 61 (ie, the same time point at which fear conditioning took place task above), 3 vehicle-treated rats and 3 L-KYN-treated rats were individually placed in the conditioning chambers and allowed to acclimate to the chambers for 2 min. Foot shock was then delivered for 0.5 s using a current of 0.1 mA. If the rat did not exhibit a behavioral response to the shock (defined as a startle, flinch, freeze, or jump as observed by an observer who was blind to condition), the current was increased by 0.05 mA and another shock applied (30 s interstimulus interval). This procedure was repeated until the rat exhibited a behavioral response to shock delivery. Analyses of freezing behavior during training and the tone test session were conducted using repeated measures ANOVA with Group as the between-subjects variable and Trial as the within-subjects variable. For the context test session, a repeated measures ANOVA was conducted using Group as the between-subjects variable and Block (ie, 64-s epoch) as the within-subject variable. Adolescent KYNA Exposure and Memory Table 1. KYNA Concentration Measured at Various Time Points during and after the Treatment Regimen Age (PND) 27 32 39 61 Corresponding time point during treatment (KYNA) in controls (pMol/g) (KYNA) in L-KYN-treated rats %increase in (KYNA) 1st day of 1st cycle 17.6 6 7.8 95.8 6 2.7* 543% 6th day of 1st cycle 28.9 6 6.5 32.1 6 3.1 111% 1st day of 3rd cycle 19.7 6 6.8 90.2 6 11.1* 457% 1st day of behavioral training 31.4 6 8.3 37.6 6 4.9 119% concentration in young rats. On the 3rd day following the first cycle of treatment, KYNA concentration returned to normal as expected (table 1, column 2; t4 = 0.4, P > .6). Importantly, L-KYN administration again increased KYNA concentration during the 3rd cycle of treatment (table 1, column 3; t4 = 5.4, P < .006). Finally, as indicated in column 4, KYNA concentration returned to control levels by PND 61 (t4 = 0.6, P > .5), which corresponds to the first day of behavioral training for rats that underwent the behavioral tasks. whereas L-KYN-treated rats spent comparable amounts of time with the novel and familiar objects. This was supported by analysis of the discrimination ratios, which Fear Conditioning Freezing behavior for one control rat and one L-KYNtreated rat was not available for analysis (resulting in n = 7 per group for the fear conditioning analyses). As shown in figure 1A, the amount of postshock freezing was comparable between control and L-KYN-treated-rats during the training session and increased as training progressed. This was confirmed by a repeated measures ANOVA, which revealed a significant main effect of Trial (F2,24 = 51.5, P < .0001). There was no significant main effect of Group (P > 0.1) nor was there a Group 3 Trial interaction (P > .6). Despite comparable freezing during training, freezing was reduced in the L-KYN group during the context test session (figure 1B). A repeated measures ANOVA revealed a main effect of Group (F1,12 = 6.2, P < .03). The Group 3 Epoch interaction was not statistically significant (P > .3). Data from the tone test session are presented in figure 1C. Rats in both groups exhibited high levels of freezing to the tone, which gradually diminished over the course of the extinction session (F15,180 = 7.0, P < .0001). There was no main effect of Group (P > .1) and no significant Group 3 Trial interaction (P > .9). Novel Object Recognition There was no significant group difference in the time spent exploring the objects during the sample session (P > .4). Mean exploration times were 126.1 6 10.4 s and 137.4 6 9.3 s for the control and L-KYN groups, respectively. Data from the object recognition test day are shown in figure 2. Control rats spent significantly more time exploring the novel object than the familiar object, Fig. 1. Effects of L-KYN Administration on Fear Conditioning. Rats treated with L-KYN during adolescence exhibited comparable levels of freezing during training (A) and during the tone test session (C) compared with vehicle-treated control rats. In contrast, freezing was reduced in L-KYN-treated rats during the context test session (B). Freezing was