A PROFILE OF NEUROGENIC ACTIVITY IN THE AGING HIPPOCAMPAL FORMATION: A CLOSER LOOK AT THE ROLE OF 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

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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 , 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.

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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.

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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 …………………………………….. 21

Conclusion………………………………………………………………. 23

FIGURE CAPTIONS…………………………………………………………… 24

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REFERENCES…………………………………………………………………. 35

APPENDICES

APPENDIX A. THE …………………………………. 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 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 for increased

voluntary wheel running.

Aging is one of the most well known, natural regulators of hippocampal

neurogenesis. By comparing 6- and 18- month old C57Bl6 mice it was found that aged

mice had less cell proliferation than younger mice, based on the number of BrdU positive cells at the 24 hours after last injection. Similarly, it was found that survival was decreased in old age since the young animals were capable of retaining more of the newly generated neurons compared to the older animals (Kempermann, Kuhn, Gage, 1998).

Thus, the reduced neurogenesis is due to less proliferation and reduced survival of newly generated neurons. Furthermore, this finding is not specific to mice; it has also been shown in rats (Kuhn, Dickinson-Anson, & Gage, 1996; Rao, Hattiangady, & Shetty,

2006). In the study by Rao et al. (2006), five age groups of Fisher 344 rats were used to Adult Neurogenesis 4

examine the decline in neurogenesis across the lifespan (approximately 24 months). The study found that the age related decline in this strain of rats starts at 7.5 months of age persisting into adulthood and the decline in neurogenesis is due to the lack of

proliferation of the neural precursor cells and not due to survival.

With the understanding that exercise promotes neurogenesis in young animals,

some researchers have investigated the ability of exercise to reverse the age-related

decline seen in neurogenesis. van Praag et al. (2005) used C57Bl6 mice and showed that six weeks of exercise in old mice (18-months) was sufficient to restore neurogenesis and cognitive functioning back to the level of a young mouse (3-months; van Praag, Shubert,

Zhao & Gage, 2005). That is, the exercised, old mouse was capable of restoring its brain to the same level of an unexercised young mouse, a still very intact state. When examined for cognitive functioning, aged runners outperformed the aged sedentary mice in the spatial memory task and also had a higher number of BrdU positive cells. This supports the role of exercise and its benefits for cognition and during aging, and

moreover suggests that exercise may be able to reverse or delay the decline in aging or

preserve the brain in a young state of higher functioning. Human data have also shown

that exercise promotes cognitive health in old age and may also prevent

neurodegenerative disease (for review, see Kramer, Colcombe, McAuley, Scalf, &

Erickson, 2005).

In the present study, hippocampal neurogenesis was investigated across the

lifespan of the senescence accelerated prone mouse (SAMP-8). Because these mice are a

good model of aging and have a well documented age-related impairment of learning and

memory (Takeda, 1999), the present study was performed to determine the time course of Adult Neurogenesis 5

the age-related decline of hippocampal neurogenesis. Additionally, the effectiveness of exercise and environmental enrichment in ameliorating the age-related decline of neurogenesis in SAMP8 was independently investigated.

Adult Neurogenesis 6

MATERIALS AND METHODS

Animals

In total, 91 male SAMP-8 mice were used for this study. All mice were bred in our

colony at Bowling Green State University and were housed and treated in accordance with the guidelines and principles set forth by the BGSU Institutional Animal Care and

Use Committee. A total of four age groups are represented: 2 month (young), 5 month

(early middle aged), 7-8 months (late middle aged) and 12 month (aged). All mice were

kept on a twelve hour light/dark cycle (lights on at 7:00 a.m.) and given food and water ad libitum.

Isolated animals (n=31) were single housed in a standard laboratory cage (17”L x

9”W x 6.5”D) with food, water, and bedding. Exercise animals (n=31) were single

housed with unlimited access to a running wheel in addition to food, water, and bedding

in the standard laboratory cage. Enriched animals (n=29) were housed in large Nalgene

bins (37.5”L x 19.75”W x 21”D or 24”L x 17.75”W x 19”D) with food, water and

bedding, along with a variety of toys and tubing. Enriched animals were housed with

litter mates to avoid excessive fighting and to promote social enrichment. Enriched

animals also had toys that were replaced with new toys or moved to new positions daily

to enhance cognitive stimulation.

Injections and Duration

All animals were given a one week acclimation period in their respective condition

(described above) before they were administered the thymidine analog 5-bromo-2’- deoxyuridine (BrdU) during weeks two and three (Figure 1). All animals were injected with 150mg/kg (15mg/mL) of BrdU. Injections were delivered twice daily (between 8:30 Adult Neurogenesis 7

a.m. and 9:30 a.m. for the first injection and the second injection two hours later), 3 times

a week with at least one day between injections. Thus, there were a total of six days and

twelve injections for each animal over a period of two weeks. Animals were sacrificed 24

hrs or two weeks after the last injection to assess cell proliferation (21 days in condition)

or cell survival (35 days in condition), respectively (Figure 1). All animals were given an

anesthetic dose of 25% urethane at 0.4mL, and perfused transcardially with saline and

then 4% paraformaldehyde. The were removed and postfixed overnight in 4% paraformaldehyde, which was replaced with 30% sucrose the following day. Following equilibration in the 30% solution, brains were mounted on a sliding microtome and cut at

50µm. The entire hippocampus was sectioned and every slice was saved for tissue analysis.

Immunohistochemistry

BrdU

BrdU labeled cells are indicative of nuclei that were in S-phase during injection of

BrdU. These darkly stained nuclei represent the post-mitotic progeny of neural precursor

cells.

One-sixth of the obtained slices were used for BrdU immunohistochemistry.

Fixed tissue was pretreated for immunocytochemistry by three five minute washes in

phosphate buffered saline (0.1M PBS), then a two hour incubation in 2N HCl at 37°C for

30 minutes to denature DNA, and incubated in 0.1M borate buffer (pH 8.5) for 10 minutes at room temperature to neutralize the acid. This was then followed by three five minute washes in PBS, a 15 minute incubation in 1% H2O2 (to reduce background

staining), and three five minute washes in 0.1M PBS. Adult Neurogenesis 8

Antibody to BrdU (mouse anti-BrdU IgG, Roche) was applied as a primary

antibody in a 1:200 dilution (in 0.5% Triton, 0.01% NaN3, 0.1M PBS) and incubated

overnight at 4ºC on a shaker. Free floating sections were then incubated for four hours at

room temperature in secondary antibody (biotinylated donkey anti-mouse IgG, Jackson

Immunoresearch) at a 1:200 dilution (in 0.5% Triton, 0.01% NaN3, 0.1M PBS). Tissue was washed with three five minute washes in 0.1M PBS, incubated overnight in Avidin

& Biotin solution (ABC Standard kit; Vector Laboratories) at 4ºC on a shaker, and then reacted with 3’3’ diaminobenzidine (DAB, 0.05% solution) reacted with glucose oxidase

(10KU, MPBio) followed by a 6x5 wash in 0.1M PBS. Tissue was then mounted onto slides, dehydrated, cleared, and coverslipped with Permount (Fisher Scientific).

Ki-67

Ki67 is a protein expressed in all stages of the cell cycle except for G0 and G1

(Kempermann, Adult Neurogenesis p.201). Using the Ki-67 antibody as a marker allows

for a true representation of proliferation as it labels all cells that were undergoing division

at the time of sacrifice and does not serve as a birth-dating process like BrdU. All tissue

was pretreated with 1% H2O2 for 15 minutes, washed in 0.1M PBS (3x5) and primary

antibody was applied, rabbit anti-Ki-67 IgG used at a 1:500 dilution (Novocastro; in

0.5% Triton, 0.01% NaN3, 0.1M PBS). The tissue was incubated in the primary antibody

at 4ºC on a shaker for 72 hours. After three five minute washes in PBS, biotinylated

donkey anti-rabbit IgG for a secondary was then applied at a 1:200 dilution (Jackson, in

0.5% Triton, 0.01% NaN3, 0.1M PBS) and the tissue incubated for a minimum of four

hours at room temperature. The tissue was then placed into Avidin & Biotin solution

overnight at 4ºC on a shaker. After three five minute washes in 0.1 M PBS, the tissue is Adult Neurogenesis 9

reacted with diaminobenzidine (DAB) reacted with glucose oxidase which is then

followed by a 6x5 wash in PBS. Tissue was then mounted onto slides, dehydtaed,

cleared, and coverslipped with Permount.

