Photoperiod, Brain Plasticity, and Behavior DISSERTATION...Photoperiod, Brain Plasticity, and...

Photoperiod, Brain Plasticity, and Behavior

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University

By

James C. Walton, B.S.

Neuroscience Graduate Studies Program

The Ohio State University

2013

Dissertation Committee:

Professor Randy J. Nelson, Advisor

Professor A. Courtney DeVries

Professor Georgia A. Bishop

Copyright by

James C. Walton

2013

ii

Abstract

Outside of the tropics, distinctive sets of adaptations have evolved

to cope with the unique demands of winter and summer on survival and

reproduction. Because these seasonal adaptations often require

significant time to develop, individuals rely upon an environmental signal

(day length) to alter gene expression in order to produce the suite of

season-specific adaptations. Photoperiodism is the ability of plants and

animals to measure environmental day length (photoperiod) to ascertain

the time of year and engage in seasonally appropriate adaptations; the

annual cycle of changing photoperiod provides the environmental switch

between seasonal phenotypes. The aim of this dissertation is to describe

the influence of photoperiod on phenotype, the distribution of

energetically expensive processes across the year, to maximize survival

and fitness in males of a small photoperiodic rodent species, white-footed

mice (Peromyscus leucopus).

In Chapter 1 I provide a review of photoperiodism and an overview

of the suite of adaptive responses at multiple levels to changing day

lengths in a variety of vertebrate species. Among the adaptive responses

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in male white-footed mice are reduced brain size, and a marked

reduction in hippocampal volume. Given that the hippocampus is critical

for spatial learning and memory, I explore the functional outcomes of

having a small hippocampus in Chapter 2 utilizing a spatial learning and

memory task. I also assess neuronal physiology in the form of long-term

potentiation in the hippocampus, the form of neuronal plasticity that is

the putative mechanism of how memories are stored in the brain. In

Chapters 3 and 4 I explore how photoperiod-mediated changes in

hormones alter hippocampal function. Given that mammals use pineal

melatonin rhythms to monitor day length, in Chapter 3 I explore the role

of melatonin in the functional and structural hippocampal changes

described in Chapter 2. Downstream of pineal melatonin signaling,

gonadal steroids are also altered in short days in this species, and the

role of gonadal steroids in photoperiod-mediated changes in the

hippocampus is explored in Chapter 4. Given that the hippocampus is

heavily interconnected with other brain structures, it follows that these

areas, and behaviors dependent upon them, are altered in short days as

well. In Chapter 5 I explore how photoperiod affects the structure and

function of the amygdala and alters amygdala-dependent fear behaviors.

Whereas Chapters 3-5 explore functional outcomes of reduced

brain volume, in Chapter 6 and 7 I explore a potential mechanism by

which brain mass and hippocampal volume may be altered. The

iv

recruitment and retention of new neurons in the brains of adults may

contribute to volumetric and mass changes; thus, I explore the effect of

photoperiod on neurogenesis in the two main neurogenic niches of adult

brains: the olfactory bulb (Chapter 6) and the dentate gyrus of the

hippocampus (Chapter 7). In conclusion, in Chapter 8 I summarize my

findings and propose future directions for research in this species into

how photoperiod affects brain plasticity and behavior, and how this

research can inform us on complex gene-environment interactions that

affect phenotypic plasticity, and thus behavior.

v

Dedication

For Andrew, Holly, and Jennifer.

vi

Acknowledgments

When considering scientists who have molded my career to date,

foremost I think of my father, Clem Walton. While I was still in

elementary school, he was never hesitant to take me along with him to

some remote Maine river at 3 AM in early April to sample whichever

species of anadromous fish was currently on their spring migratory run.

He was a consummate scientist and thinker, and was ever fascinated

with animal behavior across phyla. Thank you for impressing upon me

the joys of being inquisitive about the wonders of nature. I also thank my

mother, Katharine Walton, for her tireless patience, guidance,

understanding, and for always being there.

I owe an enormous debt of gratitude to my graduate mentor, Randy

Nelson. I thank Randy for the opportunity he gave me to do research in

his lab and for his support and guidance during my tenure here. Randy‟s

passion for science, unparalleled skill at mentorship, unique style of

motivation, and priceless sense of humor has left a lasting impression on

me. I also thank Courtney DeVries for her scientific guidance, for

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entrusting me with running her daily lab operations for several years,

and for serving on my candidacy and dissertation committees.

I thank Lou Gainey at the University of Southern Maine for

plucking me out of his Animal Physiology class and hiring me as an

undergraduate to do research in his lab, and for introducing me to

neuroscience in a seasonal context. I thank Rick Thompson at Bowdoin

College for introducing me to the areas of research that have become my

passion: behavioral neuroendocrinology and neuroethology.

I thank Georgia Bishop for serving on both my candidacy and

dissertation committees. I thank the many other scientists at Ohio State

with whom I‟ve collaborated, with special thanks to John Oberdick, Joe

Travers, Mike Bailey, Zhixiong Chen, Ben Leuner, and Sung Ok Yoon. I

also thank the scientists at other institutions with whom I have had the

pleasure of working with: Leah Pyter, Solomon Snyder, Michael

Greenberg, Balakrishnan Selvakumar, and Kristen Hoffbuhr.

To my current and former lab mates: Laura Fonken, Tracy

Bedrosian, John Morris, Kate Weil, Greg Norman, Ning Zhang, Shan

Chen, Joanna Workman, Taryn Aubrecht, Tomoko Ikeno, Noah Ashley,

Jeremy Borniger, Brant Jarrett, Adam Hinzey, Katie Stuller, and Kris

Gaier thank you for your friendship and for making the lab an

academically stimulating and entertaining place. I also thank Zach Weil

for all of the stimulating conversations, and for being himself. I would be

viii

remiss if I didn‟t thank all of the undergraduate students who have

assisted with my research. To Erika, Roxanne, Brittany, Anqui, James,

Sarah, and Amanda: thank you. I owe special thanks to Jordan Grier,

James Spieldenner, and Kara Ruder, the capable undergraduate

researchers who completed their honors theses while working with me.

I thank my family. To Jennifer, Holly, Andrew, Lisa, Kathy, Dave,

Pat, Chris, and Amanda, without your continuous support and

understanding, this undertaking would not have been possible.

ix

Vita

2001 ................................................ B.S. Biology, Summa Cum Laude,

University of Southern Maine

2009-2010 ....................................... Graduate Research Associate,

University Fellow, Department of

Neuroscience, The Ohio State

University Wexner Medical Center

2010-2012 ....................................... Graduate Research Associate,

Department of Neuroscience, The

Ohio State University Wexner

Medical Center

2012-present ................................... Graduate Research Associate,

Presidential Fellow, Department of

Neuroscience, The Ohio State

University Wexner Medical Center

x

Publications

26) Bedrosian, T.A., Herring, K.L., Walton, J.C., Fonken, L.K., Weil, Z.M.,

& Nelson, R.J. (2013) Possible feedback control of pineal melatonin

secretion. Neuroscience Letters, doi: 10.1016/j.neulet.2013.03.021.

25) Ashley, N.T., Walton, J.C., Haim, A., Zhang, N., Prince, L.A., Fruchey,

A.M., Lieberman, R.A., Weil, Z.M., Nelson, R.J. (2013) Sleep deprivation

attenuates endotoxin-induced cytokine gene expression independent of

day length and circulating cortisol in photoperiodic rodents. Journal of

Experimental Biology, PMID: 23531821.

24) Thompson, R.R., Walton, J.C. (2013). Isotocin and vasotocin

involvement in fish behavior. In: Oxytocin, Vasopressin and Related

Peptides in the Regulation of Behavior. Edited by E. Choleris, D. Pfaff, M.

Kavaliers, Cambridge University Press, ISBN 9780521190350.

23) Walton, J.C.*, Pyter, L.M.*, Weil, Z.M., Nelson, R.J. (2012)

Photoperiod mediated changes in olfactory bulb neurogenesis and

olfactory behavior in male white-footed mice (Peromyscus leucopus). PLoS

ONE, 7(8):e42743 doi:10.1371/journal.pone.0042743. *Authors

contributed equally to this work.

xi

22) Yoon, S.O., Park, D.J., Ozer, H.G, Ryu, J.C., Tep, C., Shin, Y.J., Lim,

T.H., Pastorino, L., Kunwar, A.J., Walton, J.C., Nagahara, A.H., Lu, K.P.,

Nelson, R.J., Tuszynski, M.H., Huang, K. (2012) JNK3 perpetuates

metabolic stress induced by Abeta peptides. Neuron, 75(5):824-837.

21) Walton, J.C., Haim, A., Spieldenner, J.M., Nelson, R.J. (2012)

Photoperiod alters fear responses and basolateral amygdala neuronal

spine density in white-footed mice (Peromyscus leucopus). Behavioural

Brain Research, 233(2):345-50.

20) Walton, J.C., Grier, A.J., Weil, Z.M., Nelson, R.J. (2012) Photoperiod

and stress regulation of corticosteroid receptor, brain derived

neurotrophic factor, and glucose transporter GLUT3 mRNA in the

hippocampus of male Siberian hamsters (Phodopus sungorus).

Neuroscience, 213:106-111.

19) Walton, J.C, Schilling, K., Zhu, M.X., Nelson, R.J., Oberdick, J.O.

(2012) Sex-Dependent Behavioral Functions of the Purkinje Cell-Specific

Gαi/o Binding Protein, Pcp2(L7). Cerebellum, 11(4):982-1001.

18) Workman, J.L., Manny, N., Walton, J.C., Nelson, R.J. (2011) Short

xii

days alter depressive-like responses, and CA1 hippocampal morphology

in Siberian hamsters. Hormones and Behavior, 60(5):520-8.

17) Bedrosian, T.A., Fonken, L.K., Walton, J.C., Haim, A., & Nelson, R.J.

(2011) Dim light at night provokes depression-like behaviors and reduces

CA1 dendritic spine density in female hamsters.

Psychoneuroendocrinology, 36(7):1062-9.

16) Bedrosian, T.A., Fonken, L.K., Walton, J.C., Nelson, R.J. (2011)

Chronic exposure to dim light at night suppresses immune responses in

Siberian hamsters. Biology Letters, 7(3):468-71.

15) Walton, J.C., Weil, Z.M., Nelson, R.J. (2011) Influence of photoperiod

on hormones, behavior, and immune function. Frontiers in

Neuroendocrinology, 32(3):303-19.

14) Walton, J.C., Chen, Z., Weil, Z.M., Travers, J.B., Pyter, L.M., Nelson,

R.J. (2010) Photoperiod-mediated impairment of long term potentiation

and learning and memory in male white-footed mice. Neuroscience,

175:127-32.