defined as the absence of movement (except for respiration). During training, each rat was observed every 8 s during the 64-s period preceding the first trial (BL, baseline) and during the 64-s period after each trial (PS, postshock period). For the context test, the test period was broken up into 64-s epochs and rats were observed every 8 s. For the tone test session, rats were observed every 2 s during the tone presentation. Data are mean 6 SE. 773 Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 Note: As described in the Methods, rats underwent a 3-day on, 3-day off cycle of drug treatment from postnatal day (PND) 27 through PND 53. KYNA concentration was determined from 3 vehicle-treated rats and 3 L-KYN-treated rats at each time point. *P < .05. J. K. Forsyth et al. Shock Sensitivity Fig. 2. Effects of L-KYN Administration during Adolescence on Novel Object Recognition in Adulthood. The amount of time control rats and L-KYN-treated rats spent exploring the novel and familiar objects during the test session is shown in panel A. Panel B depicts the average discrimination ratios (6SEM) of each group, calculated as the time spent exploring the novel object divided by total time spent exploring both objects. A discrimination ratio of 0.5 indicates no discrimination between the novel and familiar objects, as indicated by the dotted line. Values significantly greater than 0.5 reflect successful discrimination, ie, rats spent more time exploring the novel vs. familiar object. Control rats, but not those treated with L-KYN during adolescence, discriminated between the novel and familiar objects indicating that object memory was impaired in L-KYN-treated rats. *P < .03. indicated that the ratio for the control group differed significantly from 0.5 (t7 = 3.7, P < .01) but the ratio for the L-KYN group did not (t7 = 0.9, P > .3). In addition, the discrimination ratio for the control group was significantly higher compared with the L-KYN group (t14 = 2.3, P < .03). Analysis of the raw data also indicated that control rats spent more time with the novel object than the familiar object, but the L-KYN-treated rats did not (Group 3 Object interaction, F1,14 = 5.9, P < .03). Importantly, however, the total time spent exploring the objects during the test session did not differ between groups (t14 = 1.1, P > .3). Locomotor Activity The number of line crossings exhibited by vehicle-treated rats and L-KYN-treated rats during the habituation session of the novel object recognition task is shown in figure 3. Prior treatment with L-KYN during adolescence did not alter locomotor activity compared with rats that had been treated with vehicle (t14 = 0.7; P > .5). 774 There was no difference in shock sensitivity between the groups that had been treated with vehicle or L-KYN during adolescence. For both groups, the mean current at which a behavioral response was first detected was 0.217 6 0.017 mA. Discussion In the present study, adolescent rats were treated with L-KYN to increase endogenous KYNA concentration throughout a critical period of brain development and assess the resulting impact on cognitive function in adulthood. Two behavioral tasks were used so that multiple forms of memory could be examined. In the fear-conditioning task, rats treated with L-KYN as adolescents exhibited impaired contextual fear memory as adults, consistent with previous studies in which adults were treated acutely with L-KYN.14 The deficit in context memory was observed despite normal levels of freezing during the training session (postshock freezing), indicating that treatment with L-KYN did not simply impair performance of the freezing response. Indeed, shock sensitivity was not altered by prior treatment with L-KYN. There was also no significant difference between the vehicle-treated group and the L-KYN-treated group during the tone test session, again consistent with prior findings resulting from acute L-KYN treatment in adults.14 In the second behavioral task, rats were tested in a common novel object recognition procedure. Vehicle-treated rats exhibited robust object memory as evidenced by a greater amount of time spent