1. The Effect of a High Fat Diet and Enriched Environment in Rat Performance in the 8-Arm Radial Maze Task and the Forced Swimming Test J. H. Calderon, V. Rodriguez, B. Vergara, & I. C. Sumaya Department of Psychology, California State University, Bakersfield, CA. It has been well established that enriched environments have a positive impact on rodent behavior and brain morphology with the underlying mechanism of action thought to be hippocampal neurogenesis. Stranahan et al., (2007) found an increase in dendritic spine density in both granule neurons of the dentate gyrus as well as in various regions of the entorhinal cortex of rats. Research has shown that the negative effects of a high fat diet in rats tested in various cognitive tasks can be overcome by environmental enrichment (Wimocour, 1999). Previously, we have demonstrated that rats on a high fat diet and treated with fluoxetine show increased learned helplessness in the forced swim test compared to their controls (Sumaya et al., 2007). The purpose of our research was to investigate cognitive (spatial learning) and emotional domains (anxiety and learned helplessness) in rats fed a high fat diet, housed in enriched environments, and treated with Fluoxetine. Additionally, we sought to further investigate our previous findings showing a negative interaction between a high fat diet and acute administration of the SSRI, Fluoxetine. Background Animals: 64 male Sprague Dawley rats (4 mo old, Charles River, Wilmington, MA) were kept in a 12/12 L/D cycle with lights on at 0600 hrs, food and water provided ad libitum. Rats were handled in accordance with institutional guidelines set forth by the National Institute of Health and approved by the Institutional Animal Care and Use Committee at California State University, Bakersfield. Standard Diet, High Fat Diet: With the exception of the six day trials of the eight arm maze, in which each animal tested was given a daily ration of 10g of food, rats were fed ad libitum. The standard diet group was fed rat chow (11% fat, Purina). The high fat chow was formulated to contain 32.5% fat, 48% carbs, and 19.5% protein (AIN-93M: isocaloric with target nutrients, Research Diets, NJ, USA). Eight Arm Radial Maze: The apparatus consisted of a clear Plexiglas starburst 8-arm maze (arms: 61cm L, x 23cm W x 23cm H) whereby the arms were baited with Frootloops (Kelloggs, Cincinnati, Ohio). A day prior to testing all rats were placed in the maze for a 2-hour acclimation period. Each animal was given 15 min to complete the task. Data were recorded for number of errors in the 8 arm maze, and the time animals took to complete task (time data not shown here). Elevated Plus Maze: The elevated plus maze was used to assess animal anxiety. The maze is constructed of four Plexiglas arms that form a cross with a central square (81cm H x 114cm L x 13cm W). Rats were placed in the center square and given 300 seconds to complete the task under dim lights. Data were recorded in seconds for time spent in the open arms, closed arms, and center. Forced Swim Test: The forced swim test (Porsolt et al., 1977) measures learned helplessness via the quantification of immobility in the rat. Briefly, rats were placed individually in a clear plexiglas cylinder (25cm dia, 40cm H) containing 13 L of water at 25  C for 6 min (2 min acclimation, 4 min of testing). Each rat was judged to be immobile when it ceased to struggle and remained in a floating position motionless making movements only necessary to keep its head above water. Procedure & Enriched Environments (EE): Rats were first randomly assigned to either a standard cage (SC) (N=32, 43cm L x 20cm H x 23cm W) or an enriched environment (N=32, multilayer: 56cm L x 81cm H x 42cm W: running wheels, toys, tunnels, chew bones, chew balls, huts, planks) and fed standard rat chow. 7 weeks later, half the rats were placed on a high fat diet while the other half were kept on regular chow. Four weeks later, the rats were tested. See timeline below. Method Results Results In the cognitive domain we have shown that rats housed in standard cages and on a high-fat diet had more errors in the 8-arm maze task and took longer time to complete the task as compared to their counterparts on regular chow. However, regardless of diet, the rats housed in enriched environments showed the best performance in the 8-arm maze task and had the quickest times of completion. These data show enriched environments counteract the negative effects of a high- fat diet on spatial learning. In the emotional domain (forced swimming test) rats treated with fluoxetine on a high fat diet, and housed in standard cages had significantly more time immobile as compared to the vehicle treated supporting our previous findings of a negative interaction between a high fat diet and the SSRI. Furthermore, although rats treated with fluoxetine on a high fat diet showed more learned helplessness as compared to the vehicle treated, the enriched environment served to dampen this effect. Results from the elevated plus maze (anxiety) show that enriched environments have no effect on anxiety in rats. However, we did find diet affecting this domain. Rats fed a high fat diet showed more anxiety as compared to rats fed the standard rat chow. Taken together these data show positive behavioral effects of enriched environments on learning and memory and learned helplessness paradigms with no effect on anxiety. Supported by SRS Scholars program California State University, Bakersfield Enriched Environment Acknowledgements Figure 3a & 3b. (3a). A 2x2 ANOVA for the standard rat chow groups showed no effect. (3b) For the high fat diet group a main effect for drug was shown, F (1, 28) = 13.24, p <.05. Performance in Radial 8 Arm Maze Performance in Elevated Plus Maze Performance in Forced Swim Test Rats assigned to EE or SC High fat diet begins First day of testing in 8 arm maze for 6 consecutive days Elevated plus maze testing Forced swim test - rats treated with Fluoxetine. Day 1Day 52Day 75Day 81Day 82 Timeline of Experiment Summary of Findings Fig. 1aFig. 1b Fig. 2b Fig. 2a Fig. 3aFig. 3b Sumaya, I.C. Bailey, D.W., Catlett, S.L., & Fields, C. (2007) The Effects of the 5HT3 Agonist, 2-Methyl-5HT and the 5HT3 Receptor Antagonist, Ondansetron, in an Animal Model of Depression and Anxiety in Rats Kept on a High Fat Diet. Presented to the 37th Annual Meeting of the Society for Neuroscience. November 3-7, 2007, San Diego, CA., Neurosci. Abstr.33, Abstract #629.10, 2007. Porsolt, R., Anton, G., Blavet, N., Jalfre, M. (1977). Behavioural despair in rats: A new model sensitive to antidepressant treatments. European Journal of Pharmacology, 47, 379-391. Stranahan, A. M., Khalil, D., & Gould, E. (2007). Running induces widespread structural alterations in the hippocampus and entorhinal cortex. Hippocampus, 17, 1017-1022. Winocour, G., & Greenwood, C. E.(1999). The effects of high fat diets and environmental influences on cognitive performance in rats. Behavioral Brain Research, 101, 153-161. References Figure 1a &b. A 2X6 mixed ANOVA was conducted on error scores in the 8 arm maze. For the test of the within subjects variable (trial) a significant main effect was found F (5, 300) = 13.15, p <.001). For the between subjects effects a main effect for environment, F= (1, 60) =177.65, p =.02., a main effect for diet, F (1, 60) = 5.49, p =.02. and a significant interaction between environment and diet was shown F(1, 60) = 4.80, p =.03. Figure Figure 2a & b. 2x2 ANOVAs showed a main effect for diet, F(1, 60) = 9.13, p =.004 in the open arms, and a main effect for diet was also found for the closed arms, F(1,60) = 9.70, p =.003.