13) Fonken, L.K., Workman, J.L., Walton, J.C., Weil, Z.M., Morris, J.S.,

xiii

Haim, A., Nelson, R.J. (2010) Light at night increases body mass by

shifting the time of food intake. Proceedings of the National Academy of

Sciences USA, 107(43):18664-9.

12) Karelina, K., Walton, J.C., Weil, Z.M., Norman, G.J., Nelson, R.J.,

DeVries, A.C. (2010) Estrous phase alters social behavior in a polygynous

but not monogamous Peromyscus species. Hormones and Behavior,

58(2):193-9.

11) Bailey, M.T.*, Walton, J.C.*, Dowd, S.E., Weil, Z.M., Nelson, R.J.

(2010) Photoperiod modulates gut bacteria composition in male Siberian

hamsters (Phodopus sungorus). Brain, Behavior, and Immunity, *Authors

contributed equally to this work.) 4(4):577-84.

10) Norman, G.J., Karelina, K., Zhang, N., Walton, J.C., Morris J.S.,

DeVries, A.C. (2010) Stress and IL- 1beta contribute to the development

of depressive-like behavior following peripheral nerve injury. Molecular

Psychiatry, 5(4):404-14.

9) Walton, J.C., Waxman, B., Hoffbuhr, K., Kennedy, M., Beth, E.,

Scangos, J., Thompson, R.R. (2010) Behavioral effects of hindbrain

xiv

vasotocin in goldfish are seasonally variable but not sexually dimorphic.

Neuropharmacology, 58(1):126-34.

8) Fonken, L.K., Finy, M.S., Walton, J.C., Weil, Z.M., Workman, J.L.,

Ross, J., Nelson, R.J. (2009) Influence of light at night on murine

anxiety- and depressive-like responses. Behavioural Brain Research,

205(2):349-54.

7) Thompson, R.R., Walton, J.C. (2009) Vasotocin immunoreactivity in

goldfish brains: Characterizing primitive circuits associated with social

regulation. Brain, Behavior and Evolution, 73(3):153-164.

6) Weil, Z.M., Norman, G.J., Karelina, K., Morris, J.S., Barker, J.M., Su,

A.J., Walton, J.C., Nelson, R.J., DeVries, A.C. (2009) Sleep deprivation

attenuates inflammatory responses and ischemic cell death.

Experimental Neurology, 218(1):129-36.

5) Thompson, R.R., Walton, J.C., Bhalla, R., George, K.C., Beth, E. H.

(2008) A primitive social circuit: Vasotocin–substance P interactions

modulate social behavior through a peripheral feedback mechanism in

goldfish. European Journal of Neuroscience, 27(9):2285-93.

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4) Thompson, R.R., George, K., Walton, J.C., Orr, S.P., Benson, J. (2006)

Sex-specific influences of vasopressin on human social communication.

Proceedings of the National Academy of Sciences USA, 103(20):7889-94.

3) Thompson, R.R., George, K., Dempsey, J., Walton, J.C. (2004) Visual

sex discrimination in goldfish: seasonal, sexual and androgenic

influences. Hormones and Behavior, 46(5):646-54.

2) Thompson, R.R., Walton, J.C. (2004) Peptide effects on social behavior:

effects of vasotocin and isotocin on social approach behavior in male

goldfish (Carassius auratus). Behavioral Neuroscience, 118(3):620-6.

1) Gainey, L.F., Walton, J.C., Greenberg, M.J. (2003) Branchial

musculature of a venerid clam: pharmacology, distribution and

innervation. Biological Bulletin, 204(1):81-95.

Fields of Study

Major Field: Neuroscience

xvi

Table of Contents

Abstract................................................................................................. ii

Dedication ..............................................................................................v

Acknowledgments .................................................................................. vi

Vita ...................................................................................................... ix

Table of Contents ................................................................................. xvi

List of Tables ..................................................................................... xxiii

List of Figures .................................................................................... xxiv

Chapter 1: The Influence of Photoperiod on Hormones, Behavior, and

Brian Plasticity. ..................................................................................... 1

1. Introduction.................................................................................... 1

2. The pineal gland, melatonin, and photoperiod ................................. 4

3. Photoperiod and energy balance ...................................................... 9

3.1. Somatic responses to photoperiod ........................................... 10

3.1.2. Neuroendocrine mechanisms............................................ 11

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3.1.3. Photoperiod and central energy conservation .................... 14

3.2. Photoperiod and reproduction ................................................. 15

3.2.1. Short-day breeders ........................................................... 16

3.2.2. Long-day breeders ............................................................ 18

4. Photoperiod, affect, and non-reproductive behaviors ..................... 24

4.1. Affective responses .................................................................. 25

4.2. Non-reproductive social behaviors ........................................... 26

4.3. Learning and memory ............................................................. 30

5. Conclusions .................................................................................. 32

Chapter 2: Photoperiod, Hippocampal Long-Term Potentiation, and

Spatial Learning and Memory .............................................................. 37

1. Introduction.................................................................................. 37

2. Experimental Procedures .............................................................. 40

2.1. Animals .................................................................................. 40

2.2. Long-term potentiation ............................................................ 41

2.3. Barnes maze ........................................................................... 42

2.4. Statistical Analyses ................................................................. 44

3. Results ......................................................................................... 45

3.1. Short days reduce body and reproductive tissue mass. ............ 45

xviii

3.2. Short days impair Barnes maze performance. .......................... 45

3.3. Short days impair LTP. ............................................................ 46

4. Discussion .................................................................................... 46

Chapter 3: The Role of Melatonin in Photoperiodic Changes in

Hippocampal Plasticity. ....................................................................... 55

1. Introduction.................................................................................. 55

2. Materials and Methods.................................................................. 58

2.1. Animals .................................................................................. 58

2.2. Melatonin implants and photoperiod treatment ....................... 59

2.3. Long-term potentiation (LTP) ................................................... 60

2.3. Spatial learning and memory .................................................. 61

2.4. Hippocampal neuronal morphology ......................................... 62

2.5. Radioimmunoassay ................................................................. 63

2.6. Statistics ................................................................................. 63

3. Results ......................................................................................... 64

3.1. Melatonin assay ...................................................................... 64

3.2 Reproductive responses to MEL and SD ................................... 64


3.4. Hippocampal long-term potentiation ....................................... 65

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3.5. Morphometry of hippocampal neurons .................................... 65

3.5.1. Cell soma ......................................................................... 65

3.5.2. Dendritic material ............................................................ 66

3.5.3. Dendritic complexity ........................................................ 66

3.5.4. Spine density ................................................................... 67

4. Discussion .................................................................................... 67

Chapter 4: The Role of Sex Steroids in Photoperiodic Changes in

Hippocampal Plasticity ........................................................................ 81

1. Introduction.................................................................................. 81

2. Materials and methods ................................................................. 84

2.1. Animals .................................................................................. 84

2.2. Gonadectomy and steroid implants ......................................... 85

2.4. Tissue collection and gene expression ..................................... 86

2.5. Statistics ................................................................................. 87

3. Results ......................................................................................... 88

3.1. Reproductive tissues ............................................................... 88


3.3 Sex steroid receptor gene expression ........................................ 90

4. Discussion .................................................................................... 91

xx

Chapter 5: Photoperiod, Fear Behavior, and the Basolateral Amygdala. 99

1. Introduction.................................................................................. 99

2. Materials and methods ............................................................... 102

2.1. Animals ................................................................................ 102

2.2. Behavioral Tests .................................................................... 102

2.2.1. Passive avoidance ........................................................... 102

2.2.2. Auditory fear conditioning .............................................. 103

2.3. Sample collection and histology ............................................. 105

2.3.1. Dendritic arborization analysis ....................................... 105

2.3.2. Dendritic spine density analysis ..................................... 106

2.3.3. Corticosterone assay ...................................................... 107

2.3.4. Statistics ........................................................................ 107

3. Results ....................................................................................... 108

3.1. Physiological measures ......................................................... 108

3.2. Behavioral measures ............................................................. 108

3.2.1. Passive avoidance ........................................................... 108

3.2.2. Auditory-cued fear conditioning...................................... 109

3.3. Neuronal morphology ............................................................ 110

3.3.1. Dendritic spine density................................................... 110

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3.3.2. IL pyramidal neuronal morphology ................................. 110

4. Discussion .................................................................................. 110

Chapter 6: Photoperiod and Olfactory Bulb Neurogenesis................... 122

1. Introduction................................................................................ 122


2.1. Animals ................................................................................ 125

2.2.1. BrdU injections .............................................................. 126

2.2.2. Tissue collection and histology ....................................... 127

2.3. Experiment 2: Effects of photoperiod on dendritic morphology in

the olfactory bulb granule cell layer. ............................................. 129

2.3.1. Olfactory bulb dendritic morphology ............................... 129

2.4. Experiment 3: Effects of photoperiod on investigation of

conspecific male urine. ................................................................ 130

2.4.1. Habituation-dishabituation test...................................... 130

2.5. Statistical analyses ............................................................... 131

3. Results ....................................................................................... 132

3.1. Reproductive responses to photoperiod ................................. 132

3.2. Experiment 1 ........................................................................ 132

3.3. Experiment 2 ........................................................................ 133

xxii

3.3.1. Olfactory bulb granule cell morphology........................... 133

3.4. Experiment 3 ........................................................................ 134

3.4.1. Habituation-dishabituation ............................................ 134

4. Discussion .................................................................................. 134

Chapter 7: Photoperiod and Hippocampal Neurogenesis. .................... 148

1. Introduction................................................................................ 148


2.1 Animals ................................................................................. 151

2.2 Longitudinal assessment of neurogenesis using BrdU ............ 152

2.3 Brain histology ....................................................................... 153

2.4 Statistics ................................................................................ 155

3. Results ....................................................................................... 156

3.1. Progenitor cell 4 week survival .............................................. 156

3.2 BrdU+ cell phenotype ............................................................. 156

4. Discussion .................................................................................. 156

Chapter 8: Conclusions and Future Directions. ................................. 166

References. ........................................................................................ 172

xxiii

List of Tables

Table 4.1 Experimental groups ............................................................ 95

Table 6.1 Olfacotry bulb progenitor cell phenotypes ........................... 147

Table 7.1 Dentate progenitor cell phenotype across photoperiod......... 165

xxiv

List of Figures

Figure 1.1 Transduction of photic input to neuroendocrine output ...... 35

Figure 1.2 Photoperiodic differences in allocation of energy among

competing processes ............................................................................ 36

Figure 2.1 Photoperiodic body and reproductive tissue responses ....... 52

Figure 2.2 Photoperiod induced deficits in spatial learning and memory

in the Barnes maze .............................................................................. 53

Figure 2.3 Photoperiodic impairment of long-term potentiation in the

hippocampus ....................................................................................... 54