exploring the novel object compared with the familiar object during the test session. However, rats treated with L-KYN during adolescence spent comparable amounts of time exploring the novel and familiar objects on the test day, indicating that object memory was impaired by prior treatment with L-KYN and exposure to increased levels of KYNA during adolescence. Importantly, there were no group differences in object exploration during the sample session, indicating that the effects of L-KYN treatment during the test session were not simply due to drug-induced changes in Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 Fig. 3. L-KYN Administration during Adolescence Had No Effect on Locomotor Activity during the Habituation Session of the Novel Object Recognition Task. Data are mean 6 SE. Adolescent KYNA Exposure and Memory learning when subjects are adults.44 In addition, behavioral manipulations such as exposure to an enriched environment during development have also been shown to affect subsequent behavior.45 In a similar fashion, exposure to various stressors such as maternal separation during development can impair not only cognitive function but also brain cholinergic systems.46 The present study was not designed to identify specific brain regions that were affected by increased KYNA concentration and responsible for the observed cognitive impairments. However, the pattern of behavioral deficits observed in the fear conditioning and novel object recognition tasks may provide some insight and direction for future studies. For example, it is interesting to note that contextual fear memory but not cue-specific fear memory (ie, freezing tothe tone) was impacted by exposureto KYNAinthe present study. This was also true in our previous study in which adult rats were treated acutely with L-KYN.14 A substantial amount of data indicates that the hippocampus is critically important for contextual fear memory while cue-specific fear memory is more reliant on the amygdala,47,48 suggesting that treatment with L-KYN results in behaviorally relevant changes in KYNA production particularly in the hippocampus. Indeed, spatial memory, which is also dependent on the hippocampus, is also impaired by treatment with L-KYN,13 and a decrease in KYNA concentration has been shown to enhance spatial learning and memory.18 Similarly, prior studies indicate that object memory is largely dependent on the perirhinal cortex37 but can also involve the hippocampus depending on task parameters.49 The findings from this study also add to a growing literature that supports the notion that increased KYNA concentration may underlie neural and behavioral changes in schizophrenia. Indeed, numerous studies document context-processing deficits in schizophrenia50,51 and attribute a variety of cognitive deficits (eg, altered attention, working memory, and inhibition) to a fundamental deficit in processing contextual information.52 In a version of the Continuous Performance Test, eg, it has been shown that persons with schizophrenia failed to properly represent contexts.53,54 Together with our prior findings in adult rats acutely treated with L-KYN,14 the pattern of fear conditioning deficits in the current study is consistent with the idea that elevations in KYNA could contribute to deficits in processing contextual information exhibited by patients with schizophrenia. Importantly, the new findings reported here also indicate that prior exposure to increased KYNA concentration can influence future behavior, when subjects are tested under normal levels of KYNA. Establishing that altered concentration of KYNA, a glial-derived molecule, is capable of influencing cognitive functions that depend on intact NMDA or nicotinic receptor transmission is important from both a basic science perspective as well as a clinical perspective. Indeed, KYNA is 775 Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 exploratory behavior. Moreover, both groups of rats exhibited comparable amounts of locomotor behavior in the novel object apparatus, indicating that prior treatment with L-KYN did not simply alter locomotor activity. Although these data