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The effects of short term voluntary exercise in isolated animals Webber, Alina Jane 2006

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THE EFFECTS OF SHORT T E R M V O L U N T A R Y EXERCISE IN ISOLATED ANIMALS by ALINA JANE WEBBER B.Sc, The University of British Columbia, 2004 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Neuroscience THE UNIVERSITY OF BRITISH COLUMBIA July 2006 © Alina Jane Webber 2006 Abstract Voluntary exercise has been shown to consistently increase proliferation and neurogenesis in the dentate gyrus. Recently it was reported that social isolation can counteract the effects of voluntary exercise on cell proliferation and neurogenesis in the dentate gyrus of the hippocampus. We examined the effects of social isolation on animals engaging in voluntary exercise, looking at adult male C 5 7 B L / 6 J mice. Animals in this study were isolated for 1 week and then placed in their respective housing conditions. Exercise wheels were quasi-randomly assigned to cages, and animals were left in this environment for 11 days. On the 12 t h day, 5-bromo-2-deoxyuridine (BrdU) was injected as a marker of dividing cells. Animals were sacrificed 2 hours later to measure cell proliferation. We found no significant effects o f social isolation on cell proliferation. In contrast, voluntary exercise nearly doubled the number of B rdU labeled cells regardless of the animals housing situation. The number of cells positive for doublecortin, (DCX) , a protein found specifically in immature neurons, also significantly increased in response to voluntary exercise. Social isolation did not have a significant effect on this measure, and voluntary exercise, rather than social housing was also found to increase the complexity of the processes of immature neurons labeled by D C X . In addition, a pilot study in aged mice indicated that even aged animals that have been isolated for more prolonged periods of time have a greater survival o f B r d U labeled cells in response to voluntary exercise, although this was a non significant trend. i i Table of Contents: Abstract ii Table of Contents iii List of Tables v List of Figures vi Lists of Abbreviations vii Acknowledgements viii Chapter 1: Introduction 1 1.1 Hypothesis and Obj ectives 1 1.2 Introduction 1 1.3 History of Neurogenesis 3 1.4 Labeling Newly Dividing Cells 5 1.5 Environmental conditions, exercise and neurogenesis 7 1.6 Proposed Mechanisms behind exercise and neurogenesis 9 1.7 Voluntary Exercise and Dendritic Branch Morphology 12 1.8 Neurogenesis and Disease 12 1.9 Summary. 13 Chapter 2: The Effects of Isolation on voluntary exercise induced changes in the Hippocampal Dentate Gyrus IS 2.1. Introduction 15 2.2. Materials and Methods 16 2.2.1. Experimental Subjects 16 2.2.2. Immunohistochemistry 17 2.2.3. Quantification of Immunostaining 18 2.2.3. Corticosterone Serum Measurements 19 2.3. Results 19 2.3.1. The effects of social isolation on cell proliferation in the hippocampal dentate gyrus 19 2.3.2. The effects of social and isolated housing conditions on new neurons in the dentate gyrus 22 2.3.3. The effect of voluntary exercise and isolation on Corticosterone levels 26 2.3.4. The effects of voluntary exercise in isolated housing conditions on cell survival in the dentate gyrus of aged mice 28 2.4. Discussion 30 2.4.1. General discussion 30 iii 2.4.2. Possible limitations 35 2.4.3. Future directions 36 2.5. References 37 Appendix .; 50 iv List of Tables Table 2.1. The effects of social isolation and voluntary exercise on cell proliferation.... 21 Table 2.2. The effects of social conditions and voluntary exercise on the number of D C X labeled cells in the dentate gyrus 23 Table 2.3. The effects of social isolation and voluntary exercise on the morphology of new neurons 25 Table 2.4. Corticosterone levels (ug/dl) of animals in various housing conditions 27 2.4.2. Possible limitations 35 2.4.3. Future directions 36 2.5. References 37 Appendix 50 iv List of Abbreviations A B C Avidin-biotin-peroxidase complex BDNF Brain Derived Neurotrophic Factor BrdU 5-bromo-2-deoxyuridine DAB 3,3 -diaminobenzidine tetrahydrochloride D C X Doublecortin G C L Granule Cell Layer HPA hypothalamic-pituitary-adrenal ip intraperitoneal NGF Nerve growth factor V E G F Vascular endothelial growth factor vii A c k n o w l e d g e m e n t s First and foremost I would first like to thank my supervisor, Dr. Brian Christie for his patience, academic insight and encouragement. Your kindness, sense of humor, and positive attitude never ceased to impress me. I am truly grateful for all your support. Dr. Rankin, thank you for your encouragement and ideas. Dr. Galea, thank you for your help, comments and encouragement. Dr. Soma, thank you for your support, and generously allowing me access to your lab. I would like to thank the members of my lab, past and present. Van, thank you for your patience, guidance and your friendship. There are so many people in the lab that I have enjoyed working with and have helped with so many things: Andrea, Brennan, Carl, Chris, Cristina, Dave, Timal, Marion, Julie and Steph. I would also like to thank our neighbors, the members of the Galea lab for all their help, and occasional piece of cake. M.P. and Mr. T, you have helped out with so much. I am truly tharikful for your support, advice, keeping me profanity free, and most importantly your friendship. I really don't know what I would have done without you guys. I am also grateful for the support of my friends and family. Mom and Dad, Dan, Colleen and Aleena, thanks for your understanding and support. v i n Chapter 1: Introduction 1.1. H y p o t h e s i s a n d O b j e c t i v e s The purpose of this research was to further investigate the effects of voluntary exercise on hippocampal plasticity, with a focus on cellular proliferation and neurogenesis in the hippocampal dentate gyrus. Chapter 2 focuses on a recent report that showed the effects of social isolation counteract the positive benefits of short term voluntary exercise with respect to cellular proliferation (Stranahan et al., 2006). The aim of this study was to quantify the effects of short term voluntary exercise in both isolated and social housing conditions to see if differences exist with respect to cellular proliferation and measures of young neurons. The morphology of these young neurons was also quantified. In addition a pilot study was conducted on aged mice. Aged animals have been shown to have reduced levels of cell survival in the hippocampal dentate gyrus that can be enhanced by exercise (van Praag et al., 2005). These particular animals were aged, and had previously live for a period of at least 6 months in isolation. We looked to see if these animals would benefit from voluntary exercise, even though they have been isolated for a significant period of time. 1.2. I n t r o d u c t i o n The hippocampus is a bilateral structure situated in the medial temporal lobe with connections from several different brain regions, including processed input from many cortical regions. The hippocampus can be divided into four parts, the dentate gyrus, the cornu ammonis, now known as the CA regions, the presubiculum and the subiculum. The hippocampus is known for it's plasticity, and this structure is critical to memory consolidation (Scoville and Milner, 1957; Frankland and Bontempi, 2005). 1 Figure 1.1. The rodent hippocampal formation. The picture on the left shows the position of the rat hippocampus in relation to the whole brain. The picture on the left represents a saggital section of the hippocampus illustrating the position of the corau ammonis (CA1-CA3) and dentate gyrus in relation to one another. Graphic from Unfortunately, not much was known about the function of the hippocampus when a patient, H.M., was given a bilateral medial temporal lobe resection as an experimental treatment for his epilepsy (Scoville, 1954). H.M.'s seizures were significantly reduced, yet he was left with retrograde amnesia, or the inability to remember some events prior to his surgery. Perhaps more devastating was his severe anterograde amnesia, or the inability to form new memories. The surgery that caused this condition not only removed the hippocampus, but the amygdala and other surrounding tissue (Corkin et al., 1997), thus the exact role of the hippocampus in this case is unclear. Nonetheless, this infamous patient inspired numerous studies on the function of the hippocampus with respect to learning and memory. The hippocampus was later seen to be critically important to spatial memory. Lesions to the hippocampus produce spatial learning deficits, most commonly tested in the Morris water maze paradigm (Morris et al., 1982; Mumby et al., 1999; Debiec et al., 2002). More specific lesions to the hippocampal dentate gyrus also produce spatial learning deficits in rats (McNaughton et al., 1989; Xavier et al., 1999; Gilbert et al., 2001). Contextual fear conditioning is yet another task dependent on the hippocampus (Kim et al., 1992; Wiltgen et al., 2006), as is visual recognition memory (Prusky et al., 2004). The hippocampus is not only particularly interesting as it is critical to forms of learning and memory, but it also contains one of 2 only