Figure 3.1 Melatonin delivery from implants and reproductive responses

to extended melatonin exposure ........................................................... 73

Figure 3.2 Spatial learning and memory in the Barnes maze ................ 74

Figure 3.3 Schaffer collateral long-term potentiation ............................ 75

Figure 3.4 Dendritic material of hippocampal neurons ......................... 76

Figure 3.5 CA1 dendritic complexity .................................................... 77

Figure 3.6 CA3 dendritic complexity .................................................... 78

Figure 3.7 Dentate dendritic complexity ............................................... 79

Figure 3.8 Neuronal spine density in hippocampal neurons ................. 80

xxv

Figure 4.1 Body and reproductive tissue mass responses to steroid and

photoperiod treatment ......................................................................... 96

Figure 4.2 Photoperiod and steroid treatment interact to affect spatial

learning and memory ........................................................................... 97

Figure 4.3 Effects of photoperiod and steroids on estrogen and androgen

receptor expression in the hippocampus .............................................. 98

Figure 5.1 Photoperiod effects on reproductive tissue mass and plasma

corticosterone .................................................................................... 118

Figure 5.2 Short days enhance fear memory ...................................... 119

Figure 5.3 Effects of short days and fear conditioning on spine density in

the basolateral amygdala ................................................................... 120

Figure 5.4 Effects of short days and fear conditioning on pyramidal

neuron morphology in the infralimbic cortex ...................................... 121

Figure 6.1 Reproductive responses to photoperiod ............................. 142

Figure 6.2 Olfactory bulb photomicrographs from long day and short day

white-footed mice............................................................................... 143

Figure 6.3 Olfactory progenitor cell proliferation and survival............. 144

Figure 6.4 Olfactory bulb neuronal morphology ................................. 145

Figure 6.5 Olfactory habituation-dishabituation test .......................... 146

Figure 7.1 Longitudinal assessment of hippocampal neurogenesis

experimental design ........................................................................... 162

xxvi

Figure 7.2 Progenitor cell survival in the dentate gyrus across short day

exposure ............................................................................................ 163

Figure 7.3 Phenotype of dentate progenitor cells after 4 weeks ........... 164

1

Chapter 1: The Influence of Photoperiod on Hormones, Behavior,

and Brian Plasticity.

1. Introduction

In order to be successful, individuals must grow, survive, and

reproduce. Fitness is determined by how many offspring survive and

ultimately produce offspring that themselves survive and reproduce.

Investing in survival mechanisms depletes resources necessary for

reproduction. Conversely, reproduction requires significant resources

that may compromise survival. Thus, fitness reflects successful trade-

offs between investments in the mechanisms underlying survival and

reproduction that reflect life history strategies (Stearns 2000). Natural

selection has produced exquisite adaptations that have allowed

individuals to successfully survive and reproduce in remarkably specific

niches. Outside of the tropics, individuals have been selected to adapt to

temporal niches, as well as spatial niches, because the yearly orbit of the

Earth around the Sun drives seasonal variation in several environmental

factors that affect temperature, weather, and food availability.

Habitats may vary substantially from winter to summer and in

some cases distinctive sets of adaptations have evolved to cope with the

2

often unique demands of winter and summer on survival and

reproduction. For example, the winter set of adaptations may include a

shift of energy allocations from non-essential functions such as growth

and reproduction to those functions that are critical for immediate

survival (Demas, Drazen et al. 2003). When the odds of successful

reproduction are low, resources are shunted from reproduction and

growth into survival mechanisms such as immune function,

thermoregulation, or cellular maintenance. Consequently, over

evolutionary time, seasonal patterns in the expression of adaptations

have emerged that allow redistribution of energy resources to mediate

trade-offs between traits such as immune function and reproductive

effort (Nelson 2002). During the fall and winter, the dual challenges of

limited food availability with the need for additional energy to support

thermogenesis make reproductive efforts unlikely to be successful,

especially among small vertebrate animals; thus, these animals often

reduce the size and function of their reproductive system (Nelson 2004).

In addition to reproduction, small nontropical vertebrates also display

seasonal adjustments in body mass, adiposity, foraging, gut efficiency,

pelage, sleep, growth, immune function, as well as cognitive and affective

responses (reviewed in Prendergast, Zucker et al. 2009). Because these

seasonal adaptations often require significant time to develop,

3

individuals rely upon an environmental signal to alter gene expression in

order to produce the suite of season-specific adaptations.

Photoperiodism is the ability of plants and animals to measure

environmental day length (photoperiod), a process that underlies the so-

called biological calendar (Nelson, Denlinger et al. 2010). The biological

ability to measure day length permits organisms to ascertain the time of

year and engage in seasonally appropriate adaptations. Although the

specific mechanisms that underlie the ability to measure day length

differ among taxa, individuals that respond to day length can precisely,

and reliably, ascertain the time of year with just two bits of data: (1) the

length of the daily photoperiod, and (2) whether day lengths are

increasing or decreasing. For individuals of many species, the annual

cycle of changing photoperiod provides the environmental switch between

seasonal phenotypes. Changes in day length, while probably of little

direct importance to most animals, provide the most error-free indication

of time of year, and thus enable individuals to anticipate seasonal

conditions. Because the same photoperiod occurs twice a year (e.g., 21

March and 21 September), animals must be able to discriminate between

these two dates; many photoperiodic vertebrates have solved this

problem by developing an annual alteration between two physiological

states (reviewed in Prendergast, Zucker et al. 2009). Obviously, the

environmental switch that controls the phenotypic trajectory is important

4

in teasing out the interaction between environment and genes which

drives phenotype.

The goal of this dissertation is to focus on the influence of

photoperiod on phenotype, the distribution of energetically expensive

processes across the year to maximize survival and fitness, and the

enduring effects of early life photoperiod on adult phenotype. Phenotype

is the result of the interactions between genes and environment. In the

wild, day length often determines the phenotype of newborn small

vertebrates (Boonstra 1989; Gundersen and Andreassen 1998; Ergon,

MacKinnon et al. 2001). In mammals, photoperiodic information can

even be passed to developing fetuses in utero so that the summer or

winter phenotype can begin to develop prior to birth (Weaver, Keohan et

al. 1987). Thus, photoperiodic rodents born in the spring will grow to

adult size, undergo puberty, and become reproductive in 6-8 weeks,

whereas a sibling born in the autumn will not grow or undergo puberty

for 4-5 months (Hoffmann 1978; Frost and Zucker 1983). Day length can

be used in the laboratory as a precise environmental factor to probe gene

expression during phenotypic development.

2. The pineal gland, melatonin, and photoperiod

To understand photoperiodism, it is important to understand how

animals keep time physiologically. The pineal gland, and its hormone

5

melatonin, mediates photoperiodic time measurement in mammals

(Malpaux, Migaud et al. 2001; Hazlerigg 2010). Pinealectomy blocks

responsiveness to photoperiod in every mammalian species studied

(Hazlerigg and Wagner 2006). Information about environmental light

arrives to the brain via the lateral eyes in mammals (Nelson and Zucker

1981). A nonvisual neuronal pathway, the retinohypothalamic tract,

carries light information from melanopsin expressing retinal ganglion

cells directly to the suprachiasmatic nuclei (SCN) of the hypothalamus

(Card, Whealy et al. 1991; Lucas, Freedman et al. 1999; Simonneaux and

Ribelayga 2003). The SCN are the primary mammalian biological clocks

(Schwartz, de la Iglesia et al. 2001). From the SCN, the main pathway for

photoperiod information to the pineal gland is relayed through the

paraventricular nucleus of the hypothalamus, leaving the brain through

the intermediolateral cells of the upper spinal cord, and then through the

superior cervical ganglion. Postganglionic noradrenergic fibers project

back into the brain and innervate the pineal gland, where neural

information is transduced into a hormonal message (Chattoraj, Liu et al.

2009; Falcon, Besseau et al. 2009). Melatonin, an indole amine hormone

secreted rhythmically by the pineal gland (Axelrod, Wurtman et al. 1965),

can induce photoperiodic responses when exogenously administered in a

number of ways (Bartness, Powers et al. 1993; Goldman 2001;

Simonneaux and Ribelayga 2003). Particular aspects of the responses

6

observed following constant release implants, daily injections, or daily

infusions of melatonin led to some early controversy regarding the

significance of various temporal characteristics of melatonin secretion.

Melatonin is normally secreted in a circadian fashion, with an extended

peak occurring at night, and basal secretion during the day (Axelrod,

Wurtman et al. 1965). The duration of this nocturnal „peak‟ varies

inversely with day length in mammalian species, including humans.

Results obtained in sheep and in Siberian hamsters strongly favor the

overriding importance of the duration of the melatonin peak to transduce

photoperiodic information. When pinealectomized hamsters or sheep

were infused with melatonin on a daily basis, the types of responses

elicited depended on the duration, but not on the phase, of the daily

infusion (Carter and Goldman 1983; Bittman and Karsch 1984;

Bartness, Powers et al. 1993). Data obtained from similar studies in

several other mammalian species support the conclusions that the

duration of melatonin is the critical physiological parameter providing

photoperiod information (Badura and Goldman 1992; Saxena and Sinha

2000; Romera, Mohamed et al. 2010).

With the use of 2-[125I] iodomelatonin, melatonin binding sites have

been discovered throughout the periphery and nervous system in

vertebrates (Dubocovich and Markowska 2005). Among mammals, high

melatonin binding is commonly observed in the pars tuberalis (PT) of the

7

pituitary, the suprachiasmatic nuclei (SCN) and dorsalmedial nucleus of

the hypothalamus, preoptic area, and area postrema (Morgan, Barrett et

al. 1994; von Gall, Stehle et al. 2002; Jockers, Maurice et al. 2008;

Zawilska, Skene et al. 2009). Lesions or pharmacological blockade of 2-

[125I] iodomelatonin-binding sites in the mediobasal hypothalamus blocks

the inhibitory effects of endogenous and infused melatonin on

gonadotropin secretion (Maywood and Hastings 1995; Bae, Mangels et al.

1999; Prendergast 2010). Low densities of 2-[125I] iodomelatonin binding

have been reported elsewhere throughout the central nervous system

(CNS) and periphery (Dubocovich and Markowska 2005), and are

reported to have a wide range of functions (reviewed in Dubocovich,

Rivera-Bermudez et al. 2003), including modulation of visual function in

the retina (Dubocovich 1983; Baba, Pozdeyev et al. 2009),

immunomodulation (Haldar and Ahmad 2010), and cerebrovascular

physiology (reviewed in Delagrange, Atkinson et al. 2003).

