A PROFILE OF NEUROGENIC ACTIVITY IN THE AGING HIPPOCAMPAL FORMATION: A CLOSER LOOK AT THE ROLE OF EXERCISE AND ENVIRONMENTAL ENRICHMENT IN THE SAMP-8 Ashley M. Fortress A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS August 2007 Committee: Kevin C.H. Pang, Advisor Verner P. Bingman Dale S. Klopfer ii ABSTRACT Kevin C.H. Pang, Advisor Neurogenesis is a process of neuronal proliferation that is most prominent during development but has recently been found in adulthood. This phenomenon occurs naturally in the hippocampal formation of many mammalian species, including humans. Various factors influence neurogenesis, such as age, voluntary exercise and environmental enrichment. The present study was performed to determine the profile of the age-related decline of neurogenesis in the senescence accelerated mouse (SAMP8). Additionally, the ability of exercise and environmental enrichment to independently reverse the age-related decline of neurogenesis was investigated. Four age groups of male SAMP8 mice were examined: 2-, 5-, 7- and 12-months. Assessing both survival and proliferation using immunocytochemistry for BrdU and Ki67, it was found that: (1) exercise showed a trend for reversing the age-related decline in neurogenesis (2) environmental enrichment significantly decreased neurogenesis in the 2-month old age group and had no effect for all other age groups when compared to isolated animals (3) neither exercise nor enrichment was beneficial in promoting survival. iii With science, all things are possible. iv ACKNOWLEDGMENTS I would like to thank my academic advisor and personal mentor, Dr. Kevin Pang, for helping me set a solid foundation in both my career and in my personal life. When he took a chance on a scientifically naïve girl, he gave me the opportunity and confidence to undertake a major project on adult hippocampal neurogenesis and a career in neuroscience research. His belief in me and my work, time and again, have given me inspiration beyond measure. I would like to thank my family: my parents for the many sacrifices they had to make to get me where I have been in life and where I will go; my mom for teaching me how to appreciate the small things and for showing me that the best things in life are often not tangible; my dad for teaching me how to work hard and never give up and how to always dream big and settle for nothing less than extraordinary; and my grandparents for spoiling me with quality time filled with card games, books, and plenty of homemade baked goods - some of my fondest memories that I will cherish always. I am no one without those who built my character and gave me life, and I am forever thankful. v TABLE OF CONTENTS Page INTRODUCTION……………………………………………………………… 1 MATERIALS AND METHODS………………………………………………. 6 Animals…………………………………………………………………. 6 Injections and Duration…………………………………………………. 6 Immunohistochemistry………………………………………………….. 7 Stereological Quantification…………………………………………….. 9 Statistical Analyses……………………………………………………… 9 RESULTS………………………………………………………………………. 10 BrdU Positive Cells…………………………………………………….. 10 Ki67 Positive Cells……………………………………………………… 12 DISCUSSION…………………………………………………………………… 13 The SAMP-8 as a mouse model of aging………………………………. 13 Neurogenesis and aging………………………………………………… 15 Effects of exercise on neurogenesis……………………………….……. 16 Effects of environmental enrichment on neurogenesis…………………. 17 Stress as a possible explanation for all observations…………………… 18 BrdU versus Ki67 positive cells………………………………………… 20 Implications for adult neurogenesis…………………………………….. 21 Conclusion………………………………………………………………. 23 FIGURE CAPTIONS…………………………………………………………… 24 vi REFERENCES…………………………………………………………………. 35 APPENDICES APPENDIX A. THE HIPPOCAMPUS…………………………………. 43 APPENDIX B. NEUROGENESIS……………………………………… 47 APPENDIX C. PILOT DATA………………………………………….. 65 vii LIST OF FIGURES Figure Page 1 Injection Schedule and Time Course of Experiments........................................ 26 2 Immunocytochemistry for BrdU (2-, 5-, 7-, 12-month Isolated)............................... 27 3 Stereological Analysis of All Conditions (Proliferation, BrdU)................................ 28 4 Stereological Analysis of All Conditions (Survival, BrdU) ...................................... 29 5 Immunocytochemistry for Ki67 (2-, 5-, 7-, 12-month Isolated)................................ 30 6 Stereological Analysis of Ki67 Conditions (Proliferation)........................................ 31 7 Proliferation v. Survival in Isolated animals (BrdU)................................................. 32 8 Figure of Hippocampal Anatomy and Circuitry ........................................................ 33 9 Group Housed versus Single Housed Enriched Animals .....................................