two areas of the brain where neurons are produced well into adulthood (Kemperman, 2006). Some studies suggest that new neurons contribute to some forms of hippocampal dependent learning (Shors et al., 2001; Shors et al., 2002; Madsen et al., 2003). It has been suggested that spatial memories are improved by, or are somewhat dependent on, adult neurogenesis for their formation and consolidation (Snyder et al., 2005). Although there is a growing amount of evidence to support these claims, there is controversy as to the actual functional significance of hippocampal neurogenesis. Other findings show that neurogenesis is not correlated to learning and memory tasks (Merrill et al., 2003; Meshi et al., 2006). Meshi and colleagues used irradiation to show that the behavioral effects of environmental enrichment are unaffected by blocking neurogenesis. Radiation has been also previously used to show that the behavioral effects of antidepressants are dependent on neurogenesis (Santarelli et al., 2003). Blocking neurogenesis with radiation in general has been criticized, however, as it not only blocks neurogenesis, but disturbs the surrounding neuronal network (Monje et al., 2002; Kempermann et al., 2004a). Using a different approach to study the function of new neurons, one study looked at the variations in neurogenesis in 10 different strains of mice. Rates of neurogenesis varied significantly, and correlated with acquisition, or the slope of the learning curve, in the Morris water maze. Although there is debate about the exact significance of this phenomena, understanding what triggers neurons to grow back has exciting therapeutic potential (Briones, 2006; Gouras and Fillit, 2006; Jin et al., 2006). 1.3. History of Neurogenesis Traditionally all aspects of the brain were not considered to be plastic. Neuronal connections were once seen as fixed circuitry, and the generation of neurons in adulthood was thought to be improbable (Rakic, 1985). The first report of neurogenesis in the hippocampus came in 1962 (Altaian, 1962), yet this field of research didn't gain momentum until around 20 years later. Thereafter came reports of stem cells in the adult brain (Reynolds and Weiss, 1992; Palmer et al., 1995). Studies of neurogenesis also expanded to more species. Not only was neurogenesis shown in rats, mice, and 3 nonhuman primates (Gould et al., 1999c), but it also was shown to occur in humans, as well (Eriksson et al., 1998). Erickson's 1998 study was done on terminally ill cancer patients, who had received 5-bromo-2-deoxyuridine (BrdU) injections to mark proliferating cells for tumor staging purposes. One particular patient was 72 years old, indicating that adult neurogenesis is a phenomena that persists well into adulthood. Substantial neurogenesis occurs only in the olfactory system or the dentate gyrus where new olfactory neurons or granule cells are respectively generated (Kempermann et al., 2004a; Lehmann et al., 2005). There have been scattered and unsubstantiated reports of neurogenesis in other regions aside from the hippocampus and olfactory bulbs, in regions of the brain like the neocortex (Gould et al., 1999a; Rietze et al., 2000; Gould et al., 2001). These reports are controversial and remain to be independently verified. In the dentate gyrus, new neurons are specifically formed in a narrow band of tissue known as the subgranular zone (SGZ). In the environment of the SGZ, often referred to as the "neurogenic niche", neurogenesis occurs throughout the lifespan. Within the SGZ there are progenitor cells that are similar in appearance to radial glial cells, but give rise to new neurons (Seri et al., 2001; Filippov et al., 2003; Fukuda et al., 2003). Neurons are present in all stages of maturation here, in addition to neuronal and glial progenitor cells (Filippov et al., 2003; Fukuda et al., 2003; Kronenberg et al., 2003; Seri et a l , 2004). These new neurons, produced in the SGZ, are functional. The first demonstration of this used a green fluorescent protein (GFP) expressing retrovirus, which was used to mark new neurons in the granule cell layer. These new neurons not only had membrane properties and action potentials similar to mature neurons, but they also made functional synaptic connections (van Praag et al., 2002). Newly dividing cells destined to become neurons express early dendrites at just 1 week old. At this time they are also receiving G A B A inputs, which are excitatory until the first glutamatergic synapses develop (Zhao et al., 2006). At an approximate time point of 1.5 weeks, these neurons have extended dendrites into the molecular layer, before the formation of spines (Jones et al., 2003). It is about this time that an axon begins a path towards the CA3 region. The assumed role of dentate gyrus has been the creation of distinct patterns to the CA3 region, primarily by mossy fiber input (Treves and Rolls, 1992). Neuronal development is summarized in figure 1.2. 4 From Entorhinal Figure 1.2. Maturation of granule cells: A new cell has few projections, and is migrating to it's final position at 3 days old. 1 week following division early dendrites appear and this cell begins to receive somatic G A B A inputs at this time (not pictured). A primary dendrite extends at around 5 days later (1.5 weeks). At 2.5 weeks the neuron has a few spines, indicating it synapses with the entorhinal cortex. 2 months later this neuron develops more complex dendritic arborizations that are indicative of mature cells. This neuron also exhibits electrophysiology similar to mature neurons. Modified from (Aimone et al., 2006) 1.4. Labeling Newly Dividing Cells It is important to note that there are two interrelated aspects of hippocampal cell growth, being cellular proliferation and cellular survival (Kepperman, 2006). Cellular proliferation is simply the mitotic activity of progenitor cells, which can be measured 5 hours to days after the injection of a mitosis marker, typically BrdU, or [3H]thymidine (Cameron et al., 1993; Dayer et al., 2003). Cellular proliferation has no causal relationship to the rate at which cells survive and develop into neurons (neurogenesis). The initial proliferation of cells is required, however, for new neurons to be produced. There are several methods of measuring cell division. BrdU was developed to label S phase cells (Miller and Nowakowski, 1988), incorporating itself into DNA by acting as a thymidine analogue during DNA synthesis. One downfall of using BrdU is that it's also a mutagen that can produce morphological and behavioral abnormalities (Nagao et al., 1998; Kolb et al., 1999). Cameron and McKay found that with a single, low dose, BrdU injection the number of cells doubles between 2 and 24 hours of injection (Cameron and McKay, 2001). The S-phase of the cell cycle in proliferating cells in the dentate gyrus of C57BL/6J mice was determined to be 8 hours (Nowakowski et al., 1989). The S-phase is typically followed by a G2 phase, and in total the cell cycle of embryonic neural stem cells is thought to be 9-12 hours (Ueno et al., 2006) Regardless, various studies measure cellular proliferation at different time points prior to the administration of BrdU, perhaps the most common at 2 hours and 24 hours following a BrdU injection. (Gould et al., 1999b; Falconer and Galea, 2003; Eadie et al., 2005; Redila et al., 2006). Cell survival is measured at various time points after BrdU administration. After only 4 days after a BrdU injection the cell count is reflective of cell death as opposed to BrdU dilution (Dayer et al., 2003). The number of surviving cells typically decreases after 4 days, yet even 292 days after treatment with BrdU, labeled cells can still be seen (Dayer et al., 2003; Zupanc et al., 2005). Another marker of neurogenesis in the hippocampus is doublecortin (DCX). D C X is a microtubule associated protein found in most migrating neural precursor cells during nervous system development (Gleeson et al., 1998). D C X has been shown to be a valid marker for neurogenesis in adult rodents (Brown et al., 2003b; Kempermann et al., 2004b; Rao and Shetty, 2004; Couillard-Despres et al., 2005). Unlike BrdU, it is non-invasive, thus allowing for the study of neurogenesis in tissue that could not otherwise be labeled, as in the case of wild animals, for instance (Barker et al., 2005). D C X is expressed in cells at early stages of neurogenesis. It's expression is transient, lasting for 6 around 2-3 weeks, and up to 4 weeks (Brown et al., 2003b). During this time, as these cells mature into granule cells, they send out growth cones (Brown et al., 2003b). Perhaps another advantage of D C X over other makers of neurogenesis is that it's associated with the cytoskeleton, and these growth cones can therefore be visualized (Francis et al., 1999). It has also been shown that 90% of cells that expressed D C X in the adult hippocampus co-labeled with BrdU and early neuronal markers. There was a lack of coexpression with glial and apoptotic markers as well, suggesting that D C X is labeling healthy neurons (Rao and Shetty, 2004). D C X contributes to the formation of new neurites and microtubule reorganization (Gleeson et al., 1998; Francis et al., 1999; Shmueli et al., 2001; Ribak et al., 2004). Fully developed, more complex dendritic