Two mammalian G protein-coupled melatonin receptors, MT1 and

MT2, have been identified based on their affinity for 2-[125I] iodomelatonin

and classified via molecular cloning techniques (Dubocovich 1995;

Reppert, Godson et al. 1995). Several other receptors in this family have

been characterized (Mel1c: Ebisawa, Karne et al. 1994; GPR50: Reppert,

Weaver et al. 1996), as well as other proteins that bind melatonin (MT3:

Nosjean, Ferro et al. 2000), RZR/RORα: Becker-Andre, Wiesenberg et al.

8

1994). MT1 is the dominantly expressed subtype of melatonin receptor,

and targeted deletion of the MT1 gene in Mus disrupts prolactin release

from the PT by altering clock gene expression, implicating it in

neuroendocrine regulation of reproduction (reviewed in von Gall, Stehle

et al. 2002). Further evidence for MT1 being the putative receptor for

neuroendocrine photoperiodic responses comes from two photoperiodic

species which do not express MT2 receptors: Siberian hamsters (Weaver,

Liu et al. 1996) and sheep (Pelletier, Bodin et al. 2000). Thus, all

photoperiodic responses in these species are mediated via the MT1

receptor (Pelletier, Bodin et al. 2000; Prendergast 2010). MT1 activation

primarily inhibits adenylate cyclase and enhances phosholipase C

activation (Godson and Reppert 1997), but also has other effects through

various signal transduction pathways, depending on tissue (reviewed in

Masana and Dubocovich 2001; von Gall, Stehle et al. 2002; Reiter, Tan

et al. 2010).

At the intracellular level, melatonin, via activation of cell surface G

protein-coupled receptors, interacts with circadian clock genes in target

tissues to transmit the circadian light signal (Morgan, Messager et al.

1999; Hazlerigg and Wagner 2006; Johnston, Tournier et al. 2006;

Shimomura, Lowrey et al. 2010). Mammalian cellular circadian clocks

are tightly regulated by a suite of clock genes that generate a self

sustaining cycle via transcriptional feedback loops with approximately a

9

24 hour period. Cellular circadian rhythms are generated by a molecular

pacemaker involving dimerized CLOCK-BMAL transcription factors that

promote expression of period (PER) and cryptochrome (CRY) proteins,

which in turn feedback to inhibit CLOCK and BMAL activity (Ko and

Takahashi 2006; Takahashi, Hong et al. 2008). Melatonin influences the

rhythmic expression of several clock genes (Lincoln, Messager et al.

2002; Johnston, Tournier et al. 2006), except in the SCN, where

compensatory mechanisms may mask melatonin receptor signaling

(Shimomura, Lowrey et al. 2010). Onset of darkness and onset of light

differentially modulate clock gene expression patterns in the PT:

increasing levels of melatonin induce Cry expression, while decreasing

levels induce Per1 expression (Messager, Ross et al. 1999; Lincoln,

Messager et al. 2002); thus, the molecular circadian clock in extra-SCN

tissues can entrain to the light phase of the external environment using a

hormonal signal. These effects are indeed melatonin dependent, as

pinealectomy or deletion of the MT1 receptor abolishes these rhythms in

the PT (Messager, Garabette et al. 2001; von Gall, Garabette et al. 2002).

3. Photoperiod and energy balance

As noted above, individuals of several photoperiodic species

undergo a suite of adaptive responses when exposed to short day

lengths, putatively to allocate energy resources among energetically

10

expensive processes to survive the harsh days of winter and to maximize

fitness. Because many of these adaptations require significant time to

develop, anticipating the appropriate adaptive response using

photoperiodic information to alter physiology and reproductive state is

critical to maximize survival (and ultimately, fitness) across seasons.

3.1. Somatic responses to photoperiod

The majority of research on somatic responses to photoperiod in

mammals has been conducted in small rodents, which use different

strategies in their responses to short day exposure. Although many

temperate rodents regress their gonads in response to short days (see

discussion below on reproductive responses to photoperiod; Nelson

1987), they differ in their body mass response. Two alternate strategies

are used to survive the energetic bottleneck of winter: 1) gain body mass

via increased adiposity (Campbell, Tabor et al. 1983; Bartness and Wade

1984; Nagy, Gower et al. 1994; Kriegsfeld and Nelson 1996), or 2)

decrease body mass via decreased adiposity and regression of other

tissues (Steinlechner and Heldmaier 1982; Dark, Zucker et al. 1983;

Hoffman 1983; Dark and Zucker 1984; Wade and Bartness 1984; Blank

and Freeman 1991; Nelson, Kita et al. 1992). Increased adiposity equates

to elevated endogenous energy stores for use during times of reduced

energy intake, and decreased body mass would equate to reduced energy

11

requirement to support total body mass; both are energy conserving

strategies (Dark and Zucker 1985).

3.1.2. Neuroendocrine mechanisms

One of the hallmarks of photoperiodic responses to short days in

rodents is the reduction in circulating gonadal steroids due to the

involution of the gonads (see discussion of photoperiodic effects on the

HPG axis below). Although gonadectomy can recapitulate the appropriate

short day responses in body mass and adiposity in many rodents that

use both adaptive strategies (reviewed in Nelson, Denlinger et al. 2010),

further reductions in mass occur when gonadectomized animals are

exposed to short days (Wade and Bartness 1984). Additionally,

gonadectomized animals maintained in short days will become

photorefractory and return to long day body mass in the absence of

gonadal steroids (Hoffman, Davidson et al. 1982). Thus, gonadal steroids

alone are not the only factor contributing to photoperiodic mass

regulation.

In many species circulating leptin, which is synthesized in white

adipose tissue, correlates strongly with body fat content, suggesting that

it is the signal for monitoring adiposity. Leptin binds to receptors (Ob-Rb)

in the arcuate nucleus (ARC), which in turn sends projections of

orexigenic peptides (neuropeptide Y (NPY), agouti-related protein (AGRP)),

12

anorexigenic peptides (pro-opiomelanocortin (POMC)), and cocaine- and

amphetamine-related peptide (CART) to various nuclei in the

hypothalamus to control feeding behaviors potentially based on a

seasonal adiposity “set-point” (reviewed in Morgan and Mercer 2001;

Valassi, Scacchi et al. 2008; Henry, Blache et al. 2010). Although there

are seasonal changes in leptin sensitivity (Tups, Ellis et al. 2004; Krol,

Duncan et al. 2006), individuals are not in negative energy balance

during photoperiod-mediated weight loss (Adam and Mercer 2004).

Precisely how the hypothalamic set point is established for satiety is

unknown (Bartness and Song 2007). However, establishing the set point

may involve converging input on the gut-brain axis of photoperiodic

information (pineal melatonin), leptin signaling from the ARC, and

ascending short-term satiety information from brainstem nuclei (Helwig,

Archer et al. 2009).

Within the mediobasal hypothalamus, triiodothyronine (T3) has

been implicated in regulation of food intake (Kong, Martin et al. 2004).

One of the primary loci of photoperiodic signaling of pineal melatonin to

the neuroendocrine axis is in the mediobasal hypothalamus (see above).

Indeed, melatonin-dependent photoperiodic variation in deiodinase

expression regulates T3 levels in the mediabasal hypothalamus (Revel,

Saboureau et al. 2006; Yasuo, Nakao et al. 2006; Barrett, Ebling et al.

2007). Hypothalamic T3 levels are controlled by expression levels of

13

deiodinase II (DIO2), which converts thyroxine (T4) into the active form of

T3, and deiodinase III (DIO3), which leads to the conversion of T3 and T4

into inactive forms (Hazlerigg 2010). DIO3 is upregulated in short days,

whereas DIO2 is upregulated in long days, resulting in elevated

hypothalamic levels of active T3 during long days (see below). The

majority of research on the contribution of thyroid hormones to

photoperiodism has been in regulation of reproduction (see below); thus,

the role of thyroid hormones in photoperiodic regulation of body mass

has not been fully investigated.

In addition to hormonal and behavioral regulation of body mass,

central control over energy homeostasis and lipid mobilization is

regulated by autonomic outflow. The autonomic nervous system (ANS)

innervates both white adipose tissue and brown adipose tissue

(Heldmaier, Steinlechner et al. 1989; Bartness, Demas et al. 2002;

Bowers, Gettys et al. 2005; Leitner and Bartness 2010). Both branches of

the ANS are dynamically regulated by photoperiod (Weil, Norman et al.

2009), and the sympathetic branch of the ANS plays an important role in

lipid regulation and lipid mobilization. Melatonin receptors (MT1) are

expressed in many distributed forebrain nuclei involved in sympathetic

outflow, and timed infusion of melatonin into these nuclei, mimicking

short day levels for 5 weeks, can elicit short day body mass responses in

Siberian hamsters (Leitner and Bartness 2010).

14

3.1.3. Photoperiod and central energy conservation

The CNS is composed of the most energetically demanding cells in

the body (Ames, Li et al. 1992). Minimizing CNS metabolic demands

during times of restricted energy availability (viz., short days), without

impairing CNS function to the point of impacting long term reproductive

fitness and survival, presumably provides adaptive advantages (Jacobs

1996). Seasonal brain plasticity has been reported for all vertebrate taxa

(Tramontin and Brenowitz 2000), and photoperiodic brain plasticity has

been studied in birds, primarily in two circuits; the song control system

and the hippocampus. In support of this hypothesis of short-day energy

conservation, upon exposure to long days, brain volume and soma size

increases in several nuclei of the song system (reviewed in Jacobs 1996;

Meitzen and Thompson 2008). In white-footed mice (Peromyscus

leucopus), exposure to short days decreases hippocampal volume, with

concurrent impairment in hippocampal function (see learning and

memory below; Chapter 2; Pyter, Reader et al. 2005; Workman, Bowers et

al. 2009; Walton, Chen et al. 2011). Short day reductions in brain mass

have also been reported in meadow voles (Microtus pennsylvanicus)

(Dark, Dark et al. 1987; Galea and McEwen 1999), bank voles (Myodes

glareolus) (Yaskin 2009), and shrews (Sorex araneus L.) (Dehnel 1949;

Yaskin 1994). However, short day increases in hippocampal volume

occur in several species of wild food-caching birds (Sherry and Hoshooley

15

2010). Total brain volume also increases among long-day squirrels

(Sciurus carolinensis) (Lavenex, Steele et al. 2000). Energetic investment

in increased brain volume in short days, in these cases, may impart an

adaptive advantage by increasing spatial memory needed to locate food

caches, which outweighs the increased energetic demands of supporting

the increased neuronal volume. Alternatively, these volume increases

could simply be a result of increased hippocampal usage during caching

behavior (see Lavenex, Steele et al. 2000; Sherry and Hoshooley 2010).

However, the direct contribution of varying seasonal demands in energy

homeostasis to the evolution of these adaptations remains unspecified.