Stereological Quantification

Cell quantification was performed as described by Kempermann (original paper

1997). This method excludes the counting of cells that are in focus at the top of the plane.

The dentate gyrus was mapped under a 4x objective and was counted using a 40x

objective (0.75 numerical aperture). Only the right hemisphere was counted. For each

stain, a total of 8-12 brain sections were obtained and each section was counted in

attempt to account for the entire rostrocaudal extent of the hippocampus. Because one in

every sixth section was stained, the number of cells obtained for each stain was

multiplied by six to account for every section in the rostrocaudal extent of the

hippocampus.

Statistical Analyses

A 4 (Age) X 3 (Condition) X 2 (Duration) between subjects ANOVA was performed to

determine if age, exercise or enrichment, or amount of time post injection influenced the

number of BrdU positive cells. Significant main effects and interactions were furthered

described with post-hoc comparisons as needed using Tukey’s HSD. Similarly, a 4 (Age)

X 3 (Condition) between subjects ANOVA was performed to examine the influence of

age, exercise or enrichment on ki67 positive cells. Tukey’s HSD was used for post hoc

analyses for any significant main effects and interactions. The criterion of p < .05 was

used to determine significance. Adult Neurogenesis 10

RESULTS

BrdU Positive Cells

Overall analysis.

In general, SAMP-8 mice show a decrease in BrdU positive cells with age, F(2,61) =

27.036, p = .000. Exercise and enrichment also had an effect on BrdU positive cells, as

seen by an effect of condition, F(2,61) = 12.706, p = .000. Also, there was a significant

difference between the number of BrdU positive cells in the proliferation and survival

conditions, F(1,61) = 27.271, p = .000. Furthermore, the age-related decline in

neurogenesis was shown to be modulated by exercise and/or enrichment, F(4,61) = 5.811,

p = .000. Additionally, age differentially affected proliferating and surviving cells, as

suggested by the presence of an age by duration interaction, F(2,61) = 7.182, p = .001.

Exercise and/or enrichment did not differentially promote proliferation or survival as the

condition by duration interaction was not significant, F(2,61) = 2.192, p = .120.

Furthermore, the three way interaction (Age X Condition X Duration) was not

significant, F(4,70) = 1.657, p = .172.

Effects of age on neurogenesis.

Animals housed in standard laboratory conditions (isolated) showed a sharp decline in cell proliferation (BrdU positive cells, 24-hours following the last BrdU injection) as a part of the aging process, F(3,14) = 15.698, p = .000 (figure 2). Post hoc analysis showed that 2-month old animals showed the most robust level of BrdU positive cells. The number of proliferating cells started to decrease substantially by five months of age and remains constant throughout the remainder of the lifespan as 5-, 7-, and 12-month old animals are statistically similar (figure 3). Adult Neurogenesis 11

In contrast to cell proliferation, survival of the newly generated cells was not

altered by age. Two-weeks after the last BrdU injection, the number of BrdU positive

cells in each age group was not statistically different, F(2,10) = 1.565, p = .256 (figure 4).

Effects of exercise on neurogenesis.

In exercise animals, cell proliferation decreased with age, F(3,10) = 9.031, p = .003.

Similar to the isolated condition, 2-month old exercise animals had significantly more

BrdU positive cells (p = .01). A comparison of the isolated and exercise animals across all ages revealed that neurogenesis decreased with age, F(3,28) = 25.528, p = .000. The

influence of exercise in increasing cell proliferation just missed significance, F(1,28) =

4.094, p = .053 (figure 3), but age and condition did not interact, F(3,28) = 1.715, p =

.187 suggesting that exercise had no effect on neurogenesis for all age groups.

With respect to the two-week time period, age influenced the number of BrdU

positive cells, F(1,15) = 4.843, p = .034. However, there was no effect of exercise on

survival when compared to isolated animals, F(2,26) = 1.461, p = .256 (figure 4).

Effects of environmental enrichment on neurogenesis.

Interestingly, enriched animals showed no effect of age at the 24-hour time point,

F(3,11) = 1.697, p = .225, but a trend for a negative aging effect was present at the

survival time point, F(2,8) = 4.239, p = .056.

The two-way ANOVA comparison of isolated animals and enriched animals for

the cell proliferation revealed that living in an enriched environment significantly and

negatively impacted neurogenesis, F(1,25) = 8.158, p = .009; Tukey’s HSD revealed this

effect to be limited to the 2-month old group (p = .001) (figure 3). Adult Neurogenesis 12

In enriched animals alone, age influenced number of BrdU positive cells at two weeks post injection, F(2,10) = 4.627, p = .038. However, when the BrdU positive cells are analyzed in a two-way analysis comparing isolated animals and enriched animals at the survival time point, there was a trend for decreased BrdU positive cells with increased age, F(2,20) = 3.312, p = .057 and no effect of enrichment, F(1,20) = 3.466, p = .077

(figure 4).

Ki67 Positive Cells

Overall Analysis.

A two-way ANOVA (Age X Condition) revealed that neither exercise nor enrichment was capable of reversing an age-related decline in Ki67 positive cells. Overall, there was a decrease in Ki67 positive cells between 2-, 5-, 7-, and 12-month old SAMP-8 mice,

F(2,17) = 6.043, p = .010 (figure 5). Post hoc analyses revealed a significant difference between 2-month and 12-month old groups, F(3,16) = 3.769, p = .03, but no differences between any of the other ages. Exercise and enrichment did not significantly impact the number of Ki67 positive cells, F(2,17) = .758, p = .484. Furthermore, there was no age by condition interaction, suggesting that even though there was no effect of exercise or enrichment, all ages were impacted were impacted equivalently, F(4,17) = 1.366, p =

.287 (figure 6).

Adult Neurogenesis 13

DISCUSSION

Initial studies on neurogenesis have shown that exposure to an enriched

environment leads to an increase in hippocampal neurogenesis and furthermore, that

these environments promote cell survival (Kempermann et al., 1997, van Praag et al.,

1999). Complementary studies have shown that voluntary exercise alone has robust

effects on cell proliferation (van Praag et al., 1999; van Praag et al, 2005). Considering

that many enriched environments contain access to wheel running, we decided to separate

the two conditions (enrichment with no wheel and voluntary exercise) and compare the

results to isolated, sedentary animals. In all three conditions, we quantified the number of

BrdU positive cells at 24-hours and 2-weeks post injection, and Ki67 positive cells.

We first established that neurogenesis decreases with age in all conditions tested.

Isolated, exercised and enriched animals all showed a significant decrease in BrdU

positive cells with increasing age. For the isolated animals the age-related loss in

neurogenesis starts before five months of age. Furthermore, age did not significantly

influence cell survival in the isolated animals. Isolated and enriched animals also

independently showed a significant age-related decline in Ki67 positive cells. These

findings are consistent with previous reports on neurogenesis during senescence which

have shown that neurogenesis decreases with age (Kempermann, Kuhn, Gage, 1998) and may start to decline at early middle age (Rao, Hattiangady, & Shetty, 2006).

The SAMP-8 as a mouse model of aging

In attempt to reverse this age-related decline in neurogenesis we used two

standard behavioral paradigms (voluntary exercise and environmental enrichment) both

known to elicit neurogenesis. With the interest in aging, we used a specific mouse model Adult Neurogenesis 14

of aging, the senescence accelerated prone mouse (SAMP-8). All of the senescence

accelerated mice come from the background strain AKR/J and developed their respective sub-line anomalies upon selective breeding after noticing that certain mice died earlier than others and expressed certain age related pathologies but did not express any kind of growth retardation or overt neurological dysfunction (Takeda, 1999). The SAM is a family of approximately nine sub-lines showing a different age-related pathology (Prone;

SAMP) and four sub-lines serving as a control strain (Resistant; SAMR). The SAMP strain, when compared to the SAMR strain, shows an accelerated aging rate of about 40% with a SAMP living approximately 9.7months and the SAMR living to be approximately

16.3months (Takeda, 1999). However there is some variation within each colony; our

colony of SAMP8 has a lifespan of approximately 13-15 months in the male animal.

Within the SAMP sub-lines, the SAMP8 and SAMP-10 strains are known for aging

impairments in learning and memory. The SAMP-8 shows learning and memory

impairments (Chen, Wang, Wang, and Zhou, 2004) and abnormal circadian rhythms

(Takeda, 1999) and aging impairments that are specific to the hippocampus compared to the SAMP-10 (Miyamoto, 1997). The attractiveness of the SAMP8 animal model is that they express a variety of neurobiological changes associated with age-related pathology without an overexpression of tau or beta amyloid, as in the transgenic models. Thus, this mouse model is a more natural model of aging with respect to changes in neurochemistry,

neurotransmission, and inflammation (Butterfield and Poon, 2005).