trees that extend to the outer molecular layer are not only required for proper hippocampal function, but are suggestive of neuronal maturity and integration into the hippocampal circuit (Rihn and Claiborne, 1990; Jones et al., 2003). As D C X marks these developing neurites, their morphology can be analyzed. Chronic alcohol exposure reduces the dendritic growth of immature dendritic cells, as seen with DCX, for example (Jones et al., 2003). Levels of neurogenesis and proliferation in the hippocampus vary considerably. In a C57BL/6J mouse, around 1600 cells proliferate each day, whereas on average 800 new cells appear daily in the hippocampus of a BALB/cByJ mouse (Hayes and Nowakowski, 2002). This phenomenon has been studied in depth in rats, and an astonishing 6% of the total size of the granule cell population is generated each month. (Cameron and McKay, 2001) 1.5. Environmental conditions, exercise and neurogenesis Before the discovery that new neurons could be generated into adulthood it was known that the environment had effects on brain functioning. Perhaps the earliest profound studies on the environment's effects on the brain came from Hebb, in 1947. Hebb, better known for his neuropsychological postulate, took some of the rats he was studying home. These rats were kept as pets as his children, who were cited as experimenters in his work. These rats were housed in a more complex environment than a standard laboratory cage. These rats were then compared to rats raised in laboratory 7 conditions, and it was noted how experience clearly influenced cognitive abilities later in life (Hebb, 1947; Morris, 1999). The effects of these variables were only later studied with respect to neurogenesis (Kempermann et al., 1997; Ickes et al., 2000; Bruel-Jungerman et al., 2005; Lazic et al., 2006). The definition of an enriched environment is an interesting subject. A classic definition of enrichment is "a combination of complex inanimate and social stimulation" (van Praag et al., 2000). Standard housing typically involves animals housed together in groups. Housing animals together is one part of enrichment, as are complex environments, but this varies greatly between experiments. In one study on hippocampal neurogenesis, environmental environment meant rats were housed 10 per cage with access to a running wheel and various other objects. This particular study therefore didn't distinguish social components from environmental complexity or voluntary exercise (Matsumori et al., 2006). Yet other studies make clear distinctions about the separation of voluntary exercise and environmental environment (van Praag et al., 1999a; Faherty et al., 2003; Will et al., 2004). An enriched environment has been shown to increase neurogenesis (Paylor et al., 1992; Kempermann et al., 1997; Pham et al., 1999; Bruel-Jungerman et al., 2005). environmental environment has also been shown to improve an animals performance on hippocampal dependent tasks (Paylor et al., 1992). Adding extra objects and companions for animals affects the hippocampus, but specifically separating voluntary exercise from environmental environment makes a difference with respect to cellular proliferation (van Praag et al., 1999a; Olson et al., 2006). Voluntary exercise was first noticed by Gage's group in 1999, showing that voluntary exercise in mice increased neurogenesis, doubling the amount of surviving cells, and also causing an increase in proliferation (van Praag et al., 1999a). That year Gage and colleagues also showed that running enhances synaptic plasticity and learning and memory in addition to increasing neurogenesis (van Praag et al., 1999b). The experiments done in Gage's laboratory looked separately at the effects of various conditions on proliferation and cell survival. Some animals were exposed to learning and memory tasks in a water maze (a learner), there were swimming controls (no learning), a voluntary exercise only group, animals with an enriched environment, and lastly a control group. Throughout all of these conditions it was found that only voluntary 8 exercise resulted in an increase in proliferation, and that both an environmental environment and voluntary exercise caused similar increases in cell survival. Since voluntary exercise caused an increase in cellular proliferation, unlike environmental enrichment, yet both manipulations increased neurogenesis, this study also hinted at the possibility that there are two distinct pathways in which neurogenesis is upregulated. One of the initial common concerns about the methodology behind environmental enrichment and voluntary exercise would be that adding an exercise wheel seems similar to adding environmental environment materials. Environmental enrichment can include adding objects to an animal's cage, thus making their environment more complex, and there is likely an increase in motor activity due to exploration of these novel objects. An exercise wheel could be considered to fit this definition, too. Exercise has been shown to increase neurogenesis when mice are placed in the same conditions, the only different condition being a motile vs. non motile treadmill (Trejo et al., 2001; Llorens-Martin et al., 2006). Another study involved exercise wheels that were only able to rotate for a limited amount of time. The same wheels were kept in cages, yet they were blocked from movement for set times, and a dose dependent effect was seen on neurogenesis (Holmes et al., 2004). In studies involving voluntary exercise, animals that have access to a running wheel typically utilize it frequently. Mice in one study ran variable amounts, ranging from 9.6 km per day to 14 km per day (Valentinuzzi et al., 1997). Other studies with rats have yielded more modest results, with an average of 4.8 km/day (Farmer et al., 2004). Both studies show that the animals that utilize their running wheels do so with a startling frequency. Regardless of whether or not exercise levels were measured, the addition of an exercise wheel typically doubles levels of proliferation and neurogenesis (van Praag et al., 1999b; van Praag et al., 1999a; Couillard-Despres et al., 2005; Eadie etal, 2005). 1 . 6 . P r o p o s e d m e c h a n i s m s b e h i n d e x e r c i s e a n d n e u r o g e n e s i s There are a variety of ways in which exercise is thought to have an impact on neurogenesis. One suggested method involves changes in vasculature, and increased 9 blood flow to the brain. Exercise results in increased cerebral blood flow and volume (Yancey and Overton, 1993; Swain et al., 2003). With increased motor activity comes an increased permeability of the blood brain barrier, as well as an increase in glucose utilization (Vissing et al., 1996). This increase in blood flow and blood brain barrier permeability may perhaps more effectively deliver an increase in growth factors that are accompanied with exercise. Some of these growth factors that increase with exercise are vascular endothelial growth factor (VEGF) (Schobersberger et al., 2000), brain derived neurotrophic factor (BDNF) (Neeper et al., 1996; Russo-Neustadt et al., 1999; Farmer et al., 2004) and nerve growth factor (NGF) (Neeper et al., 1996). Thus, vascular changes as a result of voluntary exercise are suspected to play a large role in the effects of exercise and neurogenesis. The SGZ, the site of neurogenesis in the hippocampus, is a highly vascularized region. Theo Palmer's concept of a "vascular niche" refers to his observations that cell division in this area is highly linked to vasculature (Palmer et al., 2000). This group made the observation that cells divide in dense clusters around the vasculature, and that 37% of these dividing cells have endothelial markers, most of which will disappear over the following weeks. This suggested that neurogenesis is intimately associated with vasculature. Interestingly, the potent growth factor that results in angiogenesis, VEGF, also increases neurogenesis (Jin et al., 2002; Cao et al., 2004). Increasing V E G F in adult rats was seen to result in a doubling of new neurons (Cao et al., 2004; During and Cao, 2006). V E G F has recently been shown to be involved in mediating the effects of the animals environment on neurogenesis and cognition (During and Cao, 2006). Although this has yet to be further investigated, V E G F is an important regulator of neurogenesis. BDNF was first isolated from pig brains in 1982(Barde et al., 1982), known to be a neurotrophic factor, it was shown to support the survival and growth of neurons in culture (Lindsay and Rohrer, 1985). BDNF has been shown to help stimulate neurite outgrowth, and promote neuronal survival and regeneration (Wozniak, 1993; Horch, 2004). BDNF is speculated to be another one of the major mechanisms in which running influences neurogenesis, as levels of BDNF increase with excercise (Farmer et al., 2004). BDNF was directly infused into the hippocampus via osmotic pumps, and neurogenesis 10 was examined a month later. It was also found that neurogenesis was increased on the side of the BDNF infusion (Scharfman et al., 2005) Many additional factors have been linked directly or indirectly to proliferation and neuronal survival in the dentate gyrus. In addition to species differences, the amount of actively dividing cells