AN ABSTRACT OF THE DISSERTATION OF Siba

approved: _____________________________________________________________________ Kathy R. Magnusson As the aging population continues to grow the world over, age related complications become more and more apparent among the elderly population. One such complication is age associated memory impairment, which makes the elderly more dependent on caregivers early on. NMDA receptors in the brain are important for memory formation, consolidation and retrieval. Expression of NMDA receptors declines with age, which is associated with declines in memory observed during aging. Age-related changes in the protein and mRNA expression of some of the splice forms of the GluN1 (GluN1, NR1) subunit of the NMDA receptor have been seen in mice and rats. The present study was designed to determine whether individual splice forms of the GluN1 subunit of the NMDA receptor within prefrontal / frontal cortical regions contribute to memory deficits during aging and whether experience in learning tasks can influence the expression of the splice forms. mRNA expression of 4 splice forms GluN1X11, GluN1X10, GluN10XX and GluN11XX (GluN1-1, GluN1-3, GluN1-a and GluN1-b, respectively) and mRNA for all known splice forms (GluN1-pan) were examined by in situ hybridization. mRNA for the C-terminal splice forms, GluN1X11 (GluN1-1; +C1 and +C2 cassettes) and GluN1X10 (GluN1-3; +C1 and +C2’), showed significant declines during aging in several brain regions even though overall GluN1-pan mRNA expression was not significantly affected by aging. This work provides evidence that these splice forms are more influenced by aging than the subunit as a whole. There was an increase in the expression of GluN10XX (GluN1-a; -N1 cassette) splice form in the behaviorallyexperienced old mice relative to the younger groups. Old mice with the highest levels of mRNA expression for the GluN10XX (GluN1-a) splice form in orbital cortex showed the best performances in spatial working and reference memory tasks, but the poorest performances in a cued, associative learning task. These results suggest that the GluN10XX subunit splice variant may be important for spatial memory performance in the old animals. Protein expression of GluN1 subunits containing C-terminal cassettes C2 or C2’ were observed to decline with increasing age, regardless of experience. In middleage animals, higher expressions of the GluN1 subunit and C2’ cassette proteins were associated with good reference memory on initial search. In the aged animals, higher protein expression of GluN1 subunits containing C1 cassettes and the whole population of GluN1 subunits were found to be associated with better performance in the final phase of probe trials but this appeared to be due to perseveration or delays in applying an accurate search. These results provide support for the theory that there is heterogeneity in the effect of aging on the expression of the GluN1 subunits containing different splice cassettes. It also suggests that the GluN1 subunit might be most important for good reference memory during middle age. The next study was undertaken to determine if the GluN10XX splice form is required for good performance in reference memory tasks in young mice. Mice were injected with 5μl of either siRNA specific to GluN10XX, control siRNA or vehicle alone into ventro-lateral orbital regions of both sides of the brain using a stereotaxic apparatus. A fourth group of mice did not receive any injections. Five days postinjection, mice were tested for their performance in a spatial reference memory task for 4 days using the Morris water maze. There was a 10 -19% reduction in GluN10XX splice variant expression for mice after siRNA treatment in ventro-lateral orbital regions of the brain. Decline in performance in the first half of reference memory were observed in the mice receiving siRNA specific for GluN10XX splice form, as compared to the mice injected with control siRNA and/or vehicle. These results suggest an important role of the GluN10XX splice variant in orbital regions for spatial reference memory acquisition and/or consolidation in the early stages of memory training. These results suggest that there is a complex interaction between GluN1 splice form expression and performance of memory tasks during aging. Future studies designed to differentiate between involvement of splice variants in particular stages of memory formation would be helpful. Copyright by Siba Ranjan Das September 23, 2010 All Rights Reserved Influence of Aging and Behavioral Experience on Expression of GluN1 Splice Variants of the NMDA Receptor in Prefrontal Cortex of Mice Brain.