1. Long-Term Enriched Environment But Not Cage Size Improves Memory in Adulthood and Late Adulthood in Male Rats I.C. Sumaya, J.H. Calderon, V. Rodriguez, C.M. Franz & A.A. Amaya Department of Psychology, Behavioral Neuroscience Laboratory, California State University, Bakersfield, CA. It has been well established that enriched environments positively impact rodent learning and memory. Rats placed in enriched environments have shown improvement in performance in a variety of memory tasks including the Morris Water Maze (Harburger et al., 2007 ), the novel object recognition task (Bruel-Jungerman et al., 2005), and the radial arm maze task (Hoffmann et l., 2009). One of the proposed underlying mechanisms of action for improvement of memory is that of neurogenesis in the hippocampus, a region important for learning and memory in both rodents (Kempermann et al., 1997; Veena et al., 2009; Nilsson et al., 1999) and humans (Suthana et al., 2009; Goodrich-Hunsacker et al., 2009). Although many studies have investigated the effects of neurogenesis on learning and memory in rodents, much of the data reported have been collected during short-term enriched housing (2 weeks to 2 months) and in younger populations at one time point. The aim of our experiment was to investigate the effects of long-term enriched housing on spatial learning (8 arm-maze task) at two developmental time points, adulthood (6 months) and later adulthood (12 months) in rats that were born, raised, and matured in enriched environments. Additionally, we were interested in the importance of cage size within th e context of the enriched environments. That is to say, does cage size matter? Results Results Background Animals: Five pairs of Sprague-Dawley (Charles River, Wilmington, MA) male and female (2 months old) rats were randomly assigned to one of five environments for breeding. All pairs were kept in a 12L/12D cycle with lights on at 0600, fed standard rat chow ad libitum and fresh water daily. The five environments included a standard non-enriched cage (Standard NE: 19” x 10.5” x 8”h), a medium non-enriched cage (Medium NE: 25”x 16” x 14.75”h), a large non-enriched cage (Large NE: 40.5" x 18” x 20.5 h), a medium enriched cage (Medium E), and a large enriched cage (Large E). Enriched environments included running wheels, ledges, toys and edible rat shacks. Breeding: Male rats were placed in a cage with clean bedding for 24 hours prior to placing the female in the cage. The pairs were kept together for 7 days after which the males were removed from the cages. All females were impregnated and gave birth approximately 21 days later. Once the dams gave birth, and the pups were weaned, the pups were separated by gender, the mothers were removed from the environment with the offspring remaining in the environment they were born in (N=34, only report male data here). At 6 months the rats were tested in the radial 8-arm maze task (1 trial/day for 8 days) with perseveration errors and time to completion measured. Six months later, the rats were again tested. Method Over the 8 trials at 6 months, regardless of cage size, rats in the enriched environments had significantly less errors overall and completed the spatial task faster than their non-enriched counterparts. The same pattern occurred at 12 months of age. Regardless of cage size, rats in the enriched environments continued to have significantly less errors overall. Additionally, the rats in the enriched environments also completed the task quicker at both 6 months and 12 months of age. These data show that enriched environments continue to positively impact spatial learning and memory as measured in the radial arm maze into late adulthood. Our unanticipated results were that the size of the environment is not an important contributing factor in the positive impact on memory. Figure 1, 2 & 3. At 6 months, there was a main effect for environment, F(4, 28) = 5.40, p <.002, partial eta 2 =.44; a main effect of trial, F(7,196) = 8.42, p <.001, partial eta 2 =.23, and a significant environment X trial interaction, F(28, 196) = 1.79, p <.05, partial eta 2 =.20. Over the 8 trials, regardless of cage size, rats in the enriched environments had significantly less errors overall (StandardNE: 6.25+0.53 errors, MediumNE: 6.71+0.62 errors, LargeNE: 4.62+ 0.79 errors, MediumE: 2.77+0.72 errors, LargeE: 3.0+0.89 errors). Summary of Findings References Acknowledgements Environment (1 year - test at 6 mo & 12 mo) Standard Cage (19” x 10.5” x 8”h) Medium Cage (25” x 16” x 14.75”h) Large Cage (40.5” x 18” x 20.5”h) Medium Enriched (25” x 16” x 14.75”h) Large Enriched (40.5” x 18” x 20.5”h) Design: 5X8 Radial 8-Arm Maze: The maze was made of Plexiglas (arms: 61cm L, x 23cm W x 23cm H) whereby the arms were baited with fruitloops (Kelloggs, Cincinnati, Ohio). A day prior to testing all rats were placed in the maze for a 2-hour acclimation period. The arms were baited with fuitloops (Kelloggs). 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). Figure 4, 5 & 6. At 12 months, there was a main effect for environment, F(4, 22) =8.87, p <.001, partial eta 2 =.60; a main effect of trial, F(7,154) = 12.23, p <.001, partial eta 2 =.36, and a significant environment X trial interaction, F(28, 154) = 3.03, p <.001, partial eta 2 =.36. At 12 months of age the rats showed similar patterns of learning over the 8 trials. Regardless of cage size, rats in the enriched environments continued to have significantly less errors overall (StandardNE 4.77+0.41 errors, MediumNE: 4.10+0.41 errors, LargeE: 4.12+0.44 errors, MediumE: 2.35+0.41 errors, LargeE: 1.62+0.5 errors). Figure 7. At 6 months, a main effect for environment was shown, rats in the enriched environments completed the spatial task faster than their non- enriched counter parts (StandardNE: 7.03+0.89 min, MediumNE: 8.46+1.0 min, LargeNE: 5.05+1.3 min, MediumE: 3.33+1.2 min, LargeE: 3.96+1.4 min). Figure 8. At 12 months, a main effect for environment was shown, rats in the enriched environments completed the spatial task faster than their non-enriched counter parts (StandardNE: 6.22+0.63 min, MediumNE: 7.72+0.63 min, LargeNE: 5.77+0.69 min, MediumE: 3.37+0.63 min, LargeE: 4.31+0.78 min). Errors in the Radial-Arm Maze at 6 Months Errors in the Radial-Arm Maze at 12 Months Environments Fig. 1 Fig. 3 Fig. 2 Time to complete the Radial-Arm Maze at 6 Months Time to complete the Radial-Arm Maze at 12 Months Fig. 4Fig. 5 Fig. 6 Fig. 7Fig. 8 Supported by the McNair Scholars Program at California State University, Bakersfield Bruel-Jungerman, E., LaRoche, S., Rampon, C. (2005). New neurons in the dentate gyrus are involved in the expression of enhanced long-term memory following environmental enrichment. Eur. J of Neurosci., 21(2):513-21. Casey, D.E. (2006). Implications of the CATIE trial on treatment: extrapyramidal symptoms. CNS Spectr., 11:25-31. Dask, P.K., Orsi, S.A., Moore, A.N. ( 2009). Histone deactylase inhibition combined with behavioral therapy enhances learning and memory following traumatic brain injury, Neuroscience, 29;163(1):1-8. Goodrich-Hunsaker, N.J. & Hopkins, R.O. (2009). Word memory test performance in amnesic patients with hippocampal damage. 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Xu, X., Ye., L. & Ruan, Q., (2009). Environmental enrichment induces synaptic structural modification after transient focal cerebral ischemia in rats. Exp. Biol. Med., 234(3):296-305. Trial ( 1 trial a day for 8 consec days) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7 Trial 8 8-Arm Maze Statistics: A 5X8 Mixed ANOVA was used with environment as the between subjects factor and Trial as the within groups factor. Post hoc tests were performed with the Bonferroni correction. Alpha was at 0.05.