is altered by several factors, including age, prolactin, corticosteroids, and serotonin, to name a few (Kao and Lin, 1996; Reagan and McEwen, 1997; Shingo et al., 2003; Lucassen et al., 2004). Studies in this field must be done with great attention to the minor details of an animal's environment, as several factors can impact this phenomena. What might be seen as minor details, such as the texture of food, has been seen to significantly impact on proliferation of cells in the dentate gyrus (Aoki et al., 2005). One surprising aspect of voluntary exercise is that there is a somewhat paradoxical effect on levels of stress hormones. Exercise increases blood corticosterone levels (Droste et al., 2003; Colbert et al., 2006). Elevated levels of corticosterone are typically a result of stress, and in stressful situations elevated corticosterone levels have been linked indirectly to decreases in proliferation and survival of these granule cells (Tanapat et al., 1998; Tanapat et al., 2001; Wong and Herbert, 2006). The effects of exercise on the hypothalamic-pituitary-adrenal (HPA) axis, however, is complex. Exercise can also reduce anxiety related behavior (Binder et al., 2004), yet mice that exercise have been shown to have increases in stress hormones. Behavioral testing of this effect was particularly interesting. Stress tests that were physically challenging caused increases in corticosterone levels, whereas stressors that were more psychological resulted in decreased corticosterone levels in mice that had previously exercised. (Droste et al., 2003). In summary, exercise increases corticosterone, but seems to have some of the opposite effects of an increase in corticosterone; a decrease in anxiety related behavior and an increase in cell proliferation. The effects of corticosterone on the amount of BrdU labeled cells has also been shown to have a dose dependent effect (Montaron et al., 2006) 11 1.7. V o l u n t a r y E x e r c i s e a n d D e n d r i t i c B r a n c h M o r p h o l o g y Other forms of neuronal plasticity can change as a result of voluntary exercise. Reorganization at a synaptic and cellular level can occur due to external influence. For example exercise has been seen to change the density of synapses within the hippocampus (Nakamura et al., 1999; Rampon et al., 2000; Eadie et al., 2005). Voluntary exercise and BDNF have not only seen to have a profound difference on the survival of neurons, but they also have been shown to influence the cytoarchitecture of granule cells in the dentate gyrus. Voluntary exercise has been shown to increase the overall dendritic length of granule cell neurons in the dentate gyrus, and increase their complexity. It was surprising to see that voluntary exercise appeared to decrease the number of primary projections coming from these cells. This was presumably as a result of increased neurogenesis and thus more underdeveloped cells were present (Redila and Christie, 2006). Another study looked at dendritic branch morphology and length in both the CA1 region and dentate gyrus of the hippocampus. They found that length and complexity of neurons in the dentate gyrus changed as a result of the environment, but not as a result of voluntary exercise (Faherty et al., 2003). Voluntary exercise may cause changes that would intuitively seem to enhance the functioning of granule cell neurons, whereas other modifications can do the opposite. Prenatal ethanol exposure interestingly was not seen to decrease spine density in the CA1 region of rats, but changed the way these cells responded to environmental environment, as only control rats had environmental environment related increases in spine density. Defects in dendritic branch morphology were also seen in pups born to mothers with a known cause of intrauterine growth restriction (Dieni and Rees, 2003). 1.8. N e u r o g e n e s i s a n d D i s e a s e The ability to regenerate neurons in the adult brain has exciting potential applications to disease. For example increases in hippocampal neurogenesis as a result of environmental environment appears to have benefits in the mouse model of Huntington's disease (Lazic et al., 2006). Alzheimer's disease is characterized by a loss of hippocampal neurons due to toxic plaque formation, and therapies that increase neurogenesis have been suggested, 12 some with promising preliminary results (Greenberg and Jin, 2006; Jin et al., 2006; Kelleher-Andersson, 2006). Previously, I have worked with an animal model of prenatal ethanol exposure. Fetal alcohol exposure is not only the leading cause of birth defects in the United States, but the leading cause of mental retardation (Stratton, 1996). The extent of prenatal exposure to ethanol, and the timing of exposure during fetal development can have huge impacts on the severity of the symptoms. Severe enough growth, facial, and neurodevelopmental abnormalities results in the familiar diagnosis of Fetal Alcohol Syndrome (Jones et al., 1973). FAS is typically associated with a variety of neurobehavioral disorders and a reduced brain weight (Burd and Martsolf, 1989). Even at moderate doses, ethanol exposure during fetal development can cause learning and memory problems throughout the adult lifespan, and all prenatal ethanol exposure related disorders are classified as Fetal Alcohol Spectrum Disorder (Sokol et al., 2003). Ethanol causes a dramatic reduction in neurogenesis in the adolescent brain (Crews et al., 2006), although moderate ethanol intake increases neurogenesis and proliferation in the dentate gyrus (Aberg et al., 2005). Ethanol typically negatively affects neuronal cell growth in the dentate gyrus (Nixon and Crews, 2002; Crews and Nixon, 2003; Crews et al., 2004; He et al., 2005; Crews et al., 2006). It has also been shown that the negative effects of chronic ethanol exposure on neurogenesis in adult animals can be reversed by voluntary exercise (Crews et al., 2004). There are known hippocampal deficits in animals with fetal alcohol syndrome. We were able to demonstrate that voluntary exercise was enough to counteract the deficits in spatial learning and memory and long term potentiation that was seen in FAS animals without access to running wheels (Christie et al., 2005). Thus the processes that underlie voluntary exercise can have dramatic consequences in disease models. 1.9. S u m m a r y The hippocampus, a structure critical to learning and memory, is one of only two places in the brain where neurogenesis occurs in adulthood. Neurogenesis is a relatively new field of research, and factors that have been shown to increase neurogenesis are 13 often linked to improved learning and memory, although the exact role of new neurons is still debatable. Voluntary exercise has been shown several times to result in substantial increases in the proliferation of cells and amount of new neurons seen in the dentate gyrus. This is often attributed to an increase in vasculature and various growth factors produced in response to voluntary exercise. Other conditions such as disease can be associated with neuronal loss. Only recently have connections been made to treating these conditions with treatments that have been seen to increase neurogenesis. Understanding the processes controlling neurogenesis has exciting implications for treatments of disease, and further understanding circuitry involved with learning and memory. 14 Chapter 2: The Effects of Isolation on Voluntary Exercise induced changes in the Hippocampal Dentate Gyrus. 2.1. Introduction The main focus of this thesis will be to examine the effects of social isolation on hippocampal cell proliferation. This interest arose from a recent report in Nature Neuroscience that showed that social isolation can counteract the effects of short term voluntary exercise on proliferation in the dentate gyrus, causing a decrease rather than an increase in cell proliferation in some situations (Stranahan et al., 2006). It was shown that isolation prevents an increase in proliferation in response to voluntary exercise from 3 to at least 24 days of exercise (Stranahan et al., 2006). The authors suggested that social interaction somehow protects the hippocampus from the negative effects of glucocorticoids that are upregulated with voluntary exercise. This study primarily looked at the effects of voluntary exercise for 12 days, or short term exercise. Stranahan hypothesized that this effect had never been seen before for three possible reasons, mainly because the effects of voluntary exercise are normally looked at in socially housed animals. In addition, most studies look at neurogenesis only long after the animals have been in voluntary exercise conditions, rather than looking at a short term effect. Also, if animals are isolated for the duration of their time with an exercise wheel, they are isolated for no more than 3 days and nearly immediately put into voluntary exercise conditions. Their particular study isolated animals for 1 week prior to exercise, and this was considered prolonged isolation that may be required to see this effect. To examine the effects of isolation, the authors chose to use male animals as they were concerned about fluctuations in gonadal hormones affecting neurogenesis in females (Tanapat et al., 1999; Galea et al., 2006). The