Our data suggested three weeks of a voluntary exercise routine was insufficient to

reverse the age-related decline seen in cell proliferation in the SAMP-8; and furthermore,

five weeks was insufficient to promote cellular survival. This is inconsistent with other Adult Neurogenesis 15 research that has demonstrated that exercise promotes cell proliferation and survival (van

Praag, Kempermann, & Gage, 1999).

In addition to examining voluntary exercise, we also looked at environmental enrichment. We found that an enriched environment had a negative effect on cell proliferation and no effect on cell survival. These findings are not consistent with previous reports that have shown that an enriched environment is important for cell survival (Kempermann, Kuhn, & Gage, 1997; van Praag, Kempermann, & Gage, 1999).

Neurogenesis and Aging

Our studies found that although aging reduces the number of neurons proliferating, survival of newly proliferating cells was not altered by aging in SAMP8 mice. This finding is inconsistent with previous reports on neurogenesis during senescence which has shown that neurogenesis decreases in 18 month versus 6 month old

C57Bl6 mice (Kempermann, Kuhn, & Gage, 1998). To further describe this aging effect, we found a profound decline in proliferation prior to five months of age, what we are calling “early middle age” for the SAMP-8 mice which have a lifespan of approximately

14 months in our colony. In a similar study by Rao et al. (2006), they reached a similar conclusion in F344 rats after examining five age groups over a 24 month lifespan - they showed a decline in neurogenesis starting at 7.5 months of age (Rao, Hattiangady, &

Shetty, 2006). This parallels our finding of a decrease in neurogenesis at early middle age for the SAMP-8 mice, seeing as 7.5 months is before the 12 month middle age time point for the F344 rats. In this study, we were interested in whether there was a gradual decline with age or if the age-related decline would be abrupt. We found that in the SAMP-8, the Adult Neurogenesis 16 age-related decline in neurogenesis is abrupt, coming to almost complete halt at five months of age.

If it was found that the observed loss in neurogenesis at 5-months of age would precede the onset of most cognitive disruptions seen in middle age for this mouse model, then this would support the role of neurogenesis in memory. However, using the same four age groups of male SAMP-8 mice in the Morris Water Maze, we found that only 2- month old animals were significantly different from the 12-month old animals in their ability to learn the task (our unpublished data). Additionally, we know that two middle age groups (5- and 7-month old animals) performed statistically similar. Finally, with respect to retention on a short term and long term probe trial – there was no preference for target quadrant among any age group, and within that target quadrant, there was no difference between ages. So with these unpublished observations in mind, the loss of neurogenesis at 5-months of age did not predict a decline in cognitive performance at 7- months of age, but it did at 12-months of age.

Effects of exercise on neurogenesis

In the present study, exercise was shown to have no effect on neurogenesis. This result is in contrast to other work that has shown voluntary exercise to be a significant positive modulator of voluntary exercise. With respect to other studies, it is worth noting that our finding is not consistent with Kempermann et al.’s 1999 study showing an increase in BrdU-positive cells 24 hours after last injection. Other studies have also shown that exercise is capable of reversing the effects of age in other mouse strains; van

Praag et al. (2005) reported this effect in C57Bl6 mice. It is important to consider that in our study the trend for an increase in BrdU positive cells for proliferation is similar to Adult Neurogenesis 17 these other reports. It is possible that this lack of significance could be due to a difference in strain or high within age-group differences. We have no reason to believe that this is due to the activity level of the animals, as our previous work with voluntary exercise in these animals has shown that between 2-, 7-, and 12-month old animals each group runs significantly less with age (McAuley, Miller, Beck, Nagy, & Pang, 2002). If our BrdU positive cell numbers were directly dependent on activity level, one would not have expected to see 5-, 7-, and 12-month old animals with statistically similar BrdU positive cells numbers. Furthermore, if our findings were due to age-related differences in activity, then we would have expected our 2-month old animals to have significantly more BrdU positive cells than isolated animals and all other age groups showing no differences between conditions. These expectations were not observed.

Effects of environmental enrichment on neurogenesis

Other studies have demonstrated enriched environments to be a positive modulator of hippocampal neurogenesis. The first studies conducted by Kempermann et al. suggested that enrichment serves an important role in the survival of newly generated neurons (1997, 1999); and these findings were again later supported in a study conducted by Rossi et al. (2006), showing a two-fold increase in BrdU positive cells after eight weeks in enriched conditions. It should be considered that all of these studies had a running wheel present.

To our knowledge, we are the first to show that for the SAMP-8s, an enriched environment is a significant negative regulator of neurogenesis. Given that enrichment is supposed to influence cell survival, we expected a slight upregulation or no significant increase at the 24 hour time point and a significant difference at the two-week time point. Adult Neurogenesis 18

The null effects were supported; there was no upregulation and no difference at 24 hours

and also no difference at the survival time point either.

Our observed difference compared to previous publications may be due to the

separation of the experimental conditions, as we did not include a running wheel. It is

also plausible that the downregulatory effect of the enriched environment is due to

differences in strain. In support of strain differences, Levi and Michaelson (2007) examined an enriched environment on transgenic ApoE4 mice. Three week old male

ApoE4 mice were group housed for 24 weeks in standard or enriched conditions (that contained a running wheel). The authors performed immunohistochemical analyses for

NeuN, Ki-67 and Caspase-3 and found that the environment downregulated neurogenesis and increased apoptosis in the ApoE4 mice but not the ApoE3 or wild type mice. These findings are similar to ours in that we found a decrease in neurogenesis in a specific

mouse model, however our mouse model is not a transgenic line, but it is a highly inbred

mouse strain selected for its rapid aging and learning and memory impairments. Just as

the transgenic ApoE4 mouse strain reported a downregulation as a result of enrichment,

our selectivity for rapid aging could negatively influence our ability to produce new

neurons or exacerbate the apoptotic process – if that is what is really happening in this

condition (as suggested by the authors).

Stress as a possible explanation for all observations

One hypothesis that might explain why exercise had a trend for increased

proliferation and an enriched environment significantly decreases cell proliferation with

both cases not influencing cell survival compared to sedentary animals is the effect of

stress on hippocampal neurogenesis. In the isolated animal, there is a baseline level of Adult Neurogenesis 19

glucocorticoids that changes throughout the lifespan, with corticosterone increasing as

the animal ages. It has been shown that lowering corticosterone levels during middle age prevents the age-related loss in neurogenesis (Montaron et al., 2006) and that removal of the adrenal gland increased cell proliferation in both young and aged animals (Cameron

& Gould, 1994).

Animals that were in the exercise condition showed a tendency for increased cell

proliferation, but this was not significant. In a study investigating the interaction between

exercise and glucocorticoids in short term (9 days) versus long term (24days) running, it

was suggested that an extended period of exercise leads to a decrease in neurogenesis and

that this was due to elevated corticosterone as seen in long term runners (Naylor, Persson,

Eriksson, Jonsdottir, & Thorlin, 2005). Because our animals were in condition for 21

days for the proliferation condition and exercise did not reverse the age-related decline in

BrdU positive cells at this point, it may be due to the fact that our animals were in

condition for too long; nine days, as suggested by Naylor et al., may have obtained an

effect of exercise in aging SAMP-8 mice. However, we arbitrarily chose to go 21 days

versus fewer days to avoid learning and novelty effects, which may explain why Naylor

et al. found an effect of exercise on neurogenesis in short term exercise but not long term.

It should be noted that there is another side of the literature that suggests that

exercise is important for upregulating trophic factors (i.e. brain derived neurotrophic

factor; Berchtold, Chinn, Chou, Kesslak, & Cotman, 2005) and it is possible that these microenvironmental factors may be responsible for the effects of exercise on cell proliferation and/or survival. Keeping in mind that it is known that corticosterone can

downregulate neurogenesis (Montaron et al., 2006; Cameron & Gould, 1994) and that Adult Neurogenesis 20

BDNF upregulates neurogenesis (Scharfman et al., 2005), it is conceivable that BDNF

may promote this neurogenic activity by lowering levels of corticosterone. To support

this idea, it has been shown that BDNF prevents cell death caused by corticosterone

(Nitta et al., 1999). So in the exercise animals, it is possible that there was no negative

effect of exercise (as seen in Naylor et al., for example) because they were 1) not in

condition long enough to gain a significant negative effect exercise via corticosterone

and/or 2) BDNF levels were high enough to maintain corticosterone levels at a similar

(baseline) state as the isolated animals. Thus, in the exercise condition, there was an increase in BDNF levels that prevented downregulation via corticosterone and furthermore, inhibited the stress response.