3.2. Photoperiod and reproduction

Maintenance and support of reproductive function in mammals is

dependent upon activation of the hypothalamic-pituitary-gonadal (HPG)

axis. Pulsatile gonadotropin releasing hormone (GnRH) secretion from

the hypothalamus into the hypophyseal portal system stimulates

releases of gonadotropins (luteinizing hormone – LH, follicle-stimulating

hormone – FSH) from the anterior pituitary, which in turn supports

development and maintenance of mature gonads (Levine 2003; Bliss,

Navratil et al. 2010). The HPG axis can be modified at multiple levels to

regulate reproductive function, some of which are influenced by

16

photoperiod and the influence of photoperiod can vary depending on

previous photoperiod exposure.

3.2.1. Short-day breeders

Sheep (Ovis aries), in common with other ungulates and most large

seasonally-breeding mammals, are so-called „short-day breeders‟. Ewes

have relatively long periods of gestation; mating typically occurs in the

autumn and lambs are born and nursed in the spring when food and

other conditions are most conducive for survival. As day lengths decrease

in late summer, the rate of GnRH secretion increases, which eventually

stimulates increased gonadotropin secretion that initiates reproductive

function (Xiong, Karsch et al. 1997). In Suffolk ewes, seasonal

reproductive transitions appear to reflect changes in the responsiveness

of the GnRH neurosecretory system to the negative feedback of estradiol.

In common with other mammals, GnRH neurons in sheep do not express

estrogen receptor alpha (ERα) (Xiong, Karsch et al. 1997). Although ~50%

of GnRH neurons in sheep express estrogen receptor beta (ERβ) (Skinner

and Dufourny 2005), the role of these receptors in estrogen feedback

regulation of GnRH signaling remains unidentified. However, ERα may be

the putative estrogen receptor for feedback regulation of GnRH secretion

as targeted disruption of ERβ in female mice does not impair

reproduction (Lubahn, Moyer et al. 1993; Krege, Hodgin et al. 1998). The

17

ultrastructure and synaptic inputs of GnRH neurons in the preoptic area

of ewes during the breeding season receive more than twice the mean

number of synaptic inputs per unit of plasma membrane than GnRH

neurons in anestrous animals (Xiong, Karsch et al. 1997), thus the

influence of estradiol on GnRH neurosecretory activity may be conveyed

via converging afferents on GnRH neurons from: A) estradiol sensitive

glutamatergic cells in the hypothalamus, BNST, and brainstem (Pompolo,

Pereira et al. 2003), B) BNST neurons receiving input from estradiol

sensitive noradrenergic cells in the brainstem (Pereira, Rawson et al.

2010), and C) dopaminergic input from the A15 nucleus (Chalivoix,

Malpaux et al. 2010). These seasonal alterations in synaptic input are

independent of seasonal fluctuations of steroid concentrations as both

intact and ovariectomized ewes bearing estradiol implants show changes

in synaptic inputs onto GnRH neurons (Xiong, Karsch et al. 1997), but

how photoperiodic information is relayed through these pathways

remains unspecified. After mating season, sheep become photorefractory;

i.e., short days lose their stimulatory effects and mating behavior wanes.

Exposure to the long day lengths of summer is not necessary to re-

establish responsiveness to short days, suggesting the presence of an

underlying circannual cycle of photosensitivity (Robinson and Karsch

1984).

18

3.2.2. Long-day breeders

Syrian, or golden, hamsters (Mesocricetus auratus), Siberian

hamsters (Phodopus sungorus), and white-footed mice (Peromyscus

leucopus) represent the most common rodent models used in laboratory

investigations of photoperiodism. In common with most small mammals,

are so-called „long-day breeders‟. Gestation is relatively brief in these

animals; mating, pregnancy, and lactation occur during the long days of

late spring and early summer. The minimum day length that supports

reproduction is called the „critical day length‟. Critical day length is not a

fixed variable, but differs among populations of animals living at different

altitudes and latitudes (Bradshaw and Holzapfel 2001). Adult male

Syrian hamsters undergo gonadal regression when day lengths fall below

12.5 hours of light (Elliot 1981). However, Siberian hamsters, which live

at higher latitudes than Syrian hamsters, display a critical day length for

reproductive function of 16 hours of light/day (Duncan, Goldman et al.

1985). In addition to latitudinal variation in critical day length,

manipulation of photoperiod history in the lab can also influence critical

day length. Siberian hamsters held in long or short day lengths will show

correct photoperiodic reproductive responses to opposite photoperiods if

placed in intermediate photoperiods that differ by >1.5 hr from the initial

photoperiod (Prendergast, Gorman et al. 2000; Prendergast and Pyter

2009). It should be noted here that most laboratory studies of

19

photoperiodism involve abrupt transfer to different day lengths, which

does not reflect naturalistic conditions. Using simulated natural

photoperiod (SNP) exposure, to mimic gradual incremental changes in

day length, in Siberian hamsters has demonstrated that photoperiod

history is critical for determining critical day length. Critical day length

can be shifted up to 6 hours by manipulating the simulated natural

photoperiod history (Gorman and Zucker 1995). Thus, early observations

that latitude determines critical day length, while correct, should be

interpreted as photoperiod history, which changes with latitude, is

responsible for determining critical day length.

When Syrian hamsters are maintained in day lengths < 12.5 hours

of light, blood concentrations of gonadotropins and sex steroid hormones

decrease, accessory organ mass diminishes to about 10% of the original

size, and reproductive behaviors stop (reviewed in Prendergast, Zucker et

al. 2009). Male hamsters remain reproductively quiescent for

approximately 16-20 weeks, a period of time that roughly corresponds to

the duration of short days experienced in the wild during autumn and

winter. Hamsters are photorefractory during the recrudescent phase; i.e.,

gonadal condition becomes unlinked from photoperiodic inhibition.

Photorefractoriness permits attainment of fully functional gonads in the

spring before environmental photoperiods attain 12.5 hours, the day

length necessary for gonadal maintenance in the autumn. This is an

20

important adaptation that allows burrowing animals (that live in

constant dark conditions) to anticipate spring conditions with the

development of fully functional reproductive systems without long-day

exposure. Once animals become photorefractory exposure to increasing

day lengths initiates reestablishment of photoresponsiveness, but the

process takes ~10 – 15 weeks to complete after initiation (Kauffman,

Freeman et al. 2003; Butler, Turner et al. 2010).

One of the mechanisms by which the nightly duration of melatonin

secretion affects the reproductive system is by altering the steroid

negative-feedback mechanisms in the hypothalamus and pituitary gland

(reviewed in Goldman and Nelson 1993). In long-day and short-day

breeders, reduced secretion of pituitary LH and FSH during the non-

breeding season results from increased sensitivity of the hypothalamic-

pituitary axis to the negative feedback effects of gonadal steroid

hormones (Tamarkin, Hutchison et al. 1976; Legan, Karsch et al. 1977;

Turek 1977). Low concentrations of blood androgens are more effective at

inhibiting post-castration elevations in pituitary gonadotropin secretion

in hamsters and rams, when males are housed in short or long days,

respectively. A return to the lower level of sensitivity returns the animals

to a state of reproductive activity via increased pituitary hormone

secretion. A similar phenomenon has been implicated in puberty, during

which a prepubertal decrease in sensitivity to steroid negative feedback

21

leads to increased secretion of LH and FSH and activation of the

reproductive system (Sisk and Foster 2004).

In addition to changes in the sensitivity of the gonadotropin

secretion system to gonadal steroid hormones, several steroid-

independent mechanisms have also been implicated in the regulation of

seasonal changes in the rate of gonadotropin secretion. In long days,

female hamsters display ~8-10-fold increase in baseline serum LH

concentrations following ovariectomy, and LH concentrations can be

returned to baseline by administration of estrogens. After several weeks

of short-day exposure, female hamsters become anovulatory and serum

LH concentrations are very low during most of the 24 h cycle; however,

the anovulatory females display daily LH surges during the afternoon.

This pattern of LH secretion is steroid-independent, as it continues

following ovariectomy or adrenalectomy (Bittman and Zucker 1977). A

steroid-independent, but melatonin-dependent, photoperiodic LH

secretion pattern difference is also found in ewes. In ovariectomized

ewes, LH pulse frequency is suppressed in long days and recovers to

normal breeding season rate in short days. This difference is abolished

by pinealectomy, but recovered with melatonin infusions mimicking

short- and long-day photoperiods (Bittman, Kaynard et al. 1985).

Although it is currently unknown whether steroid-dependent or -

independent mechanisms are more common in photoperiodic mammals,

22

seasonal variation in circulating and pituitary concentrations of

gonadotropins has been reported for several mammalian species (Karsch,

Legan et al. 1980; Bittman, Kaynard et al. 1985; Bittman, Hegarty et al.

1990; Meredith, Turek et al. 1998).

Recent research in birds has identified two members of the

RFamide family of peptides that regulate the HPG axis, kisspeptin and

gonadotropin inhibitory hormone (GnIH), which may have similar roles in

mammals (reviewed in Greives, Kriegsfeld et al. 2008; Tsutsui, Bentley et

al. 2010). These peptides act in an antagonistic fashion on GnRH

neurons; kisspeptin stimulates GnRH release (Smith and Clarke 2007)

and GnIH inhibits GnRH release (Tsutsui, Saigoh et al. 2000). Although

the role of these peptides in photoperiodic control of the HPG axis has

not been completely specified, both cell types are responsive to

melatonin. In cultured mammalian hypothalamic neurons, expression of

kisspeptin is decreased and RFamide-related peptide (RFRP, the

mammalian homologue of GnIH) expression is increased by melatonin

(Gingerich, Wang et al. 2009). Exogenous and endogenous melatonin

drives GnIH release in birds (Ubuka, Bentley et al. 2005; Chowdhury,

Yamamoto et al. 2010), and short photoperiod increases RFRP

expression in hamsters (Paul, Pyter et al. 2009; Mason, Duffy et al.

2010). Consistent with the effects of melatonin on kisspeptin in rats

(Gingerich, Wang et al. 2009), kisspeptin immunoreactivity in the

23

anteroventral periventricular nucleus of the thalamus of Siberian and

Syrian hamsters is reduced in short days (Greives, Humber et al. 2008;

Simonneaux, Ansel et al. 2009). However, in the arcuate nucleus,

kisspeptin immunoreactivity is elevated in short-day Siberian hamsters

(Greives, Mason et al. 2007; Mason, Greives et al. 2007), but no effect of

photoperiod was reported on gene expression (Paul, Pyter et al. 2009).