…. 34 Adult Neurogenesis 1 INTRODUCTION The late 1990s saw the death of a dogma and the reintroduction of a new facet of neuroscience research when it was shown definitively that new neurons can be generated throughout the lifespan (Altman & Das, 1965; Eriksson et al., 1998). The two main areas of adult neurogenesis are the olfactory bulb and hippocampus (Winner, Cooper-Kuhn, Aigner, Winkler, & Kuhn, 2002; Altman, 1962). Because of its role in cognitive and affective disorders, the mechanism of neurogenesis in the hippocampus has generated much interest. Adult hippocampal neurogenesis occurs in the dentate gyrus via a three part process: proliferation, survival and differentiation (Kempermann, Kuhn, & Gage, 1998). Proliferation is the division of a neural precursor cell into its undifferentiatied progeny. Survival is the time period during which the cell will ultimately decide whether it will undergo apoptosis or survive. Finally, upon determining that the cell will survive, additional microenvironmental factors that are present upon migration or final location allow for the cell to determine its cellular fate; a process termed differentiation. Ultimately a cell derived from a neural precursor cell will differentiate into a neuron, or it may become a non-neuronal cell of the nervous system. However, it is important to consider that for the phenomenon of adult neurogenesis to be effective, the newly divided cells must differentiate into fully functional neurons and participate in existing circuitry. Research on adult neurogenesis has thrived well into the new millennium and has convinced many that the dogma of ‘no new neurons’ is no longer true. Furthermore, other studies have shown that neurogenesis can be regulated by macro- and microenvironmental factors, a variety of drugs, disease and age. Adult Neurogenesis 2 Environmental enrichment has a long history in affecting the brain and cognition. Of particular relevance to this study is finding that environmental enrichment enhances the volume of the hippocampus (Rosenzweig, 1966). For many years, it was thought that the increase in neuron number was due to enhanced survival of neurons generated during development. However, one of the first studies examining enrichment on adult hippocampal neurogenesis compared adolescent C57Bl6 mice in an enriched environment versus standard laboratory housing (Kempermann, Kuhn, & Gage, 1997). For 40 or 68 days, mice in standard laboratory housing were exposed to the normal food, water and bedding, whereas those in enriched housing had toys, tubes, a running wheel, nesting material, food, water and bedding. In this study, enrichment was found to enhance the survival of proliferating neurons (57% more compared to standard housed animals), even though the number of proliferating cells were similar in the two conditions. The authors were one of the first to conclude that environmental enrichment promotes new neurons in the hippocampus, a process that was due to enhanced survival of new neurons, but not proliferation. In a follow up study on neurogenesis, the effects of exercise and environmental enrichment were compared (van Praag, Kempermann, & Gage, 1999). Both exercise and environmental enrichment promoted the survival of BrdU positive cells, but interestingly – only the exercise condition promoted the proliferation of BrdU positive cells. Unfortunately, this study included a running wheel in the environmental enrichment condition, so the effect of only an enriched environment could not be determined. The study by van Praag et al. (1999) also suggested that 40 or 68 days of physical exercise can also influence hippocampal neurogenesis. Studies on voluntary wheel Adult Neurogenesis 3 running have revealed that exercise promotes neurogenesis in young animals. Recent studies also showed that 45 days of running was sufficient to stimulate neurogenesis in 3- month old C57Bl6 mice (van Praag, Shubert, Zhao, & Gage, 2005). Another study looked at the effects of exercise on BrdU positive cells in mice selectively bred for increased voluntary wheel running in 6-8 week old mice of the background strain Hsd:ICR. They found that over the 40 day running period, the number of BrdU positive cells was correlated with running distance in the background strain and animals in the selected condition had more BrdU positive cells than the background strain. Furthermore, both conditions that ran on running wheels had significantly more BrdU positive cells than animals that did not have access to a running wheel (Rhodes, et al., 2003). This suggests that in these young mice, exercise exerted robust effects as seen by the increased neurogenesis in animals given access to wheels and in animals bred