For the enriched condition, there were two factors that worked together to

downregulate neurogenesis or one factor acted predominantly over the other; these two

factors being physical activity and dominance hierarchies. Social stress or lack of

physical activity compared to the exercise condition lead to decreases (or lack of an

increase) in BDNF that prevented the downregulation of glucocorticoids. Given that this

condition had less NG than controls and exercise animals independently, it is likely that

the social component of the condition lead to stress and a decrease in neurogenesis, as the control animal was single-housed.

BrdU versus Ki67 positive cells

It is important to consider the differences between BrdU positive cells and Ki67

positive cells. In this study, three cell fates are to be considered: two weeks and 24-hours

after last injection (BrdU survival and proliferation respectively) and lastly ki67 positive

cells (a measure of true proliferation at time of sacrifice). Because both BrdU measures Adult Neurogenesis 21 had some component of survival due to multiple injections over the course of twelve days, it is important to distinguish between BrdU proliferation and Ki67 proliferation.

Ki67 represents a snapshot of cells dividing only at the time of sacrifice; BrdU 24-hour represents an accumulation of cells dividing for a 2-hour period over each of twelve injections; BrdU two week represents cells that are existent two weeks after last injection.

For isolated animals, measures of proliferation (Ki67) and survival (BrdU 2 weeks) were statistically similar across the lifespan when analyzed independently. In a similar study analyzing proliferation across the lifespan, Ki67 positive cells showed a significant decline with increased age (Rao, Hattiangady, Shetty, 2006). Though we did not obtain significance in our isolated animals alone when assessing Ki67, there was a trend for an age-related difference (p=.06). However with respect to BrdU versus Ki67, the fact that both show no changes across ages independent of one another suggests that cells proliferate and survive at the same rate. It is important to note that even though older animals have less BrdU 24-hour positive cells than younger animals, they are retaining the same percentage as the young animals at the survival time point (figure 7).

Implications for adult neurogenesis

Finally, it is important to consider the plausible role that adult neurogenesis might play and why researchers are so interested in studying this phenomenon. Because of the precise location of neurogenesis (the hippocampus) and the known brain location affected by learning and memory and aging in general, it is conceivable that these new neurons may be important for learning and memory. That in mind, many researchers look for ways to potentiate neurogenesis throughout the lifespan or reintroduce it at middle-age so that the cognitive decline associated with aging may be prevented or reduced. Adult Neurogenesis 22

To this extent, much work has been done on the role of adult neurogenesis in

learning and memory (Shors, Townsend, Zhao, Kozorovitskiy, & Gould, 2002; Bizon,

Lee, & Gallagher, 2004; Snyder, Hong, McDonald, & Wojtowicz, 2005). Aged Long

Evans rats reportedly show an inverse relationship between BrdU+ cells and Morris

Water Maze performance, with a higher number of BrdU+ cells found in animals with

poorer spatial memory (Bizon, Lee, & Gallagher, 2004). Conversely, in aged Sprague

Dawley rats, it has been shown that both survival and proliferation via BrdU+ cells is

correlated with improved spatial memory (Drapeau, Mayo, Aurousseau, Le Moal, Piazza,

& Abrous, 2003). Given these opposite correlational relationships between BrdU positive

cells and spatial memory performance, studies were done using two methods that prevent proliferation of either neurons (methylazoxymethanol; MAM) or all cells entirely

(gamma irradiation) in attempt to elucidate the role of neurogenesis in memory. When

the anti-mitotic agent MAM is administered to Sprague Dawley rats, the subsequent

decrease in BrdU+ cells is not correlated with a decreased performance in the Morris

Water Maze (Shors et al., 2002) and short term memory (24 hours) is not impaired.

Interestingly, when young Long Evans rats are subject to gamma irradiation, long term

spatial memory (two weeks) is impaired, but the ability to learn the task or recall at short

term (1 week) was not impaired (Snyder et al, 2005). Unfortunately, Shors et al. (2002)

did not perform a test of long term memory, but if the authors had done the long term

memory assessment and found that there was an impairment in long term memory, it

would support the idea that neurogenesis is important for learning and long term memory.

Adult Neurogenesis 23

Conclusion

We found that the SAMP-8 model shows a sharp decline in neurogenesis across

the lifespan that starts before 5 months of age. Though two well known attempts to

reverse this age-related decline were made, neither exercise nor enrichment were

sufficient to do so; exercise was did not significantly upregulate and environmental

enrichment significantly downregulated neurogenesis. With respect to proliferation and

survival and putting new neurons into existing circuitry, neither experimental condition was capable of promoting the survival of new neurons. Furthermore, survival is the same across all age groups in the isolated animals as well. We suggest that these findings are due to a stress-related mechanism dependent upon the glucocorticoids or a difference in the selectivity of the strain.

Adult Neurogenesis 24

FIGURE CAPTIONS

Figure 1. Injection schedule for all animals. Animals were in condition for 21 days for proliferation studies and 35 days for survival studies.

Figure 2. Immunocytochemistry for BrdU. There is a significant age-related decline in

BrdU positive cells at 24-hours after last injection and this decrease occurs before five

months of age. From the age of five months and beyond, the number of BrdU positive

cells are statistically equivalent. Age was not shown to alter the likelihood of cell

survival, measured 2-weeks post injection.

Figure 3. Quantification of BrdU positive cells reveals that cell proliferation significantly

decreases before five months of age and remains statistically the same for 5-, 7-, and 12-

month old animals (blue). Furthermore, this age-related decline in neurogenesis was not

reversed by exposure to three weeks in a voluntary exercise routine (red). Animals

exposed to an enriched environment for three weeks were shown to have significantly less BrdU positive cells than isolated animals at 2-months of age (gold). Error bars represent standard errors of the mean (SEM).

Figure 4. In the isolated animals, age was not shown to influence cell survival. Similarly,

when compared to isolated animals, exercise nor enrichment were capable of promoting

cell survival. Error bars represent SEM.

Adult Neurogenesis 25

Figure 5. Immunocytochemistry for Ki67 (arrows point to darkly stained Ki67 positive

cells). Ki67 positive cells show a significant decline across the lifespan of the SAMP-8;

2-month old animals had significantly less Ki67 positive cells than 12-month old animals.

Figure 6. Stereological quantification of Ki67 positive cells. Ki67 positive cells show a

significant age-related decline with 2-month old animals being significantly different

from 12-month old animals. This age-related decline was not reversed by three weeks in

an enriched environment. Error bars represent SEM.

Figure 7. The ratio of BrdU Positive cells at 24-hours versus two weeks (BrdU proliferation versus survival). The ratio represents (mean survival/mean proliferation) x

100.

Figure 8. Drawing showing the one hemisphere of the hippocampus and the trisynaptic loop (Fuster, 1995, p.26).

Figure 9. Comparison of group housed and single housed enriched animals at the 24-hour time point. Animals were not statistically different at 5- and 7-months of age. Error bars represent SEM.

Adult Neurogenesis 26

FIGURES

Figure 1.

Adult Neurogenesis 27

Figure 2.

Adult Neurogenesis 28

Figure 3.

Adult Neurogenesis 29

Figure 4.

Adult Neurogenesis 30

Figure 5.

Adult Neurogenesis 31

Figure 6.

Adult Neurogenesis 32

Figure 7.

Adult Neurogenesis 33

Figure 8.

Adult Neurogenesis 34

Figure 9.

Adult Neurogenesis 35

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Adult Neurogenesis 43

APPENDIXES

APPENDIX A. THE HIPPOCAMPUS

Hippocampal anatomy

The hippocampus is a bilateral structure that spans the rostrocaudal extent of the in mammals and equivalent structures to the hippocampus do exist in non-

mammalian animals. The hippocampal formation consists of the dentate gyrus,

hippocampus proper, subiculum, presubiculum, parasubiculum and entorhinal cortex. The hippocampus proper can be subdivided into the CA1, CA2 and CA3 areas; these same

regions can be divided into regio inferior (now CA3 and CA2) and regio superior (now

called CA1) with the current terminology implemented by Lorente de No (Andersen,

Morris, Amaral, Bliss, & O’Keefe, 2007).