As noted above, thyroid hormones have also been implicated in

photoperiodic regulation of the HPG axis in both mammals and birds

(Yoshimura, Yasuo et al. 2003; Freeman, Teubner et al. 2007; Nakao,

Ono et al. 2008). Hypothalamic T3 levels are higher in long days and

hypothalamic implants of T3 can block short day photoperiodic responses

in Siberian hamsters (Barrett, Ebling et al. 2007). Exposure to long days

increases thyroid stimulating hormone (TSH) release from the PT, and

the increased TSH release from the PT acts in a paracrine manner to

induce DIO2 expression in the tanycytes lining the third ventricle in the

mediobasal hypothalamus, resulting in elevated T3 availability (Hanon,

Routledge et al. 2010; Yasuo, Yoshimura et al. 2010). Reciprocal

expression of DIO2 and DIO3 in the mediobasal hypothalamus thereby

regulates thyroid hormone activity in the hypothalamus, and expression

of these genes is regulated by melatonin (see discussion above;

Watanabe, Yasuo et al. 2004; Revel, Saboureau et al. 2006; Yasuo,

Yoshimura et al. 2007; Ono, Hoshino et al. 2008; Yasuo, Yoshimura et

24

al. 2010). Conservation of this mechanism suggests that it may represent

an ancestral mechanism of seasonal timing driven by photoperiod

(Hazlerigg and Loudon 2008; Hazlerigg 2010).

4. Photoperiod, affect, and non-reproductive behaviors

Although many seasonal behaviors in photoperiodic mammals are

specifically associated with reproduction (see above), there are several

behavioral traits, such as affective responses and aggression, that are

modulated by photoperiod independent of gonadal steroids.

Immunological, hormonal, and neural factors, all of which are modulated

by photoperiod, converge to modify behavioral output. Affective disorders

are generally considered maladaptive; however, photoperiodic changes in

affect may represent an adaptive strategy to conserve energy during the

energetic bottlenecks encountered during the short days of winter (e.g.,

Nesse and Williams 1994; Nesse 2000; Wehr, Duncan et al. 2001).

Additionally, conservation of energy during the short days of winter may

also be the putative mechanism driving photoperiodic modulation of

aggression (see below). Not only are these traits influenced by

photoperiod in mammals that respond reproductively to photoperiod,

behavioral responses to photoperiod are also observed in mammals that

do not respond reproductively to photoperiod, which has implications for

developing animal models of human pathologies with a seasonal

25

component, such as seasonal affective disorders and depression

(Workman and Nelson 2010).

4.1. Affective responses

Observable and abnormal emotional states, such as excessive

elation or sadness, define affective disorders (Rubin, Dinan et al. 2002).

Behavioral responses which comprise human affective disorders, such as

altered food intake and reduced motivation (depressive-like), and

increased anxiety and fearfulness (anxiety-like), can also be observed in

rodents in a laboratory setting. Until recently, there was little evidence

that photoperiod itself could alter affective behaviors, even though it has

been known for decades that there is a correlation between season and

affective disorders in humans (Frangos, Athanassenas et al. 1980; Parker

and Walter 1982; Lewy, Sack et al. 1988). Recent research has now

identified several rodent species that display changes in affective

behaviors that are induced by changes in photoperiod.

In reproductively photoperiodic rodents, exposure to short days

induces changes in affective behaviors that are independent of changes

in reproductive hormones. Exposure to short days increases anxiety-like

and depressive-like responses in collared lemmings (Weil, Bowers et al.

2007) and Siberian hamsters (Prendergast and Nelson 2005; Pyter and

Nelson 2006). These photoperiodic behavioral changes develop early

26

during short day exposure (Prendergast and Nelson 2005), and can

persist after maximal gonadal regression (Pyter and Nelson 2006; Weil,

Bowers et al. 2007), supporting the idea that the influence of photoperiod

on affect can be independent of circulating gonadal steroids. Further

support of this idea comes from a growing body of evidence showing that

animals that do not respond reproductively to photoperiod also display

photoperiodic responses (Nelson 1990; Sumova, Bendova et al. 2004).

Short-day exacerbation of depressive-like and anxiety-like behaviors have

been reported in both nocturnal rodents (rats (Molina-Hernandez and

Tellez-Alcantara 2000; Benabid, Mesfioui et al. 2008; Prendergast and

Kay 2008)), and diurnal rodents (e.g., Nile grass rats (Ashkenazy-

Frolinger, Kronfeld-Schor et al. 2010), and sand rats (Ashkenazy, Einat

et al. 2009; Ashkenazy, Einat et al. 2009)). These affective responses to

short days are unambiguously linked directly to pineal melatonin

secretion duration (Ashkenazy, Einat et al. 2009), which may model the

extended duration of melatonin secretion observed in humans with

seasonal affective disorder (Wehr, Duncan et al. 2001).

4.2. Non-reproductive social behaviors

It is presumably adaptive for organisms to differentially display

complex social behaviors such as aggression and non-reproductive

affiliation at different times of the year. Aggressive behaviors occur

27

because of competition for limited resources, whereas affiliative

behaviors occur when long term survival probability is increased by

sharing resources with conspecifics to liberate physiological resources for

other processes. Both aggression and affiliation display seasonal

variation, influenced by photoperiod, in reproductive and non-

reproductive contexts.

Rodents, such as wood rats (Neotoma fuscipes) (Caldwell,

Glickman et al. 1984), rat-like hamsters (Cricetus triton) (Zhang, Zhang et

al. 2001), Siberian hamsters (Jasnow, Huhman et al. 2000; Demas,

Polacek et al. 2004; Scotti, Place et al. 2007), California mice

(Peromyscus californicus) (Silva, Fry et al. 2010), and Syrian hamsters

(Mesocricetus auratus) (Garrett and Campbell 1980; Fleming, Phillips et

al. 1988; Badura and Nunez 1989; Jasnow, Huhman et al. 2002),

increase aggression in short days. Elevated aggression in short days may

be adaptive to protect limited resources or territories during the winter

non-breeding season (Tinbergen 1957; Schwabl 1992; Demas, Polacek et

al. 2004). In males, testosterone concentrations are positively correlated

with aggression during the breeding season (Wingfield 2005; Nelson

2006; Landys, Goymann et al. 2010). Elevated aggression in short days,

however, is typically independent of testosterone (Garrett and Campbell

1980; Caldwell, Glickman et al. 1984; Jasnow, Huhman et al. 2000;

Zhang, Zhang et al. 2001; Jasnow, Huhman et al. 2002; Demas, Polacek

28

et al. 2004). Although male aggression has been studied extensively,

female aggression has received far less scientific scrutiny (Nelson and

Trainor 2007). Nonetheless, elevated non-reproductive aggression in

females also varies in response to photoperiod in rat-like hamsters

(Zhang, Zhang et al. 2001), Siberian hamsters (Scotti, Place et al. 2007),

California mice (Silva, Fry et al. 2010), and Syrian hamsters (Fleming,

Phillips et al. 1988).

The precise mechanisms underlying short-day increases in

aggression in both sexes remain unidentified; however, recent studies

have identified some potential factors. Nitric oxide, a gaseous

neurotransmitter, has been implicated in male aggression (Nelson,

Demas et al. 1995). Short photoperiod reduces nNOS expression, the

enzyme producing nitric oxide in neurons, in the amygdala of male

Siberian hamsters, and nNOS expression is negatively correlated with

aggression in short days (Wen, Hotchkiss et al. 2004). Melatonin, a

physiological signal of photoperiod, may directly modulate aggression.

Laboratory house mice (Mus musculus) display increased territorial

aggression when given a short day like regimen of melatonin (Paterson

and Vickers 1981). Short-day alterations in aggression have been

directly linked to pineal melatonin in Syrian hamsters (Fleming, Phillips

et al. 1988; Badura and Nunez 1989), Siberian hamsters (Scotti, Schmidt

et al. 2009), and fat sand rats (Ashkenazy, Einat et al. 2009). Although

29

testosterone concentrations are at their nadir in short days (see above), it

is possible that testosterone synthesized and converted to estrogen de

novo in the brain can influence short-day aggression (Soma 2006).

Photoperiod and estrogen receptor distribution also interact to modulate

short-day aggressive behaviors (Trainor, Lin et al. 2007; Trainor,

Rowland et al. 2007), but how activation of this pathway occurs in short

days remains unspecified. Melatonin can also interact with the HPA axis,

which has been implicated in the modulation of aggression (Haller and

Kruck 2003). Exposure to short days alters HPA axis reactivity (Pyter,

Adelson et al. 2007) and the HPA axis interacts with melatonin to

modulate aggression (Paterson and Vickers 1981; Demas, Polacek et al.

2004).

In contrast to increased aggression, some rodent species display

reduced aggression in short days (Andrews and Belknap 1993; Beery,

Loo et al. 2008; Ashkenazy, Einat et al. 2009). To reduce

thermoregulatory demands during the harsh days of winter, several

rodent species form communal nests (Madison, FitzGerald et al. 1984;

Wolff and Durr 1986; Andrews, Phillips et al. 1987; Andrews and

Belknap 1993). Conspecific tolerance in these communal nests

necessarily requires reduced territorial aggression and increased

affiliative behavior. The oxytocin (OT) and vasopressin (AVP) family of

neuropeptides has been implicated in the control of diverse social

30

behaviors, including affiliation and aggression (reviewed in Goodson

2008; Veenema and Neumann 2008). Generally in mammals, increased

oxytocin binding is associated with increased affiliative behavior (Lee,

Macbeth et al. 2009) and increased vasopressin binding is associated

with aggressive behavior (Bester-Meredith, Young et al. 1999; Ferris

2005); thus, this system is poised to be a target for photoperiodic

modulation of these behaviors. Indeed, pineal melatonin can modulate

neurohypophysial AVP and OT neuronal activity (reviewed in Juszczak

2001). Photoperiodic differences in oxytocin receptor distribution

underlie increases in affiliation in short-day female meadow voles

(Parker, Phillips et al. 2001; Beery and Zucker 2010), but the role of

vasopressin in mediating photoperiodic changes in aggression are less

clear (see Albers, Rowland et al. 1991; Bittman, Jetton et al. 1996;

Caldwell and Albers 2003). Thus, although photoperiod and pineal

melatonin modulate non-reproductive behaviors, the systems are

distributed and the effects are species-specific.

4.3. Learning and memory

In addition to the social and affective behavioral changes described

above, photoperiod can modulate learning and memory, potentially a

functional result of altered hippocampal morphology (see above). In

rodents, seasonal variation in spatial learning and memory has been

31

reported in fox squirrels (Sciurus niger) (Waisman and Jacobs 2008), deer

mice (P. maniculatus) (Galea, Kavaliers et al. 1994; Galea, Kavaliers et al.

1996), and white-footed mice (P. leucopus) (Pyter, Reader et al. 2005;

Pyter, Adelson et al. 2007; Workman, Bowers et al. 2009; Walton, Chen

et al. 2011). In a naturalistic setting, seasonal variation in hippocampal

and brain volume is positively correlated with seasonal spatial navigation

and spatial memory requirements (Lavenex, Steele et al. 2000; Yaskin

2009). Indeed, in deer mice, seasonal territory maintenance life history

positively correlates with photoperiodic variation in spatial learning and

memory, leading to sex and population based differences in spatial

learning and memory performance across seasons (Galea, Kavaliers et al.