requirement of social housing for cell proliferation in the dentate gyrus is thought to be involved with the HPA axis, as social housing resulted in decreased levels of corticosterone (Stranahan et al., 2006). One recent study has also linked social isolation to depression as isolated rats have reduced BDNF levels, an important factor for neuronal survival in the hippocampus that has also been linked to depression (Lehmann 15 et al., 2005; Kalueff et al., 2006; Scaccianoce et al., 2006). Although Scaccianoce and colleagues found decreased BDNF in isolated animals, interestingly plasma corticosterone was unaffected. Since the discovery that voluntary exercise increases neurogenesis, many other studies have been published examining this phenomena in further detail. Research groups have looked at individually housed rats (Bjornebekk et al., 2005; Eadie et al., 2005; Redila et al., 2006) and group housed rats (Briones et al., 2005; Komitova et al., 2005; Briones, 2006). The C57BL/6J mouse is also a commonly, and an arguably more popular animal model for this field of research as well. Some U K estimates suggest mice account for 80% of all research animals (Hunt and Hambly, 2006). Voluntary exercise and neurogenesis has been studied in group housed male mice (Fabel et al., 2003; Kitamura et al., 2003; Kronenberg et al., 2005), group housed female mice (van Praag et al., 1999b; van Praag et al., 1999a; Brown et al., 2003a) and isolated mice, as well (Holmes et al., 2004; van Praag et al., 2005). If social isolation counteracts the effects of short term voluntary exercise, this is not only an interesting area of study, but an important methodological warning for future research. The aim of this experiment was to quantify the effects of voluntary exercise in isolated and socially housed conditions. 2 . 2 . M a t e r i a l s a n d M e t h o d s 2.2.1. Experimental Subjects Male C57BL/6J mice were ordered from Charles River. The mice arrived at the age of two months (23-25g) and were acclimatized to the laboratory in isolation for 1 week in standard cages. Animals were maintained on a 12-hour light/dark cycle and given free access to food and water. Following a week of isolation after acclimatization, to meet Stranahan's criteria of prolonged isolation (Stranahan et al., 2006), animals were either: kept isolated, kept isolated with access to an exercise wheel, socially housed, or socially housed with access to exercise wheels. Social conditions were defined as 3 animals per cage. These groups were named social runner (n=6), social non-runner (n=3), isolated runner (n=7). and isolated non-runners (n=7). Running wheels were 11 cm in diameter, thus all animals for the experimental part of our study were moved to larger cages 16 (46 x 24 x 20 cm). Group housed animals were carefully monitored for signs of aggression. If any animals displayed aggression, that group housed cage was removed from the study. All of the procedures used in these experiments were in accordance with the Canada Council on Animal Care and were approved by the University of British Columbia Animal Care Committee. For a pilot study on aged mice, males were 1.5-2 years old, and were assigned to isolated runner (n=4) or isolated non-runner (n=4) conditions. They had been previously housed in isolation for at least 6 months prior to being assigned to exercise or sedentary conditions. The younger animals were injected with BrdU on day 12 of experimental treatment. This mitotic marker was administered in a single i.p. injection of 200mg/kg of BrdU at a volume of 20 mg/kg. Animals were injected 2 hours following the start of their dark cycle, and were given a Urethane overdose 2 hours later. Animals were removed from their cage for and average of 30 seconds for the BrdU injection. Blood samples were collected from the right ventricle, and mice were then transcardially perfused with 30 ml of 0.9% saline, followed by 30 ml of 4% paraformaldehyde. Brains were removed and stored in 4% paraformaldehyde for 24 hours, then transferred to 30% buffered sucrose until saturated. Following this, 40pm coronal sections were obtained for the entire hippocampus using a Leica VT1000 vibratome. The protocol for older animals was identical with the exception that older animals were injected with BrdU two times in one day, 2 hours following the start of the light cycle, and 2 hours before their night cycle. Following access to running wheels and were perfused four weeks later as a measure of cell survival. 2.2.2. Immunohistochemistry Immunohistochemistry to detect BrdU labeled cells was performed as previously described (van Pragg et al., 1999a; Eadie et al., 2005). To block endogenous peroxidase activity, free floating sections were immersed in .6% H2O2 in Tris-buffered saline (TBS; 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5) for 30 minutes. Sections were then placed in 17 50% formamide/2x SSC (0.3M NaCl and 0.3M sodium citrate) at 65°C, rinsed for 5 minutes in 2x SSC, then incubated in 2N HC1 at 37°C for 30 minutes. Sections were then placed in 0.1 M boric acid (pH 8.5) for 10 minutes. The following steps denatured DNA. The tissue was then rinsed six times in TBS for a total of 90 minutes, and incubated in a mixture of TBS++ (TBS, 0.1% Triton X-100, and 3% donkey serum). Sections were then incubated with a biotinylated anti-BrdU (MAB3262B Chemicon, Temulcula, CA) which was diluted to a concentration of 1:100 in TBS++ overnight. After being rinsed in TBS, an avidin-biotin-peroxidase (ABC Elite Kit) was applied for 2 hours. Tissue was rinsed and treated with a peroxidase detection kit (DAB Kit, Vector). The sections were then mounted on 2% gelatin coated slides. A cresyl violet stain was applied to clearly mark the granule cell layer, and prior to that tissue was coverslipped for microscopic analysis. D C X Immunohistochemisty: Sections were washed in PBS and incubated at room temperature for 16 hours in a 1:1000 dilution of goat anti-DCX polyclonal primary antibody (Jackson ImmunoResearch Labs). This was diluted in 0.1 PBS, 0.5% Triton-X, and 2% normal rabbit serum. Sections were washed 3X for 10 minutes in 0.1M PBS before the addition of a solution containing a 1:1000 dilution of anti-goat biotinylated antibody. This was allowed to incubate for 1 hour at room temperature. Tissue was rinsed and treated with the same A B C and DAB kits for the same times as mentioned above. The sections were then mounted on 2% gelatin coated slides and dehydrated in progressively increasing concentrations of ethanol before being coverslipped. 2.2.3. Quantification of Immunostaining From each animal, every 6 th coronal section, 200pm apart were taken throughout the extent of the hippocampus. Labeling of the slides was done so that the experimenter was blind to the groups, and BrdU immunopositive cells were manually counted. The SGZ was defined as the area within two cell bodies (~20um) of the inner edge of the molecular layer. The hilus resides in between this area. The entire left and right dentate gyrus was counted per section. Only round, nuclei that appeared intact were counted. Split nuclei cells that had not finished cytokinesis were counted as one cell for a 1 8 conservative estimate, and counts were done at a magnification of 100X. Images were captured via an Olympus camera and generated in ImagePro Plus. DCX immunopositive cells were also counted manually using the same equipment. Cells were counted only if individual membranes could be observed at 100X, thus resulting in a conservative estimate in animals with many D C X + cells, as they tended to overlap. 30 cells per animal were chosen quasi-randomly for measures of nodes. A node was defined as a point in which two early dendritic branches intersected. To pick these cells the dentate gyrus from each slice was visualized, and cells that had processes that were distinguishable from other Only neurons in the SGZ were counted, as some had migrated to the IGZ, which is indicative of increased maturity (see figure 1.2). Nodes were defined as the branch points of the immature dendrites. Neurons were not counted if they had cut off dendrites or were obscured by a neighboring cell body. An average of 60 40pm sections were obtained per mouse hippocampus. An average number of cells per 40 pm slice was obtained, and multiplied by an estimate of the average number of slices that made up the hippocampus. On average 72 slices of hippocampus were obtained per animal 2.2.3. Corticosterone Serum Measurements: Total corticosterone levels (bound plus free) in was measured by radioimmunoassay. A corticosterone 3H RIA kit was used to determine plasma corticosterone levels (MP Biomedicals). An adapted protocol was utilized to obtain corticosterone levels (Weinberg and Bezio, 1987; Gabriel et al., 2002). To absorb and precipitate free steroids after incubation, Dextran-coated charcoal was used. Samples were analyzed using a Scintisafe Econo2. (Fisher Scientific, Ltd). 2 . 