The hippocampus is known for its characteristic tri-synaptic loop (figure 8). In

this circuit, the input from the entorhinal cortex (EC) to the dentate gyrus (DG) forms the

first synapse; the dentate gyrus axons then synapse on to CA3 dendrites to form the

second synapse; axons from CA3 then synapse onto CA1 dendrites to form a circuit

where the CA1 axons transmit their signals to subiculum. Because new neurons are

generated in the dentate gyrus, it is possible that the addition of new neurons here allows

for more neurons to participate in this trisynaptic loop.

For the purposes of hippocampal neurogenesis, it is important to consider the area

that gives rise to new neurons. Located within the hippocampal formation is dentate

gyrus (DG). Within the DG is the subgranular zone (SGZ) of the granule cell layer (GCL)

where neural precursor cells are located. The GCL is the area that stains darkest in a nissl

stain where it also presents two blades: one that is located between the CA3 and CA1 Adult Neurogenesis 44

field called the subpyramidal blade and one opposite to this called the infrapyramidal

blade; the point where the two blades meet (the apex) is called the crest. The SGZ of the

DG is where neurogenesis occurs.

Normal aging versus pathological aging

Hippocampal atrophy as normal aging

With age, the hippocampal formation starts to atrophy and the ventricles start to

enlarge. Because the hippocampus is highly regarded for its role in memory, the

relationship between hippocampal atrophy as seen in the aging population and the

cognitive deficits also witnessed as one ages, it is no surprise that hippocampal atrophy is implicated in the learning and memory impairments seen in aging.

To explore such interests, researchers investigated possible causes for the loss of

hippocampal cell volume and how it relates to memory performance because it was

thought that loss of cells was responsible for cognitive decline. In the 1996 study by

Rapp and Gallagher it was found that there was no difference in total hippocampal cell

number or granule cell number between young and aged rats despite aged rats having

poorer performance on a spatial memory task (Rapp & Gallagher, 1996). Interestingly,

the number of cells did not differ as a function of age or cognitive ability. In a follow up

study to investigate volumetric changes, it was found that rodents show hippocampal

atrophy similar to humans, that is limited to the middle molecular layer (MML) of the

hippocampus (Rapp, Stack, Gallagher, 1999). So if there is no neuronal loss to explain

the volumetric changes, this leaves other possibilities available: loss of glial cells, change in dendritic structure, and/or loss in (functional) . Adult Neurogenesis 45

In order to assess synaptic changes in the hippocampal formation with aging,

Geinisman et al. examined axospinous synapses and spatial learning in young and aged

rats (Geinisman et al. 2004). The findings were similar to those found for absolute cell

number – the number of synapses in the CA1 subfield stratum radiatum did not change

with age and did not predict cognitive ability. The author suggests that the learning

impairments without changes in cell or synapse number might be due to a decrease in

functional synapses – i.e., the functional AMPA receptor is missing (but a functional

NMDA receptor present) and there is no response to glutamate for an LTP-like

mechanism. However, while this may explain why there is a cognitive decline associated

with aging as a result of hippocampal impairment, it does not explain the loss of

hippocampal volume; other explanations include glial cell loss or loss of dendritic

arborization.

Hippocampal atrophy as pathological aging

In the case of pathological aging, defined as mild cognitive impairment, , or most severely, Alzheimer’s disease, hippocampal atrophy predicts the degree of cognitive decline (Jack et al., 1998). Furthermore, there is a degree of cell loss associated with this volumetric loss, unlike that seen in normal aging. In pathological aging there is a higher degree of neurofibrillary tangles that appear as a result of cell death and an increase in beta amyloid plaques that arise as a sign of cell toxicity and severity of illness

(Andersen et al., p. 797); whereas in normal aging, these protein aggregates are present, they are not present to a pathological extent. Thus, in the diseased brain, it is important to consider that the patterns of cell loss and overall structural atrophy differ.

Conclusion: The hippocampus Adult Neurogenesis 46

Adult neurogenesis is a phenomenon restricted to two brain regions, one of which is the hippocampus. This same brain area is largely responsible for our everyday ability to recall information in the form of declarative memory. Though the source of the atrophy in the hippocampus is arguable and some factors have been ruled out (loss of neurons or loss of synapses), this degree of atrophy is still being used as a predictor for severity of illness in aging individuals. Because individuals who present with signs of cognitive decline associated with aging, mild-cognitive impairment or Alzheimer’s disease all suffer from varying degrees of hippocampal atrophy, the ability to stimulate or retain neuronal production may be of great value if these neurons do truly participate in learning and memory themselves whether or not they actually change the structural volume. Adult Neurogenesis 47

APPENDIX B. NEUROGENESIS

History of neurogenesis

Joseph Altman at MIT was the first to discover adult neurogenesis in the

hippocampal formation in the early 1960s (Altman, 1962). He found mostly glial cells

but a few cells that expressed the morphology of neurons that incorporated 3H thymidine,

suggesting the division of neurons. These neurons have now been identified as

hippocampal granule cells. Later, in their 1965 paper, Altman and Das (1965) were the

first to show what many are rediscovering 40 years later: neurogenesis shows an age dependent decline, and that these cells may be derived from a precursor cell population.

Unfortunately, due to the far reaching implications of their findings, much of the scientific community disregarded the ideas and blamed it on poor technical issues.

After Altman and Das, Fernando Nottebohm renewed faith in adult neurogenesis

in 1983 with his work in the sexually dimorphic high vocal center (HVC) of song birds.

Nottebohm noticed that the HVC was the largest during the spring season in males during

times of song performance (Nottebohm, 1981). To account for this change in volume,

Goldman and Nottebohm used the 3H thymidine to estimate the number of neurons

present and the amount of cellular proliferation (Goldman & Nottebohm, 1983).

Nottebohm then used electrophysiology, horseradish peroxidase staining, and electron

microscopy to verify that neurons were proliferating (Paton & Nottebohm, 1984). These

results supported previous studies conducted by Altman and Das, however the scientific

community was still not convinced of the idea that neurons could be generated

postnatally or if they could divide it was limited to non-mammalian animals. Adult Neurogenesis 48

In the early 1990s, adult neurogenesis started to ease its way into scientific

literature with a series of publications by Elizabeth Gould and colleagues that related

stress to the downregulation of neurogenesis via corticosterone. Gould was the first to

introduce immunohistochemical double labeling methods with neuron-specific enolase

(NSE) to confirm the presence of neurons while still using the tritiated thymidine

(Cameron, Woolley, McEwen, & Gould, 1993). Shortly after Gould’s breakthrough, the

thymidine analog 5-bromo-2’-deoxyuridine (BrdU) became accepted as a marker of

proliferative cells and replaced the more complicated 3H thymidine. This drug allowed

for more efficient labeling and immunohistochemical detection that is still widely used

today.

In 1998 one of the more interesting publications surfaced and truly sparked the

death of the dogma of ‘no new neurons’ – Peter Eriksson working with Fred Gage and

others demonstrated that adult neurogenesis occurs in humans. The subjects were cancer patients who were diagnosed with squamous cell carcinoma of the tongue, larynx, or pharynx and were given an intravenous infusion of BrdU to monitor tumor growth. Upon death, a total of five patients consented to donate their brains to the researchers (Eriksson et al., 1998). This study found that neurogenesis continues throughout the lifespan of the adult human, much like that in rodents and birds. Furthermore, with the publication of the results from human data and the accumulating evidence in animals, the scientific community began to accept the idea of new neurons being formed throughout the lifespan and started investigating the functional significance and environmental regulation of adult neurogenesis.

Adult Neurogenesis 49

Process of neurogenesis

Neurogenesis starts with the neural precursor cells, which are derived from stem

cells. More specifically, pluripotent stem cells give rise to multipotent progenitor cells

that then produce neural precursor cells (NPCs). According to Emsley, Mitchell,

Kempermann, and Macklis (2005), NPCs are undifferentiated, mobile, proliferatively active, and able to produce mature neurons and glia. In vivo, NPCs can be detected by the immunocytochemical marker Ki67, a non-specific marker of all proliferating cells. The post-mitotic daughter cells of the NPCs can then be labeled with the thymidine analog

BrdU, which labels all newly divided cells. Newly divided cells can then be visualized immunocytochemically with antibodies to BrdU, and double labeling with NeuN can be used to visualize newly formed mature neurons. It is worth noting that the time from NPC division to the cell maturation state takes approximately four weeks in most mammals

(Gould & Tanapat, 1997).