1994; Galea, Kavaliers et al. 1996). When male white footed mice, which

have seasonal variation in territory size, are brought into the lab where

there are no territories to maintain, a similar decrease of hippocampal

volume observed during winter in the wild is observed in laboratory short

days (Pyter, Reader et al. 2005), indicating an underlying photoperiodic

mechanism in brain morphological plasticity. Supporting this conjecture,

exposure to short days alone impairs spatial learning and memory, alters

dendritic morphology of hippocampal neurons (Pyter, Reader et al. 2005),

and impairs long term potentiation (Walton, Chen et al. 2011), the

putative mechanism for memory formation in the brain (Bliss and

Collingridge 1993).

32

5. Conclusions

The putative mechanism central to the evolution of photoperiodism

in animals is the distribution of energetically challenging activities across

the year to optimize reproductive fitness while balancing the energetic

tradeoffs necessary for seasonally-appropriate survival strategies. For

long-day breeding mammals, the costs of territorial behavior, pregnancy,

and lactation constrain reproduction to coincide with the onset of long

days when environmental conditions are most conducive to survival. For

short-day breeding mammals, lactation appears to be the major energetic

cost associated with reproduction; thus, breeding has evolved to occur in

fall. Gestation continues throughout the winter and birth and lactation

occur in the lengthening days of spring, when food and weather

conditions are most conducive to survival. Thus, for both long- and

short-day breeders, the energetic costs of supporting reproduction and

lactation outweigh the benefits of the investment during times of low

reproductive success (viz., winter). Being able to accurately predict future

events to allocate energy among competing physiological systems to

maximize fitness and survival necessarily requires endogenous

mechanisms to permit physiological anticipation of annual conditions.

Day length provides the most noise free environmental signal for

organisms to monitor and accurately predict time of the year.

33

Day length is an unambiguous environmental signal. This signal

can be used in the lab to explore the mechanisms by which the

environment and genes interact to impart phenotype. Photoperiod

manipulation is advantageous over other accepted methods of studying

gene – environment interactions, where differential responses to

“environment” (which entail an unlimited number of variables) are

identified, and then comparative genomic methods are used to identify

underlying genetic susceptibility factors (see http://www.niehs.nih.gov/).

This unique approach allows isolation of a single environmental factor

(duration of light) that can be controlled in a laboratory setting, and

investigation of how this one factor interacts with the genome to affect

multiple behavioral, cognitive, and physiological processes across diverse

species. Using non-traditional animal models to investigate how

photoperiodic modulation of physiological and behavioral systems

interacts to impart phenotype may contribute to our understanding of

clinical disorders and other pathologies in a translational setting.

In mammals, melatonin is the hormonal signal of day length.

Although acute administration of melatonin can directly impact aspects

of physiology and immune function, altering the duration of pineal

melatonin secretion drives enduring changes in many physiological

systems, including the HPA axis, the HPG axis, the brain-gut axis, the

autonomic nervous system, and the immune system. This plastic and

http://www.niehs.nih.gov/

34

complex interplay among these systems is intimately regulated at all

levels by melatonin. Thus, melatonin is the fulcrum mediating

redistribution of energetic investment among physiological processes to

maximize fitness and survival. Whereas many advances have been made

in the past several decades, identification of the diversity of systems and

multimodal sites of action of melatonin in photoperiodic animals is an

ongoing process. Much of the progress in biology during this time has

focused on molecular biology, it is now important to use modern

molecular approaches and precise environmental factors to study the

development of phenotype. Thus, continued research in the mechanisms

of how light interacts with genes via melatonin hormonal signaling can

provide novel and important insights into conserved molecular

mechanistic themes underlying the relationship between phenotype and

genotype.

35

Figure 1.1 Photic input is transduced by melanopsin-expressing retinal

ganglion cells via the retinohypothalamic tract (RHT) to the suprachiasmatic

nuclei (SCN). Output from the SCN is relayed through the paraventricular

nucleus of the hypothalamus (PVN), to the intermediolateral cells of the upper

spinal cord (IML), then to the superior cervical ganglion (SCG), and

postganglionic innervation of the pineal gland regulates melatonin synthesis.

Pineal melatonin carries photoperiodic information to distributed systems

throughout the body, where it acts both directly and indirectly to regulate

endocrine, neuronal, immunological, and behavioral processes. 1

36

Figure 1.2 Hypothetical illustration of photoperiodic differences in allocation of

energy among competing processes. During the long days of summer, animals

are in positive energy balance due to increased availability of resources (food).

Thus, energy is available to support somatic growth and reproduction. During

the short days of winter, energy balance tips to negative. Reproduction and

somatic growth processes are curtailed, and available energy is allocated

differentially among the immune system, thermoregulation, and cellular

metabolism. Elevated energetic constraints and thermoregulatory demands in

the short days of winter have led to the evolution of torpor and hibernation to

promote survival by further reduction in the energetic demands of the

remaining active processes. 2

37

Chapter 2: Photoperiod, Hippocampal Long-Term Potentiation, and

Spatial Learning and Memory

1. Introduction

One important goal of neuroscience is to understand the

mechanisms underlying brain plasticity. Toward that end, substantial

research has been conducted using in vivo and ex vivo chemical and

genetic manipulations of standard laboratory rodent research models

(Fisher 1997; Wells and Carter 2001; Smale, Heideman et al. 2005).

However, as discussed in Chapter 1, natural selection has produced

many vertebrate species that display plasticity in physiology and

behavior across seasons as adaptive mechanisms for survival. Seasonal

changes in physiology and behavior, driven by the annual cycle of

changing day length (photoperiod), presumably evolved to allocate

resources among competing energetically expensive physiological

processes in individuals living outside of tropical latitudes (Nelson,

Denlinger et al. 2010). Among these energy-coping tradeoffs are

morphological and functional changes in the brain which have been

studied extensively in non-mammalian vertebrates such as reptiles

(Delgado-Gonzalez, Alonso-Fuentes et al. 2008), fish (Zhang, Xiong et al.

38

2009; Walton, Waxman et al. 2010), and birds (Nottebohm 2004; Ball

and Balthazart 2010). Most photoperiodic brain plasticity studies have

been conducted in birds, focusing primarily on brain regions comprising

the song control system (Meitzen and Thompson 2008; Ball and

Balthazart 2010), and the hippocampus (Sherry and Hoshooley 2010).

Compared to birds, few mammalian models of photoperiodic brain

plasticity exist (Hofman and Swaab 2002). Studies of naturally-

occurring examples of brain plasticity may provide novel insights into the

regulatory mechanisms underlying such processes.

White-footed mice (Peromyscus leucopus), are photoperiodic

rodents indigenous to the eastern and southern United States and

eastern Mexico (King 1968). This species has evolved a suite of adaptive

responses to survive the harsh conditions of winter, including reducing

the size of the reproductive system, as well as the brain, that are induced

by exposure to short days (Pyter, Reader et al. 2005). Changes in brain

morphology in P. leucopus following exposure to short day lengths are

accompanied by impaired spatial learning and memory in the Morris

water maze (Pyter, Reader et al. 2005; Pyter, Trainor et al. 2006;

Workman, Bowers et al. 2009).

Spatial learning and memory is correlated with hippocampal long-

term potentiation (LTP; (Shapiro and Eichenbaum 1999; Lynch 2004)),

such that LTP is induced in the hippocampus during learning (Whitlock,

39

Heynen et al. 2006), inhibition of LTP impairs performance in spatial

learning tasks (Morris, Anderson et al. 1986), and LTP maintenance in

the hippocampus is required for retention of spatial memory (Pastalkova,

Serrano et al. 2006). LTP, a measure of increased efficiency of synaptic

transmission following high-frequency stimulation (Bliss and Gardner-

Medwin 1973), is the strongest candidate for a synaptic mechanism by

which new memories are formed and stored in the brain (Bliss and

Collingridge 1993; Malenka 2003; Lynch 2004). Although a complete

understanding of how LTP translates into memory storage remains

unspecified, to better understand the mechanisms underlying the short-

day impairment of spatial learning and memory, we examined the role of

photoperiod on in vitro hippocampal LTP in P. leucopus. Additionally, to

confirm and extend previous findings on spatial learning and memory in

P. leucopus using the Morris water maze, I examined the role of

photoperiod on spatial learning and memory in the Barnes maze, a dry-

land, and more ecologically-relevant, alternative to the Morris water

maze.

40

2. Experimental Procedures

2.1. Animals

Thirty four adult male Peromyscus leucopus, from our breeding

colony at the Ohio State University, were singly housed upon weaning

and held in long days (LD, 16L:8D) until sexual maturity (>60 days).

Upon reaching adulthood, mice were randomly assigned to either LD (n =

15) or short days (SD, 8L:16D; n = 19). Mice were maintained in

photoperiod for 10 weeks to establish photoperiod-induced changes prior

to testing (Pyter, Reader et al. 2005; Workman, Bowers et al. 2009).

Eighteen mice were used for LTP analysis (LD n = 9; SD n = 9) and the

remainder were used for behavioral testing in the Barnes maze and

assessment of reproductive tissue mass (LD n = 6; SD n = 10). Two mice

that failed to regress their gonads in response to SD exposure were

excluded from Barnes maze and reproductive tissue mass analysis.

Mice were housed in standard polycarbonate cages (32x18x14 cm),

maintained at constant temperature (21 ± 4°C) and relative humidity (50

± 5%), provided ad libitum access to filtered tap water and food (Harlan

Teklad 8640, Indianapolis, IN, USA), and received care from the Ohio

State University Laboratory Animal Resource staff for the duration of the

study. All procedures were approved by the Ohio State University

Institutional Animal Care and Use Committee and comply with

41

guidelines established by the National Institutes of Health published in

Guide for the Care and Use of Laboratory Animals (Institute of Laboratory

Animal Resources, U.S. 1996).

2.2. Long-term potentiation

Late during the light phase, to coincide with the time of behavioral

testing (see below), transverse hippocampal slices (400 µm) were

prepared from male Peromyscus that had been exposed to LD (n = 9) and

SD (n = 9). Slices were maintained at 28°C in an incubation chamber

bubbled with mixture of 95% O2 and 5% CO2. Following 90 min

incubation, one slice was then transferred to a recording chamber affixed

to a microscope (model E600FN, Nikon) with a custom stage (Syskiyou

Instruments, Grants Pass, OR) with continuous perfusion (2 ml/min) of

oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM) 124

NaCl, 4 MgSO4, 4 KCl, 1.0 Na2HPO4, 4 CaCl2, 26 NaHCO3, 10 D-

glucose. A bipolar stimulating electrode (67 µm, NiCr) was placed in the

stratum radiatum to stimulate the Schaffer collateral–CA1 pathway. Field

excitatory post-synaptic potentials (fEPSPs) were recorded (model 2400,

AM Systems) with a glass electrode (1-2 MΩ) filled with aCSF.