3 . Results 2.3.1. The effects of social isolation on cell proliferation in the Hippocampal dentate gyrus. After only 11 days of access to running wheels, regardless of housing conditions, proliferation in the dentate gyrus was increased to values approximately double that of 19 other animals. An A N O V A and post hoc tukey analysis was performed, showing that voluntary exercise significantly increased proliferation (/7(i,i7)=71.178, p<0.00001). The differences between social and isolated animals was not significant (F(i;i7)=0.77574), and there was no significant interaction between social/isolated conditions and the effects of voluntary exercise (^(1,17)—0.1179). Cell counts and standard error of the mean are shown in table 2.1 and figure 2.1. Pictures, taken at 10 um, of the hippocampal dentate gyrus from an animal in each group are shown in figure 2.2. Weights of the mice not differ before or after the 12 days of the isolated runner, isolated non-runner, social runner, and social non-runner conditions. Mean weights at the time of BrdU injection for each group were as follows; Isolated runner: 24.0 ± 0.4g, Isolated non-runner: 24.9 ± 0.8g, Social runner: 25.5 ± 0.8g, Social non-runner: 24.7 ± 1.2g. 1800 1600 1400 1200 1000 • Isolated Runner (n=6) • Isolated Non-runner (n=6) H Social Runner (n=6) • Social Non-runner (n=3) Housing Condition Figure 2.1. The effects of social isolation and voluntary exercise on cell proliferation. Regardless of whether or not animals were isolated, voluntary exercise approximately doubled rates of cell proliferation. There was no effect of social conditions in rates of proliferation. Error bars are representative of the standard error of the mean (SEM). * denotes significance (pO.00001). 20 # BrdU labeled Cells # BrdU labeled cells Condition N per 40um section per hemisphere Isolated Non-runner 7 26.0 ± 2.4 778 ± 74 Isolated Runner 7 49.4 ± 2.4 * 1484 ± 7 4 * Social Non-runner 6 27.6 ± 1.2 826 ± 38 Social Runner 3 42.4 ± 3.2 * 1591 ± 9 7 * Table 2.1. The effects of social isolation and voluntary exercise on cell proliferation. The number of BrdU positive cells per dentate gyrus in each group was rounded to 1 decimal point. The standard error of the mean is also given. * denotes significance (pO.00001). Figure 2.2. BrdU labeled cells in the dentate gyrus. Images were taken at a magnification of 10X, and the scale bar represents 100pm. Brdu labeled cells appear dark. A) The dentate gyrus of an isolated non-running animal. B) An isolated running animal. C) A socially housed non-running animal. D) A socially housed, running animal. Photos are not necessarily representative of exact cell counts, but were included to depict the trend seen. 21 2.3.2. The effects of social and isolated housing conditions on new neurons in the dentate gyrus. The number of new neurons produced was only seen to depend on whether or not the animals had access to an exercise wheel, and was independent of the social situation. The number of D C X positive cells per dentate was analyzed with A N O V A and post hoc tukey test. Voluntary exercise significantly increased the number of D C X + cells seen in the dentate gyrus (F(i;i7)= 102.24, p<0.00001). Interestingly isolated animals with or without access to a running wheel had slightly increased amounts of D C X + cells in the dentate gyrus. Socially housed animals had an average of 448 ± 20 D C X + cells per 40um slice, whereas isolated animals had an average of 552 ± 26 D C X + cells. This difference however, was not significant (/r(i,i7)= 3.6864). There was no significant interaction between voluntary exercise and housing conditions (F(\tny= 0.327). Exact cell counts, graphs, and photos can be seen in figure 2.3, table 2.2, figure 2.4 and 2.5. Further analysis of the D C X + cells revealed that the average numbers of nodes, or branch points that a new neuron has increases with voluntary exercise, yet no effects or interactions with regards to social conditions were seen. The effect of voluntary exercise, analyzed with an A N O V A and post hoc tukey test, was significant (F(\,\9)= =76.698, p<0.00001). There were no significant effects of social isolation on the morphology of these new neurons ( F ^ i ^ =3.914), and there was also no interaction between isolation and exercise (F(i ;i9)= =0.181). A cell with one primary process only was described as having 0 nodes, and the average counts of nodes for each group is graphed in Fig. 2.5. Average node counts are given in Table 2.3. 22 35000 • Isolated Runner (n=6) • Isolated Non-runner (n=6) E3 Social Runner (n=6) • Social Non-runner (n=3) Housing Condition Figure 2.3: The effects of social isolation and voluntary exercise on neurogenesis. The only factor that significantly affected the amount of DCX + cells was voluntary exercise. Error bars are representative of the SEM. There was no effect of social conditions in rates of neurogenesis. Error bars are representative of the standard error of the mean (SEM). * denotes significance (pO.OOOOl). # of DCX labeled cells # of DCX labeled cells Condtion N per 40pm section per hemisphere Isolated Runner 6 958 ± 4 6 * 2 8 7 6 2 ± 1 4 1 0 * Isolated Non-runner 6 552 ± 26 16618 ± 7 9 5 Social Runner 6 900 ± 42 * 27046 ± 1310* Social Non-Runner 3 448 ± 20 13446 ± 5 5 9 Table 2.2. The effects of social conditions and voluntary exercise on the number of DCX labeled cells in the dentate gyrus. Cell counts are rounded up to the nearest whole number, and the standard error of the mean is given in each case. * denotes significance (pO.OOOOl). 2 3 Figure 2.4. The effects of social isolation and voluntary exercise on the number of DCX labeled cells in the dentate gyrus. Photomicrographs were taken using a 40X objective. The scale bar is indicative of a distance of 50p.m. Photographs correspond to: A) Isolated non-runner, B) Isolated runner, C) Socially housed non-runner, and D) Socially housed runner. The number of D C X positive cells increased significantly as a result of voluntary exercise (p<0.00001), and was independent of the housing scenario. 24 Figure 2.5. DCX positive cells, as seen from a 100X objective lens: An isolated cell from a non-running animal (A), and a cluster of cells from a running animal (B). Cells in which processes could be clearly distinguished, were not broken, and were in the same position in the SGZ, were analyzed for measures of morphology. Scale bars are indicative of 10pm. Condition N # of Nodes per DCX positive cell Isolated runner 7 1.385 ± 0 . 0 5 9 * Isolated non-runner 7 0.742 ± 0.066 Social runner 6 1.494 ± 0 . 0 5 7 * Social non-runner 3 0.911 ± 0 . 0 8 9 Table 2.3. The effects of social isolation and voluntary exercise on the morphology of new neurons. The average number of nodes per D C X labeled cell was taken, and rounded to three decimal places. A node is defined as a branch point between two processes. A cell with one process and no branches would have 0 nodes. Social or non-social housing did not make a significant difference on morphology, unlike voluntary exercise. * indicates a significant difference (p<0.00001). 25 • Isolated Runner (n=7) • Isolated Non-runner (n=7) E3 Social Runner (n=6) • Social Non-runner (n=3) Housing Condition Figure 2.6. The effects of social isolation and voluntary exercise on the morphology of new neurons. The two groups that engaged in voluntary exercise show significant differences from the groups that did not have access to an exercise wheel. Social housing for the duration of exercise had no effect on neuronal morphology. Error bars are representative of the SEM. * denotes significance (p<0.00001). 2.3.3. The effect of voluntary exercise and isolation on Corticosterone levels Corticosterone levels showed a trend in that runners have increases in corticosterone, and that isolated animals had decreases in corticosterone. None of these differences were significant. There was no effect of exercise on corticosterone levels (F(i,i9)= 1.443), or social conditions on corticosterone levels (F(ii9)= 0.66788). Furthermore there was no significant interaction between exercise and social housing conditions on corticosterone levels (F(i;i9)= 0.007). Measures were taken around 4 hours after the onset of the night cycle just prior to perfusions. Corticosterone levels in each group are shown in figure 2.7 and table 2.4. 26 30.00 0.00 • Isolated Runner (n=7) • Isolated Non-runner (n=7) • Social Runner (n=3) • Social Non-runner (n=6) Housing Condition Figure 2.7. Corticosterone levels (ug/dl) of animals in various housing conditions. There was a trend for animals that exercise to have increases in corticosterone. None of these differences were seen to be significant. Corticosterone Condition N levels (ug/dl) Isolated runner 7 16.47 ± 4 . 0 0 Isolated non-runner 7 11.88 ± 2 . 8 7 Social runner 6 20.16 ± 3 . 9 0 Social non-runner 3 14.89 ± 5 . 0 4 Table 2.4. Corticosterone levels (ug/dl) of animals in various housing conditions. This table shows levels of corticosterone and N values for each group. Individual variation regardless of housing condition was more pronounced. 