When considering the timeline of precursor cell to mature neuron, it is important

to distinguish between the processes of proliferation and survival. Proliferation represents

a quantity of cells that have recently undergone cell division and are still considered immature. These cells have not differentiated and have not integrated into cellular networks. The amount of cellular proliferation can be influenced by endogenous factors

such as age or extrinsic factors such as voluntary exercise. To alter the function of the

hippocampus, it is important to consider the percentage of these proliferated cells that die

or survive. Within approximately four weeks (Gould and Tanapat, 1997), neurons are

starting to form and gain the ability to participate in neural networks. These new neurons

serve the purpose of replacing nonfunctional neurons or simply serve an additive role by Adult Neurogenesis 50

enhancing the quality of a particular circuit. Therefore, one way to enhance the

population of newly formed neurons is to increase proliferation, as long as the percentage

of surviving cells remains the same. Alternatively, treatments that enhance survival of

cells, with proliferation being the same, would also result in addition to the neural circuits. Because proliferation and survival are likely to be different processes with different factors influencing each, it is important to understand the mechanisms of each.

For the proposed studies, animals will be sacrificed to assess either “proliferation”

or “survival.” Proliferation studies are those that will examine the cell quantity at a time

point too early for differentiation (24 hours after last injection). The number of proliferating cells will be assessed by counting the cells with immunoreactivity for either

BrdU or Ki67. BrdU immunopositive (BrdU+) cells will represent an accumulation of all cells that have passed through S-phase of the cell cycle within two hours of injection of the BrdU in vivo (Cameron & McKay, 2001). Thus, BrdU+ cells are non-specific for phenotype and include both the post mitotic cells such as neurons and glia, and the proliferative NPCs that are actively dividing. Similarly, Ki67 is a protein whose function

is still unknown but is present when cells are in all active phases of the cell cycle, which

includes the M-phase. So, antibodies to Ki67 are capable of labeling cells that are mitotic

(such as the NPCs).

“Survival” is characterized by the amount of proliferating cells that become

functional and is defined here by a mature cellular state in which the neuronal phenotype

is expressed (2 weeks after last injection). A typical combination of markers used to

assess this state is a BrdU/NeuN double label. Cells that show co-expression of these two

markers indicate newly generated cells (BrdU) that have become mature neurons (NeuN) Adult Neurogenesis 51 over time. Thus, it suggests that a certain percentage of the proliferating cells are actually surviving.

Factors regulating neurogenesis

Cell proliferation in the dentate gyrus can be regulated by both macro- and microenvironmental factors and given the tremendous amount of evidence for varying factors shown to regulate adult neurogenesis, only a few examples will be mentioned in this section. Macroenvironmental factors such as environmental enrichment and voluntary exercise have been shown to upregulate cell survival in addition to proliferation. Microenvironmental factors, when administered intracerbroventricularly or systemically can also regulate neurogenesis. Examples include: hormones, serotonin, and growth factors. It should be noted, however that the macroenvironmental factors are likely to work through microenvironmental mechanisms. Finally, there are certain factors at each level that are capable of downregulating and upregulating neurogenic activity at the level of the neural precursor cell, as this process is bidirectional; aging and stress are examples of processes that lead to downregulation of neurogenesis while trophic factors and antidepressants upregulate neurogenesis.

Macroenvironmental factors

Environmental enrichment.

Enrichment studies have long been studied for their ability to induce cellular reorganization and changes in brain structures. Evidence by Greenough et al. has shown that complex environments leads to increased dendritic branching and number of overall dendrites (Green, Greenough, & Schlumpf, 1983). Other studies have shown an increased hippocampal volume associated with enrichment (Rosenzweig, 1966). These studies Adult Neurogenesis 52

suggest that neural plasticity can be a result of cognitive activity and not just physical

activity alone or in conjunction with cognitive activity, because these environments

promote social, visual and tactile stimulation but are not physically involved.

Considering that cognitive stimulation was capable of promoting changes in

cortical structures even at the cellular level, the first studies investigating means of

eliciting neurogenesis consisted of environmental enrichment paradigms (which under

current standards consists of olfactory, visual, tactile and social stimulation). One of these first studies compared isolated and enriched young female C57Bl6 mice over the course of 40 days (Kempermann, Kuhn, Gage, 1997). The authors found that enriched animals had 310,000 granule cells compared to the 270,000 in control animals, and when comparing the effect of enrichment on BrdU positive cells alone – enrichment promotes cellular survival but not proliferation, as the effect was significant only at four weeks and not 24 hours post injection. This suggests that from the perspective of early studies on enrichment and neurogenesis, environmental enrichment was capable of promoting cellular survival.

In a follow up study to the 1997 paper, Kempermann et al. examined whether

enrichment was capable of reversing an age-related decline in neurogenesis

(Kempermann, Kuhn, Gage, 1998). In this study, 6-month (middle age) and 18-month old

(senescent) C57Bl6 mice were used and house with the same laboratory conditions as in

the earlier study and the experiment lasted for either 40 or 68 days. The authors found

that there was a significant decrease in BrdU positive cells between the age groups.

Furthermore, in agreement with the previous study, it was found that enrichment had no

effect on cell proliferation, but it did have an effect on survival – but only in the 6-month Adult Neurogenesis 53 and not the 18-month old animals. Finally, enrichment promoted an increase in NeuN positive cells compared to controls, suggesting that a greater percentage of newly born cells in the enriched animals are adopting a neuronal cell fate and surviving. This concludes that an enriched environment compared to standard laboratory conditions, is capable of reversing the age-related decline in neurogenesis.

Voluntary exercise.

Voluntary exercise has been widely studied in the neurogenesis literature for its ability to robustly increase cell proliferation in the hippocampus. This technique was first employed by van Praag et al. (1999), who found that mice exposed to voluntary wheel running produced significantly more BrdU positive cells than controls. This significant effect of exercise was present at both 24 hours and four weeks after last injection, suggesting that voluntary exercise plays an important role in cell proliferation and survival (van Praag, Kempermann & Gage, 1999). Though van Praag et al. (1999) showed that exercise was capable of exerting a neurogenic effect on three month old mice, a follow up study has shown that exercise is also sufficient to reverse the age- related decline in neurogenesis (van Praag, Shubert, Zhao, & Gage, 2005).

With the growing amount of literature on exercise and neuroplasticity, different research groups are taking different approaches to study the effects of exercise on neurogenesis in attempt to ascertain the causal link between exercise and neurogenesis.

Thus, in order to control for the variability in different strains and, within each, the different ages or likelihood of running, some studies have used an approach that allows the wheel to be locked after a certain distance is reached. Naylor et al. (2005) used spontaneously hypertensive rats which are known for their ability to run much longer Adult Neurogenesis 54

distances compared to other rats strains. Animals in the first part of this study were 14

weeks of age at the start of testing, and at the beginning of testing they were divided into

short term (9 days) and long term (24 days) running conditions. In the second experiment,

the long term running condition was further divided into three groups: runners, locked

wheel runners (allowed 6km/24hr period), and non-runners. The effects of exercise on

neurogenesis from this study yielded interesting results: In the first experiment, non-

runners showed less BrdU positive cells than short-term runners; and in the second

experiment long-term runners had significantly less BrdU positive cells compared to non-

runners but this negative effect of exercise was blocked when the exercise wheels were

locked to prevent over-activity. These findings suggest that exercise has an effect on

neurogenesis at first exposure, but after continuous exposure (or chronic exercise), the

effects of exercise on neurogenesis are absent. That is, the relationship follows an

inverted U-shape. The authors suggest that a reason for this may be due to increased

activation of the hypothalamic-pituitary-adrenal (HPA) axis, as the long-term runner

showed a 65% increase in adrenal gland weight compared to the short-term runner. It is also possible that there are novelty effects from simply having a wheel in the environment.

To further support the findings that exercise is beneficial when limited, another

study has demonstrated that female mice (6-8 weeks of age) bred for increased voluntary

wheel running (“selected mice”) do not show a correlation between BrdU positive cells

and distance run, supporting the evidence for the inverted U-shape described above

(Rhodes et al., 2003). However, the control strain did show a correlation between the

number of BrdU positive cells and distance run that was present for the extent of the Adult Neurogenesis 55

study (40 days), suggesting that moderate exposure to exercise can exert positive effects

on hippocampal neurogenesis. This suggests that it is possible to have too much exercise

and that much like other things, exercise is better in moderation.