suggest that exposure to high levels of KYNA during adolescence resulted in subsequent memory deficits as young adults, an alternative explanation is that L-KYN-treated rats still had elevated levels of KYNA during behavioral testing, despite the fact that drug treatment ended 8 days prior. However, our results suggest that this was not the case. Indeed, KYNA concentration on PND 61 (first day of behavioral training) was comparable between rats previously treated with vehicle or L-KYN. An additional concern was that L-KYN may not increase KYNA concentration in juvenile rats like it does in adult rats. This was also addressed, and the results indicate that KYNA concentration was increased 5-fold after the first injection of L-KYN. Finally, the findings also demonstrate that the novel treatment regimen of 3 days on/3 days off drug was able to keep increasing KYNA concentration throughout the treatment period. This was a concern since a prior study in which L-KYN was administered daily revealed an inability of L-KYN to affect KYNA concentration after 3 days, purportedly due to changes in metabolism.31 Together, the present findings support the notion that exposure to elevated concentration of KYNA during adolescence can result in cognitive dysfunction later in life. Although there have been no published studies examining the levels of KYNA during adolescence in persons eventually diagnosed with schizophrenia, there is growing evidence of abnormalities associated with schizophrenia that may likely result in changes in KYNA concentration early in life. For example, specific genotypes are thought to result in increases in tryptophan dioxygenase mRNA and protein in the brains of persons with schizophrenia, which lead to elevations in L-KYN and KYNA concentration.21–24 Indeed, levels of KYNA in the brain are normally very low early in life,40 thus excessive blockade of NMDA-Rs and/or a7 nAChRs by increased KYNA concentration could alter brain development in persons with schizophrenia because these receptors are critically involved in neural plasticity and development.4,5 Whether the cellular and physiological effects of KYNA are mediated primarily through NMDA–Rs or a7 nAChRs remains to be conclusively resolved. The observation that altering the concentration of KYNA during a critical time in brain development can affect behavior later in life is consistent with a substantial body of research indicating that chemical manipulations at an early age impact cognitive function in adulthood. For example, studies in humans as well as laboratory animals indicate that perinatal exposure to nicotine impairs learning and memory later in life41,42 and has lasting effects on the expression of nAChRs.43 Likewise, altering the availability of choline during development affects J. K. Forsyth et al. Funding National Alliance for Research on Schizophrenia and Depression Young Investigator Award (to D.J.B.); Howard Hughes Medical Institute Life Science Internship (to C.O.A.). Acknowledgments The Authors have declared that there are no conflicts of interest in relation to the subject of this study. 776 References 1. Schwarcz R, Pellicciari R. Manipulation of brain kynurenines: glial targets, neuronal effects, and clinical opportunities. J Pharmacol Exp Ther. 2002;303:1–10. 2. Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev. 1993;45:309–379. 3. Hilmas C, Pereira EFR, Alkondon M, Rassoulpour A, Schwarcz R, Albuquerque EX. The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J Neurosci. 2001;21:7463–7473. 4. Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors. Science. 1993;260(5104):95–97. 5. Broide RS, Leslie FM. The alpha7 nicotinic acetylcholine receptor in neuronal plasticity. Mol Neurobiol. 1999;20(1): 1–16. 6. Bast T, Zhang WN, Feldon J. Dorsal hippocampus and classical fear conditioning to tone and context in rats: effects of local NMDA-receptor blockade and stimulation. Hippocampus. 2003;13:657–675. 7. Gould TJ, Higgins JS. Nicotine enhances contextual fear conditioning in C57BL/6J mice at 1 and 7 days post-training. Neurobiol Learn Mem. 2003;80:147–157. 8. Erhardt S, Blennow K, Nordin C, Skogh E, Lindstrom LH, Engberg G. Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurosci Lett. 2001;313:96–98. 9. Nilsson LK, Linderholm KR, Engberg G, et al. Elevated levels of kynurenic acid in the cerebrospinal fluid of male patients with schizophrenia. Schizophr Res. 2005;80:315–322. 10. Schwarcz R, Rassoulpour A, Wu H-Q, Medoff D, Tamminga CA, Roberts RC. Increased cortical kynurenate content in schizophrenia. Biol Psychiatry. 2001;50:521–530. 11. Barry S, Clarke G, Scully P, Dinan TG. Kynurenine pathway in psychosis: evidence of increased tryptophan degradation. J Psychopharmacol. 2009;23:287–294. 12. Erhardt S, Schwieler L, Nilsson L, Linderholm K, Engberg G. The kynurenic acid hypothesis of schizophrenia. Physiol Behav. 2007;92:203–209. 13. Chess AC, Simoni MK, Alling TE, Bucci DJ. Elevations of endogenous kynurenic acid produce spatial working memory deficits. Schizophr Bull. 2007;33:797–804. 14. Chess AC, Landers AM, Bucci DJ. L-kynurenine treatment alters contextual fear conditioning and context discrimination but not cue-specific fear conditioning. Behav Brain Res. 2009;201:325–331. 15. Shepard PD, Joy B, Clerkin L, Schwarcz R. Micromolar brain levels of kynurenic acid are associated with a disruption of auditory sensory gating in the rat. Neuropsychopharmacology. 2003;28:1454–1462. 16. Erhardt S, Schwieler L, Emanuelsson C, Geyer M. Endogenous kynurenic acid disrupts prepulse inhibition. Biol Psychiatry. 2004;56:255–260. 17. Chess AC, Bucci DJ. Increased concentration of cerebral kynurenic acid alters stimulus processing and conditioned responding. Behav Brain Res. 2006;170:326–332. 18. Potter MC, Elmer GI, Bergeron R, et al. Reduction of endogenous kynurenic acid formation enhances extracellular glutamate, hippocampal plasticity, and cognitive behavior. Neuropsychopharm. 2010;35:1734–1742. 19. McClure MM, Barch DM, Flory JD, Harvey PD, Siever LJ. Context processing in schizotypal personality disorder: Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 synthesized and released only by astrocytes1,2 and is unique in that it is the only known endogenous antagonist of NMDA-Rs and nAChRs. Thus, KYNA may serve as an important regulator of neuronal excitability and plasticity. This is consistent with contemporary views of astrocytic function in the nervous system. It has been suggested, eg, that glia and so called ‘‘glio-transmitters’’ have an underappreciated and active role in modulating normal information processing and cognitive function.55–57 This is quite different from the more traditional support roles ascribed to astrocytes in controlling extracellular ionic concentration and the availability of nutrients needed by neurons. From a clinical perspective, the growing evidence that increases in KYNA concentration result in cognitive deficits reminiscent of those associated with schizophrenia may inform the development of new therapies for cognitive dysfunction in psychiatric disorders. Indeed, cognitive impairment in schizophrenia is notoriously difficult to treat with traditional antipsychotics,58 and there is increased interestindevelopingkynurenergiccompoundsforatherapeuticuse.59–62 Forexample,itmaybepossibletoreduceKYNA concentration by targeting the enzymes responsible for its synthesis (eg, kynurenine aminotransferases59). It has also been shown that common cyclooxygnease-2 (COX-2) inhibitors, such as parecoxib, decrease KYNA concentration.63 Relatedly, there is evidence that nicotine can alter KYNA concentration64 supporting the possibility that nicotine may be used by persons with schizophrenia to counter cognitive deficits induced by KYNA. Thus, the development of kynurenergic compounds as adjunctive therapies for psychosis may also be useful in combating co-occurring psychopathology and substance abuse. In addition, individuals infected with HIV-1 can develop HIV-associated dementia, in addition to exhibiting reactive astrocytes and increases in kynurenine, kynurenic acid, and its biosynthetic enzyme kynurenine aminotransferase.65 These findings lend support to the notion that upregulation of kynurenine and KYNA may contribute to symptoms that are similar to those observed in schizophrenia and extend the implications of our data to cases in which the source of this upregulation is immune activated. Adolescent KYNA Exposure and Memory 20. 