27 2.3.4. The effects of voluntary exercise in isolated housing conditions on cell survival in the dentate gyrus of aged mice. Aged animals were isolated for at least six months prior to being assigned to the isolated runner and isolated non-runner housing conditions. They showed no significant effect of voluntary exercise on cell survival in the dentate gyrus. There is a similar trend with respect to a doubling in cell counts after exposure to voluntary exercise. Each group had an n of 4, and the aged group had an average of 19 ± 7 cells per hemisphere and the aged, exercising group an average of 44 ± 11 cells per hemisphere. Using a students T-test, there was no significant difference seen between groups, (p=0.06). Cell counts are plotted in Figure 2.6. A picture depicting cell survival is shown in figure 2.9. Unfortunately, as aged isolated mice are rare, we were forced to work with a significant variation in weight. Weights ranged from 50-32g, although on average, the isolated runner groups weighed 44 ± 2g, and the isolated non-runner group weighed 43 ± 4g. 6 0 -r ffi a w 5 0 "35 o •D a> 10 3 4 0 Q) a. n E m as o 3 0 -o • o Q . m hip 2 0 -o be 1 0 -E 3 Z 0 • Isolated Runner (IR) • Isolated Non-runner (INR) H o u s i n g C o n d i t i o n Figure 2.8 The effects of voluntary exercise on survival of cells in the hippocampus of isolated, aged mice. The error bars represent the SEM. A marginally significant difference (p=0.06) was seen between the aged animals and aged animals that had access to an exercise wheel. 28 Figure 2.9 A BrdU labeled cell at 20X, and 100X. This was included to illustrate cell survival. This picture was taken from a younger C57BL/6J mouse, immunolabelled at the same time as the aged animals as a precautionary control, as surviving cells are much more frequent in younger animals. The scale bar in the larger photo is indicative of 50pm, whereas the scale bar in the magnified image is 10pm. 29 2 . 4 . Discussion 2.4.1. General discussion In this study, we have shown that in even in isolated conditions, animals exhibit an increase in proliferation and the number of new neurons in the hippocampal dentate gyrus in response to voluntary exercise. Not only that but these new neurons showed more branch points, or nodes, in response to voluntary exercise. The addition of a social component to the housing conditions had no significant effects on any of our measures. As seen in other studies, cell counts doubled after the addition of an exercise wheel (van Praag et al., 1999a; Eadie et al., 2005). The fact these positive effects of exercise occurred in isolation goes against current research (Stranahan et al., 2006), yet these results were not entirely unexpected. Previously we studied the effects of prenatal ethanol exposure and voluntary exercise on cell survival and proliferation in the hippocampal dentate gyrus (Redila et al., 2006). These rats had been bred in the laboratory, and were therefore not only well acclimatized to laboratory conditions, but had only ever known social housing conditions their entire life. They were then isolated for two weeks before being assigned to exercise or sedentary conditions for one week. Cell proliferation was measured using BrdU as described. These animals, by definition, had been previously isolated for twice as long as what had previously been described as "prolonged isolation", and had been exercising for only 7 days, not 12 (Stranahan et al., 2006). Even when nearly halving the amount of exercise and doubling the amount of isolation, we were still able to see benefits with respect to cellular proliferation in isolated conditions. It was previously suggested that isolation delayed the benefits of exercise due to HP A axis overactivity caused by voluntary exercise, and buffered by social interaction (Stranahan et al., 2006). Voluntary exercise is known to increase corticosterone levels (Droste et al., 2003; Fediuc et al., 2006; Stranahan et al., 2006). Interestingly isolation and varying group sizes has been seen to not significantly effect corticosterone levels of male mice (Hunt and Hambly, 2006). This is consistent with our findings. Our measures of corticosterone were also taken at the time of perfusion, which was approximately 4 hours after the onset of the dark cycle. In mice that exercise, increases in corticosterone 30 are typically seen only the onset of the dark cycle, which is thought to be a result of the HP A axis adapting to an increase in metabolic demand when the mice typically begin exercising (Droste et al., 2003). Corticosterone levels varied between animals, yet we were unable to find significance between groups in our study. The fact that both running groups showed a trend of increased corticosterone could be due to the fact that they may have had an increased response to restraint, anesthesia, or perhaps a slow decline in the increased corticosterone at the onset of the light cycle. Further research would be needed to address this. As this study conflicts with current research suggesting that isolation delays the positive effects of exercise on hippocampal cell proliferation in rats, a difference in our experimental methods could be important (Stranahan et al., 2006). We have previously looked at these effects in rats, which were Stranahan's experimental subjects, and found no delay in the positive effects of exercise with isolation (Redila et al., 2006). The experimental subject is therefore an unlikely factor. In both of our studies, mice and rats also experienced similar to greater periods of isolation (Redila et al., 2006; Stranahan et al., 2006). Thus the duration of isolation would likely not be a factor. Interestingly the animals that exercised in Stranahan's study experienced decreases in proliferation that were further decreased with additional BrdU injections, specifically in the exercise group. This suggests that the method of injection may have caused a decrease in proliferation in animals that exercise. Stranahan mentioned that their animals were in a restraint for 1 to 1.5 minutes for the duration of the BrdU injection, not including the time taken to place animals into restraints. Our animals were only removed from their cages for 30 seconds for a BrdU injection. This includes the time taken to pick up, put down, partially restrain (using one hand), and inject the animals. Restraint, a well known stressor (Fontella et al., 2005; Van den Hove et al., 2005; Tahera et al., 2006; Veldhuis et al., 2006), was therefore not minimized in this experimental protocol. I hypothesize that that this difference in injection protocol may account for the difference in results. A few studies have looked at stress, the HPA system, and voluntary exercise. Voluntary exercise has been shown to affect corticosterone levels in response to stress, but the directionality of this depends on the type of stressor. Levels of corticosterone in voluntary exercise animals increased in response to physical stress, but decreased in 31 response to a mild psychological stressor (Droste et al., 2003). Specifically, introduction to an identical clean cage was shown to increase corticosterone, but not as much in the exercise group. This was classified as a mild psychological stressor, and it would be interesting to see if isolation has similar effects in these circumstances. Restraint or forced swim tasks, types of physical stressors, can cause a relative increase in corticosterone levels in animals that exercise in comparison to controls that are also restrained (Droste et al., 2003). Another report did not see this pattern of increased corticosterone in exercising animals, but found that voluntary exercise was associated with increased adrenal sensitivity to restraint stress, (Fediuc et al., 2006). One study showed an exaggerated increase in corticosterone 10 hours after restraint in exercising animals (Adlard and Cotman, 2004). Increased corticosterone is linked to decreases in hippocampal cell proliferation (Tanapat et al., 1998; Tanapat et al., 2001; Wong and Herbert, 2006). And social interaction is thought to buffer the negative effects of increased corticosterone in exercising animals (Stranahan et al., 2006). The effects of exercise on the HPA axis are somewhat paradoxical. Exercise increases corticosterone, yet exercise has been associated with many positive effects including increased neurogenesis (van Praag et al., 1999a; van Praag et al., 2005), improved spatial learning and memory (van Praag et al., 1999b), and in humans moderate exercise has been linked to improved well-being, and anxiolytic effects that protect against consequences of stress (Moses et al., 1989; Salmon, 2001). In animals voluntary exercise is also linked to decreased anxiety related behaviors on the open field maze and elevated plus maze (Binder et al., 2004; Fulk et al., 2004) In summary, exercise activates the HPA axis on some levels, but other data would suggest that exercise may have stress relieving properties. Stressful situations can result in the near immediate downregulation of cell proliferation in the hippocampus by an adrenal hormone dependent mechanism (Falconer and Galea, 2003; Namestkova et al., 2005; Chen et al., 2006). Animals must be handled in some form to measure proliferation with BrdU, and a single BrdU injection is an acceptable measure of proliferation (Gould et al., 1998; Eadie et al., 2005; Liu and Martin, 2006). If animals exposed to voluntary exercise are experiencing an exaggerated corticosterone response to restraint stress, and the stress response can result in an 32 immediate decrease in cell proliferation, then in theory restraining animals to measure proliferation with BrdU may cause a more pronounced downregulation in proliferation specifically in the exercise group. Every precaution