Microenvironmental factors

Hormones.

Testosterone

Little work has been done to investigate the role of testosterone in regulating hippocampal neurogenesis. Because testosterone is synthesized from the same metabolic pathway as estrogens, many researchers have started to study the effects of estrogens for

its effects in both males and females. However, a few studies have examined testosterone

exclusively and a considerable amount of work has been done in the songbird. In adult

songbirds, a brain area called the high vocal center (HVC) shows fluctuations in

neurogenesis with times of song learning. After times of cell death, there is an increase in

testosterone and cell proliferation and these levels are highest after new song syllables are

learned (Alvarez-Buylla & Kirn, 1997). In another study, male prairie voles showed an

increase in proliferation after injections of testosterone or estradiol (but not after

injections of dihydroxytestosterone; Fowler, Freeman & Wang). However, this increase

in proliferation of BrdU positive cells was not found in the hippocampus, but instead was

found in the amygdala and the authors believe this effect to be due to estrogenic

mechanisms and may be specific to this species.

Estrogen

With respect to estrogen and neurogenesis, there has been a considerable amount

of research conducted on this hormone. This is most likely due to the increased likelihood Adult Neurogenesis 56

of age-related cognitive decline seen in women compared to men, the presence of

menopause in women, and the increasing popularity of hormone therapies in women. To

look at the exact effects of estrogen on cell proliferation, a study by Mazzucco et al.

(2006) found that administration of estradiol to ovariectomized rats increased the amount of cell proliferation in the dentate gyrus of the hippocampus. It is worth noting though, that the authors did not compare the intact rats to ovariectomized rats across the lifespan.

Additionally, the authors used two types of estrogen receptor agonists and found that both the ERα and ERβ agonists were capable of eliciting cell proliferation at different doses, but they were not as effective as estradiol alone (Mazzucco, Lieblich, Bingham,

Williamson, Viau, & Galea, 2006).

Serotonin.

Depression has long been studied for its potential role in affecting the

hippocampus by causing hippocampal atrophy (Bremner et al., 2000). Because of the

therapeutic potential of antidepressants and the precise location of adult neurogenesis,

there have been speculations that adult neurogenesis may also mediate the efficacy of

certain serotonin-based antidepressants (for review see Kempermann, 2002)The potential

involvement of serotonin in adult neurogenesis has been shown in both loss of function

and gain of function experiments. It was shown in the late 1990s that neurogenesis can be

modulated by serotonin. Brezun and Daszuta (1999) injected the serotonin neurotoxin

5,7-DHT into the raphe nuclei to deplete serotonin production. Eight days after lesioning,

immunohistochemical analyses were performed to analyze the number of BrdU positive

cells present in the dentate gyrus of these young adult female rats. The authors found that

damaging the serotonin system also significantly decreased the number of BrdU positive Adult Neurogenesis 57

cells in dentate gyrus. This suggests that serotonin may play a regulatory role in adult

neurogenesis. To add to this, Gould (1999) reviews the potential role of serotonin and

specifically the 5HT1A receptor in modulating neurogenesis; where stimulating the 5HT1A receptor stimulates proliferation as well.

Growth factors.

There are several growth factors that are often studied with respect to neurogenesis: insulin-like growth factor (IGF-1), fibroblast growth factor (FGF-2), and brain derived neurotrophic factor (BDNF) – all of which are considered to act as positive modulators of adult neurogenesis. Because much of this study focused on cell proliferation versus survival and exercise versus proliferation, the remainder of this section will be focused on BDNF to avoid any great, unnecessary detail.

BDNF has been studied from two different approaches: first, because BDNF as an environmental factor has been shown to promote cell maintenance and survival, some suggest that BDNF might be the mechanism by which new neurons adopt a permanent neuronal phenotype and become fully functional. Second, because exercise is known to have robust effects on cell proliferation and survival, other researchers are interested in whether BDNF is the mechanism underlying the proliferative component of this behavioral activity. Regardless, many are interested in BDNF for its potential ability to answer the ultimate question of “what causes adult neurogenesis?”

To best describe the role of BDNF in neurogenesis, it is a suitable approach to again describe gain of function and loss of function studies. In attempt to investigate whether increasing levels of BDNF would stimulate neurogenesis, Riluzole (a synthetic compound that works via blockade of sodium channels) was administered to monitor its Adult Neurogenesis 58

effects on BDNF and neurogenesis (Katoh-Semba et al., 2002). The authors found that

BDNF and BrdU positive cells were increased in the hippocampus. However, the effects on BrdU positive cells were not long lasting, as Riluzole did not promote cell survival. In

another study, the authors were interested in investigating how local infusion of BDNF

directly into hippocampus would influence neurogenesis (Scharfman et al., 2005). The

study found that BDNF infusions stimulated neurogenesis but there was also an increase

in the number of ectopic (hilar) granule cells. This is consistent with the finding that

Katoh-Semba et al. found despite the fact that Riluzole was administered systemically

and capable of working centrally and peripherally whereas the BDNF infusions only

worked centrally.

Due to recent advances in the field of neuroscience and an increased interest adult

neurogenesis, publications describing the mechanism behind this phenomenon are just

starting to surface. One important point to consider is that a loss of BDNF might have no effect on cell proliferation but instead might have an effect on survival – if the functional role of BDNF is truly for cell maintenance and survival. Further research needs to be conducted to unravel the correlational relationship between BDNF and neurogenesis to determine if BDNF is what causes adult neurogenesis or if increased BDNF is a result of neurogenesis.

Stress

Stress, and its actions via the glucocorticoids, has always been considered to play

a large role in the developmental process because of its ability to change much of the way

the body interacts with everyday stimuli. A considerable amount of work highlighting the

negative effects of stress on the hippocampus via the glucocorticoids and the Adult Neurogenesis 59

corticosteroid receptors has been done by Robert Sapolsky. To summarize the 1999

review by Sapolsky, he suggests several ways in which stress can harmfully impact the hippocampus: disruption of learning and memory, inhibition of neurogenesis, atrophy of neuronal processes, endangerment of hippocampal neurons, and neurotoxicity (Sapolsky,

1999).

Studies examining the effects of stress (working through the glucocorticoids) on

neurogenesis were some of the first conducted when this phenomenon was being

reintroduced in the late twentieth century. Cameron and Gould used [3H] Thymidine and

neuron specific enolase (NSE), a marker for mature neurons to monitor the effects of

corticosterone levels on neurogenesis using both loss of function and gain of function

techniques. They found that adrenalectomy (ADX) increased neurogenesis by decreasing the amount of corticosterone levels and that treatment with corticosterone decreased neurogenesis in the dentate gyrus (Cameron & Gould, 1994). To behaviorally show that stress can induce the same effects on neurogenesis as changes in corticosterone levels, a resident-intruder paradigm was studied in monkeys and BrdU positive cells were quantified (Gould, Tanapat, McEwen, Flugge, & Fuchs, 1998). The authors found that compared to controls, stress significantly reduced BrdU cell proliferation. This suggests

that stress downregulates neurogenesis and that these effects are due to the actions of corticosterone.

Aging

The natural aging process is known to cause a decrease in neurogenesis

(Kempermann et al., 1998; Kuhn et al., 1996; Rao et al., 2006). Because the

corticosterone was one of the first mechanisms discovered to regulate neurogenesis and Adult Neurogenesis 60

because of the known sensitivity of the glucocorticoids receptors in the hippocampus in

aging, one approach to investigate the reason for this age-related decline has been to

study the interaction between the glucocorticoid system, aging and neurogenesis. It has

been demonstrated that there is an increase in corticosterone as a rodent ages and that this

increase is likely responsible for the age-related decline in neurogenesis (Naylor et al.,

2005). Futhermore, the authors found that an adrenalectomy at 10 months of age (mid-

life in the Sprague-Dawley rats used) will increase neurogenesis compared to intact, age-

matched controls (Naylor et al., 2005). This suggests that working with the stress/aging

hypothesis supported by Sapolsky (1999; see above) and the work of Elizabeth Gould,

Bruce McEwen, and others, that there is a potential explanation for the role of

glucocorticoids contributing to age-related hippocampal changes including

downregulation in neurogenesis.