21. 22. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Dere E, Huston JP, De Souza-Silva MA. The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neurosci Biobehav Rev. 2007;31:673–704. 38. Blanchard RJ, Blanchard DC. Passive and active reactions to fear-eliciting stimuli. J Comp Physiol Psychol. 1969;68:129–135. 39. Fanselow MS. Conditional and unconditional components of post-shock freezing. Pavlov J Biol Sci. 1980;15:177–182. 40. Moroni F, Russi P, Carlá V, Lombardi G. Kynurenic acid is present in the rat brain and its content increases during development and aging processes. Neurosci Lett. 1988;94:145–150. 41. Vaglenova J, Parameshwaran K, Suppiramaniam V, Breese CR, Pandiella N, Birru S. Long-lasting teratogenic effects of nicotine on cognition: gender specificity and role of AMPA receptor function. Neurobiol Learn Mem. 2008;90:527–536. 42. Huizink AC, Mulder EJ. Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring. Neurosci Biobehav Rev. 2006;30:24–41. 43. Dwyer JB, Broide RS, Leslie FM. Nicotine and brain development. Birth Defects Res C Embryo Today. 2008;84:30–44. 44. Lamoureux JA, Meck WH, Williams CL. Prenatal choline availability alters the context sensitivity of Pavlovian conditioning in adult rats. Learn Mem. 2008;5866–5875. 45. Hoffmann LC, Schütte SR, Koch M, Schwabe K. Effect of ‘‘enriched environment’’ during development on adult rat behavior and response to the dopamine receptor agonist apomorphine. Neuroscience. 2009;158:1589–1598. 46. Aisa B, Gil-Bea FJ, Marcos B, et al. Neonatal stress affects vulnerability of cholinergic neurons and cognition in the rat: involvement of the HPA axis. Psychoneuroendocrinology. 2009;34:1495–1505. 47. Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci. 1992;106:274–285. 48. Maren S, Anagnostaras SG, Fanselow MS. The startled seahorse: is the hippocampus necessary for contextual fear conditioning? Trends Cogn Sci. 1998;2:39–42. 49. Albasser MM, Poirier GL, Aggleton JP. Qualitatively different modes of perirhinal-hippocampal engagement when rats explore novel vs. familiar objects as revealed by c-Fos imaging. Eur J Neurosci. 2010;31:134–147. 50. Quinn JJ, Loya F, Ma QD, Fanselow MS. Dorsal hippocampus NMDA receptors differentially mediate trace and contextual fear conditioning. Hippocampus. 2005;15:665–674. 51. Barch DM, Carter CS, MacDonald AW, III, Braver TS, Cohen JD. Context-processing deficits in schizophrenia. Diagnostic specificity, 4-week course, and relationships to clinical symptoms. J Abnorm Psychol. 2003;112:132–143. 52. Cohen JD, Servan-Schreiber D. Context, cortex and dopamine: a connectionist approach to behavior and biology in schizophrenia. Psychol Rev. 1992;99:45–77. 53. Cohen JD, Barch DM, Carter C, Servan-Schreiber D. Context-processing deficits in schizophrenia: converging evidence from three theoretically motivated cognitive tasks. J Abnorm Psychol. 1999;108:120–133. 54. Stratta P, Daneluzzo E, Bustini M, Prosperini P, Rossi A. Processing of context information in schizophrenia: relation to clinical symptoms and WCST performance. Schizophr Res. 2000;44:57–67. 55. Seifert G, Schilling K, Steinhauser C. Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat Rev Neurosci. 2006;7:194–205. 777 Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 23. evidence of specificity of impairment to the schizophrenia spectrum. J Abnorm Psychol. 2008;117:342–354. Waters FAV, Mayberry MT, Badcock JC, Michie PT. Context memory and binding in schizophrenia. Schizophr Res. 2004;68:119–125. Miller CL, Llenos IC, Dulay JR, Barillo MM, Yolken RH, Weis S. Expression of the kynurenine pathway enzyme tryptophan 2,3-dioxygenase is increased in the frontal cortex of individuals with schizophrenia. Neurobiol Dis. 2004;15:618–629. Miller CL, Llenos IC, Dulay JR, Weis S. Upregulation of the initiating step of the kynurenine pathway in postmortem anterior cingulate cortex from individuals with schizophrenia and bipolar disorder. Brain Res. 2006;1073–1074:25–37. Miller CL, Llenos IC, Cwik M, Walkup J, Weis S. Alterations in kynurenine precursor and product levels in schizophrenia and bipolar disorder. Neurochem Int. 2008;52:1297–1303. Miller CL, Murakami P, Ruczinski I, et al. Two complex genotypes relevant to the kynurenine pathway and melanotropin function show association with schizophrenia and bipolar disorder. Schizophr Res. 2009;113:259–267. Ricceri L, Hohmann C, Berger-Sweeney J. Early neonatal 192 IgG saporin induces learning impairments and disrupts cortical morphogenesis in rats. Brain Res. 2002;954:160–172. Philpot BD, Sekhar AK, Shouval HZ, Bear MF. Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex. Neuron. 2001;29:157–169. Dalcin KB, Rosa RB, Schmidt AL, et al. Age and brain structural related effects of glutaric and 3-hydroxyglutaric acids on glutamate binding to plasma membranes during rat brain development. Cell Mol Neurobiol. 2007;27:805–818. Harrop C, Trower P. Why does schizophrenia develop at late adolescence? Clin Psychol Rev. 2001;21:241–265. Bennett MR. Synapse formation and regression in the cortex during adolescence and in schizophrenia. Med J Aust. 2009;190:S14–S16. Guo L, Evans GC, Bucci DJ. Behavioral effects of increased endogenous kynurenic acid concentration: implications for cognitive dysfunction in schizophrenia. Program No. 546.15. 2009 Neuroscience Meeting Planner. Online. Chicago, IL: Society for Neuroscience. 2009. Vecsei L, Miller J, MacGarvey U, Beal FM. Effects of kynurenine and probenecid on plasma and brain tissue concentrations of kynurenic acid. Neurodegeneration. 1992;1:17–26. Pi LG, Tang AG, Mo XM, Luo XB, Ao X. More rapid and sensitive method for the simultaneous determination of tryptophan and kynurenic acid by HPLC. Clin Biochem. 2009;42:420–425. Zhao J, Gao P, Zhu D. Optimization of Zn2+-containing mobile phase for simultaneous determination of kynurenine, kynurenic acid and tryptophan in human plasma by high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878:603–608. Arenos JD, Musty RE, Bucci DJ. Blockade of cannabinoid CB1 receptors alters contextual learning and memory. Eur J Pharmacol. 2006;539:177–183. Maren S, Aharonov G, Fanselow MS. Neurotoxic lesions of the dorsal hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res. 1997;88:261–274. Bevins RA, Besheer J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. Nat Protoc. 2006;1:1306–1311. J. K. Forsyth et al. 778 61. Erhardt S, Olsson SK, Engberg G. Pharmacological manipulation of kynurenic acid: potential in the treatment of psychiatric disorders. CNS Drugs. 2009;23:91–101. 62. Costantino G. New promises for manipulation of kynurenine pathway in cancer and neurological diseases. Expert Opin Ther Targets. 2009;13:247–258. 63. Schwieler L, Erhardt S, Nilsson L, Linderholm K, Engberg G. Effects of COX-1 and COX-2 inhibitors on the firing of rat midbrain dopaminergic neurons—possible involvement of endogenous kynurenic acid. Synapse. 2006;59:290–298. 64. Rassoulpour A, Wu HQ, Albuquerque EX, Schwarcz R. Prolonged nicotine administration results in biphasic, brainspecific changes in kynurenate levels in the rat. Neuropsychopharmacology. 2005;30:697–704. 65. Wang T, Rumbaugh JA, Nath A. Viruses and the brain: from inflammation to dementia. Clin Sci. 2006;1:393–407. Downloaded from https://academic.oup.com/schizophreniabulletin/article/38/4/769/1866311 by guest on 22 June 2022 56. Auld DS, Robitaille R. Glial cells and neurotransmission: an inclusive view of synaptic function. Neuron. 2003;40: 389–400. 57. Pascual O, Casper KB, Kubera C, et al. Astrocytic purinergic signaling coordinates synaptic networks. Science. 2005;310:113–116. 58. Hill SK, Bishop JR, Palumbo D, Sweeney JA. Effect of second-generation antipsychotics on cognition: current issues and future challenges. Expert Rev Neurother. 2010;10:43–57. 59. Wonodi I, Schwarcz R. Cortical kynurenine pathway metabolism: a novel target for cognitive enhancement in Schizophrenia. Schizophr Bull. 2010;36:211–218. 60. Fülöp F, Szatmári I, Vámos E, Zádori D, Toldi J, Vécsei L. Syntheses, transformations and pharmaceutical applications of kynurenic acid derivatives. Curr Med Chem. 2009;16: 4828–4842.