should be taken to minimize this. As studies in our laboratory make an effort to minimize the confounding variable of restraint stress when proliferation is being measured with BrdU, this idea explains the conflict between my results and current research. Thus, I propose that social isolation does not delay the benefits of voluntary exercise on cell proliferation, and that social conditions actually do not interfere with the effects of voluntary exercise to increase cell proliferation. Prolonged social isolation did not counteract the effects of voluntary exercise. I predict that a physical stressor, however, would counteract the positive effects of voluntary exercise on proliferation, which is an interesting area of future investigation. I would also predict that this type of stress may be buffered somewhat by social housing conditions, as evidence of this has been seen before (Stranahan et al., 2006). Our results showed a nonsignificant trend that corticosterone levels were increased in socially housed male mice. We did have to remove one group of animals from our study, as one of the animals displayed aggressive behavior. Although it is not uncommon to group housed male mice for studies such as this, the group housed males were monitored carefully for signs of aggression (Briones et al., 2005; Komitova et al., 2005; Briones, 2006). Male mice, like their female counterparts, and rats, commonly display aggressive behavior for various reasons (Giammanco et al., 2005). Male mice however have been shown to be found to display the least amounts of aggression in groups of 3-5 animals, as was used in our study (Van Loo et al., 2001). Not only do male mice prefer to be socially housed when given preference tests, but interestingly this preference is seen regardless of their social status, and is stronger than a preference for more standard environmental enrichment (Van Loo et al., 2003; Van Loo et al., 2004). In a small scale sense this is perhaps a more enriched condition. Male C57BL/6J mice not only prefer social housing conditions but show a preference for novel male mice (Moy et al., 2004). In other studies of environmental environment however the actual amounts of additional animals and novel objects added to the cage are typically much greater (Paylor et al., 1992; Rampon et al., 2000). One would imagine that if 33 environmental enrichment increases neurogenesis (Lazic et al., 2006; Olson et al., 2006), and that social housing is often a part of enrichment (Rampon et al., 2000; Lazic et al., 2006; Meshi et al., 2006), that we might have seen an increase in neurogenesis due to social housing for 11 days as outlined in our experiement. This was not the case, perhaps due to the fact that the animals only had social contact for a short period of time, and no other components involved in environmental enrichment, such as extra objects, were added. In addition, factors that have been linked to neurogenesis often show correlations on Morris Water maze performance (van Praag et al., 1999b; Cao et al., 2004; Snyder et al., 2005; Olson et al., 2006). Interestingly, male mice that have been socially isolated for long periods of time showed no deficits in water maze learning when compared to aged matched socially housed animals (Voikar et al., 2005). This is consistent with our results showing that different social conditions did not change the production of new neurons. What was particularly striking about the effects of short term voluntary exercise was the degree to which it changed the number and morphology of immature neurons. After only 11 days with access to an exercise wheel, the number of new neurons doubled. It should be noted that D C X positive cells overlapped greatly in animals in the voluntary exercise conditions. In the methods it was noted that cells were counted individually, and that only when there was a distinguishable membrane were cells counted. As cells were more obscured in the voluntary exercise groups, membranes were much more difficult to distinguish, providing a conservative measurement for cell counts in these animals. After division, as seen in figure 1.2, a primary dendrite extends from neurons after a week to a week and a half. These animals had only been running for 11 days, and one might assume that this would create many more immature neurons with fewer nodes. Interestingly we found the opposite, and I would imagine that this is due to an effect that allows voluntary exercise to upregulate factors that promote dendritic outgrowth during neuronal development in addition to increasing the number of new neurons produced. As voluntary exercise has been shown to have an effect on the morphology of neurons with a Golgi-cox analysis, this result agrees with previous findings (Redila and Christie, 2006). 34 Results of the pilot study on aged mice were not significant, although the average number of cells per dentate gyrus was doubled. A lack of significance is likely due to the increased variation in the ages and weights of these animals, as mentioned in the methods. Mice over 1 year are difficult to acquire, thus we were forced to work with a small and population size that had greater than ideal variations in age and weight. Proliferation, DCX, and other measures were not examined in these animals due to this lack of significance. Other groups were able to show that voluntary exercise is beneficial in aged, isolated, mice shortly after this pilot study, however (van Praag et al., 2005). Using a student's T-Test, the p-value was 0.06. These results are marginally significant, indicating that animals isolated for 6 months prior to voluntary exercise conditions might benefit from the effects of exercise. These animals were injected with BrdU two weeks after the assignment to experimental conditions, and survival was assessed at a 4 week time point. Prolonged periods of isolation have been seen to significantly downregulate BDNF, a compound that is important to cell survival (Scaccianoce et al., 2006). Even though this may be the case, voluntary exercise was still seen to have a benefit that could be considered marginally significant. We have found in a separate unpublished study that BDNF has no impact on cell proliferation, if this is a link between the benefits of exercise in isolation. Prolonged isolation was defined by Stranahan as isolation during exercise, in addition to a week of isolated conditions prior to that. These animals had been isolated for 6 months prior to exercise. This further shows that the benefits of exercise can be seen even in periods of more prolonged isolation. 2.4.2. Possible limitations These findings show that animals housed in isolation can benefit from the effects of short term proliferation, however there are more controls that need to be done to fully examine the benefits of social companionship. In our laboratory, we commonly house animals individually, which is common for studies of neurogenesis in mice (Holmes et al., 2004; Bjornebekk et al., 2005; Eadie et al., 2005; van Praag et al., 2005). As this was standard, all the animals in our laboratory were housed in isolation and all animals experienced a period of isolation. A social component of this study was only added at the time exercise wheels were added. Perhaps the period of isolation prior to exercise 35 affected the animals. These mice were then re-housed with their littermates. Male C57BL/6J mice show a preference for social novelty, spending more time in social conditions involving new male mice (Moy et al., 2004). In addition the corticosterone measures in these mice were taken under anesthesia, a process which may cause stress. In addition, the levels of corticosterone were examined at a single timepoint, shortly after the onset of the dark cycle, where exercising animals have been shown to have increases in corticosterone. Recent studies have looked at corticosterone in group and individually housed male mice, and have suggested that an analysis of fecal corticosterone levels are the most ideal measurement, as the animals are not subjected to undue stress (Hunt and Hambly, 2006) 2.4.3. Future directions In this work, I have presented a hypothesis on why our results conflict with current research was presented. It was proposed that restraint at the time of the BrdU injection may have resulted in the potential masking of the positive effects of exercise, due to increases in corticosterone. One future direction would be to examine the effects of restraint at the time of a BrdU injection in animals that exercise and their sedentary controls. This could be compared to an injection protocol that minimized restraint, and a group of animals that received no injection, as endogenous markers of proliferation can be examined, such as Ki67, rather than utilizing BrdU (Del Bigio, 1999). In addition to this, one could futher quantify the effects of various social situations on the effects of exercise. The animals we have looked at all experienced some form of previous isolation prior to only 11 days of experimental conditions. It is possible that this period of isolation affected the animals, and that animals housed in social conditions prior to exercise may have further increases in neurogenesis. 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