Finally, it should be considered that any mechanism for a downregulation in neurogenesis, especially aging, has the potential to be explained through simple cellular kinetics. Because the proliferation of these new neurons and other cells is limited by the activity of the neural precursor cells that are more “stem-cell like,” the number of cell divisions that these cells can go through must ultimately be limited (under natural circumstances). This was first described by Leonard Hayflick describing cellular senescence, a theory proposing that somatic cells have the ability to divide through

roughly fifty population doublings with each successive doubling resulting in a

shortening of telomere length that ultimately leads to apoptosis (Hayflick, 2003). At the

time of postnatal development starting at a very young age, humans have only

approximately 20 population doublings left (Fu, Lu, & Mattson, 2002) for the next 75 Adult Neurogenesis 61

years of the expected lifespan. Some research on telomerase (the enzyme responsible for

the lengthening of the telomeres for successive cell divisions) has been conducted with

BDNF and FGF, but no concrete evidence has been drawn (Fu, Lu, & Mattson, 2002; reviewed in Mattson, Fu, & Zhang, 2001). But what it could suggest is that the decrease

in neurogenesis with age is due to a decline in activity at the stem-cell level, and that

certain microenvironmental factors (when manipulated), may be capable of sustaining the

regenerative capacity of these cells.

Functional implication for neurogenesis: Memory

The idea that adult neurogenesis only robustly occurs in two areas out of many in

the mammalian brain is astounding (which two, but remember you also mentioned

amygdala above). The exact reason(s) why this cellular phenomenon only occurs in the

hippocampus and olfactory bulb and not others is still unknown. It could be speculated

that it is due to microenvironmental properties such that these environments, particularly the hippocampus, are more conducive to cell maintenance: rich in growth/trophic factors.

Other areas not as susceptible to cell death may not have evolved to have these microenvironmental properties and thus do not facilitate adult neurogenesis. In terms of the functional significance of neurogenesis, it mostly remains to be elucidated. Some evidence has suggested that hippocampal neurogenesis is involved in depression (Duman,

Nakagawa, & Malberg, 2001) and/or plays a role in memory (Snyder, Hong, McDonald

& Wojtowicz, 2005).

The role of neurogenesis in memory is still highly debated. Aged Long Evans rats reportedly show an inverse relationship between BrdU+ cells and Morris Water Maze performance, with a higher number of BrdU+ cells found in animals with poorer spatial Adult Neurogenesis 62

memory (Bizon, Lee, & Gallagher, 2004). Conversely, in aged Sprague Dawley rats, it

has been shown that both survival and proliferation via BrdU+ cells is correlated with

improved spatial memory (Drapeau, Mayo, Aurousseau, Le Moal, Piazza, & Abrous,

2003). However, was suggested by Bizon et al. (2004) that the aged brain has been

pressured into a more pathological state and is attempting to recruit more neurons for

more efficient functioning, but was unable to reach the threshold level necessary to

perform as well as the young animals. So though Bizon et al. (2004) found an inverse

relationship and Drapeau et al. (2003) found a positive relationship, the authors suggested

that perhaps the aged Long Evans rats in the first study were having to “try harder” to

perform as well as the younger animals (Bizon et al. 2004) when compared to the aged

Sprague Dawley rats in the second study - though no strain differences in neurogenesis

have been extensively detailed. Other differences may be due to the time of injection of

the BrdU, as the actual task may influence the rate of proliferation.

In an attempt to further investigate the relationship between neurogenesis and

memory, studies were done using two methods that prevent proliferation of either

neurons (methylazoxymethanol; MAM) or all cells entirely (gamma irradiation) to

determine the role of neurogenesis in memory. When the anti-mitotic agent MAM is

administered to Sprague Dawley rats, the subsequent decrease in BrdU+ cells is not

correlated with a decreased performance in the Morris Water Maze (Shors, Townsend,

Zhao, Kozorovitskiy, & Gould, 2002) and short term memory (24 hours) is not impaired.

Interestingly, when young Long Evans rats are subject to gamma irradiation, long term

spatial memory (two weeks) is impaired, but the ability to learn the task or recall at short

term (1 week) was not impaired (Snyder et al., 2005). Unfortunately, Shors et al. (2002) Adult Neurogenesis 63

did not perform a test of long term memory, but if the authors had done the long term

memory assessment and found that there was an impairment in long term memory, it

would support the idea that neurogenesis is important for learning and memory.

To add to the complexity of the situation, there is some debate about the effect of performing tasks on the fate of proliferating cells. For example, stress is one factor that is

well known to downregulate neurogenic activity (Mirescu & Gould, 2006), and placing the animal in the water maze (or similar task) and having the animal try to locate a platform without any knowledge of purpose can be frustrating and frightful. Additionally, it is also widely accepted that physical activity upregulates neurogenesis (van Praag,

Kempermann, & Gage, 1999; van Praag, Christie, Sejnowski, & Gage, 1999), and the amount of physical activity involved in swimming compared to voluntary exercise remains to be explored. Furthermore, learning is also considered to influence neurogenesis, where learning may actually promote the survival of some newly generated neurons. As an example, Leuner et al., (2004) showed by using trace eyeblink conditioning that paired training increased the number of BrdU+ cells by 40% compared to animals in the unpaired condition. Though it should be stated that this could be a stress effect because animals who know when the shock will arrive (paired condition) will have less stress than the animals that do not have predictable control (unpaired condition).

However, in a recent report by Van der Borght et al. (2005), it was found that learning the

Morris Water Maze task did not influence the number of BrdU+ cells in either Wistar or

Sprague Dawleys, it only influenced the number of PSA-NCAM positive cells, a protein expressed by immature granule cells. Additionally, Ehninger and Kempermann (2006) suggest that, in mice, while both exercise and stress may influence neurogenesis during Adult Neurogenesis 64

the water maze, the two effects may cancel each other and still produce a net effect. In

summary, this suggests that must use caution when planning within subject designs using

behavioral and cellular measures involving BrdU as the task at hand is capable of

influencing the cell activity as well.

Collectively, there is a growing amount of evidence both for and against the role of new neurons in memory, both causal and regulatory. However, it is worth noting that it is unlikely that the new neurons are formed and used for the memory task immediately.

Rather, it is more plausible that new neurons are produced naturally and further regulated by other means and then kept in a reserve pool for later functioning. It would then be the case that, perhaps, hippocampal dependent tasks recruit mature granule cells to be used for the learning and memory task. This is supported by the fact that new neurons take approximately four weeks to become fully functional and develop into mature granule cells and 4-10 days to extend axons into CA3 along the mossy fiber tract (Hastings, Seth,

Tanapat, Rydel, & Gould, 2002). To add to this interest, evidence by Synder et al. (2005) provides that neurons that are 4-28 days old are responsible for the formation of a long term memory. Adult Neurogenesis 65

APPENDIX C. PILOT DATA

We had anticipated that enrichment would promote a slight upregulation of BrdU positive cells at the 24-hour time point and a significant difference at the two-week time point. Instead, we observed a significant downregulation at 24-hours and no effect at two-weeks in group housed animals compared to single housed isolated animals.

There is a vast amount of evidence implicating the negative role of stress on hippocampal neurogenesis (Cameron & Gould, 1994; Gould et al., 1998; Sapolsky,

1999). Furthermore, only dominant, but not subordinate, males were found to have increased cell survival when put in an enriched environment, (Kozorovitskiy & Gould,

2004). The authors provide that this finding may be because the subordinate male was not capable of gaining the effects of the enriched environment in the same respect as the dominant male was.

Considering that for our enrichment study our males were group housed for three weeks (24-hour/proliferation measure) and we did not control for dominance hierarchies other than keeping animals with littermates, we decided to compare group housed versus single housed enriched environments. Because this was a pilot study and all ages of animals were not available to us, we decided to use 5-month (n=3) and 7-month old animals (n=3), with the assumption that if an effect was found with these ages, then we would perform similar studies in 2-month old animals. All manipulations and experimental methods were the same as described above. Single housed animals were kept in the standard laboratory cages and group housed animals were kept in the larger bins as previously described. Adult Neurogenesis 66

Using a two-way ANOVA comparing BrdU positive cells, we found that there

was no difference between group housed and single housed enriched animals, F(1,10) =

.413, p = .535 (Figure 9). Also, consistent with our previous enrichment data, the two- way analysis showed no differences between ages, F(1,10) = .720, p = .416. Both groups

showed no effect of an enriched environment and no interaction was present, F(1,10) =

.440, p = .522. Had an effect of condition been present to elude to an effect of single

housed enrichment promoting more neurogenesis than group housed enrichment, this

would have suggested that our original findings were due to social stress negatively

impacting neurogenesis.