Input/output curves were established by recording fEPSPs in response to

different stimulating intensity (from 20 to 100 µA). Baseline synaptic

responses were recorded with a test stimulus delivered at 0.033 Hz (0.1

42

ms pulse duration). The stimulus strength was adjusted so that it gave

rise to fEPSPs with slope values between 30-40% of the maximal

response recorded during the input/output measurements (Selcher et

al., 2003; Nguyen, 2006). Following a 30-min recording of stable

responses (less than 10% variation), two trains of tetanus stimuli (100 Hz

for 1 sec, 5 min apart) were delivered to induce long-term potentiation

(LTP). The mean percent change of fEPSPs slope was calculated from one

slice from each animal. The slope calculations for each animal were

normalized to the mean of the control responses recorded during the 30

min prior to tetanus stimulation.

2.3. Barnes maze

The Barnes maze, (122 cm diameter) with 18 escape holes (9.5 cm)

placed every 20° around the perimeter (ENV-563-R, MedAssociates, St.

Albans, VT, USA), was completely surrounded with a 60 cm high white

polycarbonate barrier to prevent escape. The blind escape holes were

blocked by black panels, and the target escape hole was visually the

same as the blind holes, but contained a black escape box (38.7 x 12.1 x

14.2 cm). Distinct visual cues (black 2 dimensional geometric shapes, 20

– 25 cm) were attached to the upper edge of the surround at the 4

compass points and visual cues distal to the maze were present along the

walls of the room. Light intensity measured at the maze surface was

43

1200 lux, which was approximately twice the average intensity of

illumination measured 100 cm from the floor in the vivarium housing

rooms (LD=599 lux, SD=600 lux).

To avoid disruption of photic circadian cues, all testing was

performed late in the light phase and animals were returned to their

vivarium rooms prior to the onset of darkness. Testing consisted of 5

days of acquisition training followed by a single probe trial 24 h after the

last training trial. Each acquisition day consisted of one session/animal,

3 trials per session, with an inter-trial interval of 5 min. For acquisition

training, all mice were brought into the testing room and allowed to

acclimate for 30 min before the start of testing. Mice were moved from

their home cage to the testing arena in an opaque plastic beaker covered

with a small net to avoid direct handling. Each trial consisted of carefully

placing the mouse in the center of the maze from the opaque plastic

beaker. Each mouse was allowed to search for the escape box for 120 s.

If the mouse had not found the escape box by 120 s, it was gently guided

to it using a small net. The mouse was then allowed to remain in the

escape box for 60 s, and then returned to its home cage for a minimum

of 45 s. To minimize olfactory cues, the surface of the maze was cleaned

with 70% EtOH at the conclusion of testing of each mouse, and each day

the maze was rotated 90° counter clockwise, with the escape box location

and location of visual cues remaining constant throughout testing. The

44

probe trial consisted of a single 90 s trial with the former escape box

removed and replaced with a blind box. Twenty-four hours after the

conclusion of behavioral testing, animals were killed and reproductive

tissues were collected and weighed.

All behavior was recorded and scored using The Observer software

(XT 8.0; Noldus, Leesburg, VA, USA) by an observer uninformed as to the

treatment of the mice. For training trials, latency to escape and number

of errors were recorded. An error was defined as an investigation of a

blind escape hole where the entire head of the mouse broke the plane of

the edge of the escape hole. For the probe trial, latency to escape hole,

number of errors, and time in quadrant of escape hole were measured.

Learning criterion was defined as making an average of ≤5 errors for 3

trials in one day (Bredy, Lee et al. 2004).

2.4. Statistical Analyses

Repeated measures ANOVAs were used to compare Barnes maze

performance across days and LTP across time between LD and SD

groups. Within days, a priori comparisons of photoperiod were conducted

using two-tailed Student‟s t-tests. Two-tailed Student‟s t-tests were also

used for other comparisons of behavioral and reproductive tissue mass

data between LD and SD groups. All comparisons were considered

statistically significant when p < 0.05.

45

3. Results

3.1. Short days reduce body and reproductive tissue mass.

Exposure to SD reduced body mass (t(12) = 3.142, p < 0.05; Figure

2.1A), mass of paired testes (t(12) = 9.620, p < 0.001), epididymides (t(12) =

6.080, p < 0.001), and seminal vesicles (t(12) = 8.460, p < 0.001; Figure

2.1B).

3.2. Short days impair Barnes maze performance.

Compared to LD mice, exposure to SD increased errors in finding

the escape hole across days during acquisition training (F(1,7) = 6.775, p <

0.05; Figure 2.2A). There was no effect of photoperiod on latency to

escape the Barnes maze across days (F(1,7) = 0.230, p > 0.05; Figure

2.2B). However, both groups of mice displayed learning as the latency to

escape the Barnes maze decreased over days of training (SD: F(1,4)=

5.231, p < 0.05; LD F(1,4)= 10.996, p < 0.05; Figure 2.2B). SD exposure

decreased the time spent in the quadrant of the former escape hole

during the probe trial (t(12) = 2.774, p <0.05), so that the SD mice spent

no more time in the target quadrant than expected by chance (25%,

Figure 2.2C), without significantly increasing total errors during the

probe trial (t(12) = -1.727, p = 0.11; Figure 2.2D).

46

3.3. Short days impair LTP.

Two trains of tetanic stimuli induced long-term potentiation of

fEPSP slope in the Schaffer Colleteral-CA1 pathway of hippocampal slices

60 minutes post-tetanus in LD (n = 9, 172.4 ± 10.6%). LTP was also

induced at a significantly decreased level in SD hippocampal slices 60

min post-tetanus (n = 9, 136.2 ± 7.0%; Figure 2.3). Compared to LD

exposed mice, SD exposure decreased the level of LTP induced at all time

points after the paired tetanus stimuli until the end of the experiment

(F(1,15)= 6.682, p < 0.05; Figure 2.3).

4. Discussion

The present study establishes that short days decrease the

amplitude of LTP in the CA1 region of the hippocampus in vitro and

impair spatial learning and memory in the Barnes maze. The observed

behavioral impairment in short days confirms and extends the previous

reports that short days impair spatial learning and memory in the Morris

water maze in male P. leucopus (Pyter, Reader et al. 2005). Additionally,

the decrease in LTP in SD exposed male P. leucopus provides a functional

correlate of the short day induced reduction in hippocampal volume and

dendritic spine density (Pyter, Reader et al. 2005). It is well established

that LTP is associated with spatial learning and memory, and thus our

data reveal that LTP can be modified by day length.

47

Day length is encoded physiologically by the nightly duration of

pineal melatonin secretion so that short days are encoded by a relatively

long duration of elevated melatonin secretion, whereas long days are

encoded by a relatively short duration of sustained melatonin secretion

(Carter and Goldman 1983; Bittman and Karsch 1984; reviewed in:

Bartness, Powers et al. 1993; Reiter 1993; Prendergast, Nelson et al.

2009). Although the precise role of melatonin on hippocampal function

remains obscure, several studies have indicated that pharmacological

treatment with melatonin blocks LTP in the CA1 region of the

hippocampus (Feng, Zhang et al. 2002; Wang, Suthana et al. 2005;

Ozcan, Yilmaz et al. 2006; Talaei, Sheibani et al. 2010), as well as

impairs spatial learning and memory performance (Collins and Davies

1997; Feng, Zhang et al. 2002; Cao, Wang et al. 2009). Thus, our data

appear to be consistent with the attenuating effects of melatonin on LTP

and spatial learning and memory, i.e., SD corresponds to extended

melatonin exposure. However, one critical difference between the present

study and previous studies is the assessment of naturalistic melatonin

rhythms (current study) compared to the direct effects of exogenous

melatonin on LTP and learning and memory. Although melatonin was not

assayed in the current study, it is the duration of pineal melatonin

excretion, but not absolute melatonin concentration at a specific time

point, that is the critical signal transducer underlying the short-day

48

photoperiodic responses in P. leucopus (Dowell and Lynch 1987; Carlson,

Zimmermann et al. 1989). Inbred laboratory rodents have been selected

to be nonresponsive to photoperiod and many strains have blunted or

absent pineal melatonin rhythms (Ebihara, Marks et al. 1986; Goto,

Oshima et al. 1989; Stehle, von Gall et al. 2002; Simonneaux, Sinitskaya

et al. 2006). Thus, results from inbred laboratory strains of mice and rats

should be interpreted with caution and studies using naturally selected

species can produce reliable and valid data on this issue.

Melatonin may also have indirect effects on LTP as demonstrated

in the current study. Hippocampal slices were prepared and tested

during the light phase when pineal melatonin production is at its nadir,

and thus the photoperiodic differences reported here persist in the

absence of circulating or exogenously administered melatonin.

Furthermore, it has been demonstrated that circadian rhythms in CA1

LTP and neural excitability exist independent of melatonin (Chaudhury,

Wang et al. 2005). Thus, it is possible that the effects of photoperiod we

report here may not be melatonin-dependent, but could be a result of

altered circadian rhythms induced by changes in photoperiod. This

hypothesis will be tested in Chapter 3.

Underlying the photoperiodic reduction of brain mass and function

in this species are reductions in the mass of reproductive tissues and

testosterone (current study; Pyter, Neigh et al. 2005; Pyter, Reader et al.

49

2005; Pyter, Trainor et al. 2006; Workman, Bowers et al. 2009).

Testosterone impairs CA1 LTP in rats (Harley, Malsbury et al. 2000) and

alters hippocampal synaptic plasticity in Mus (Sakata, Tokue et al. 2000).

Although species differences among rodents in the effects of testosterone

on hippocampal function and LTP remain unspecified, the present study

argues against testosterone impairing hippocampal LTP in white-footed

mice, as the SD mice (low testosterone) reduced LTP. SD deficits in

spatial learning and memory can be rescued with testosterone, however

removal of testosterone from LD mice did not impair spatial learning and

memory, and neither testosterone nor photoperiod altered androgen

receptor (AR) expression in the hippocampus (Pyter, Trainor et al. 2006),

although brains of naïve mice, and not those tested behaviorally, were

assessed for AR expression in this study. Taken together, interpretation

of these results suggests that the mechanism underlying photoperiodic

behavioral and physiological plasticity in the hippocampus of white-

footed mice may be independent of gonadal steroids, however we cannot

rule out this possibility due to potential de novo synthesis of steroids in

the brain (for review see Schmidt, Pradhan et al. 2008). The role of

gonadal steroids will be further explored in Chapter 4.

Induction of Schaffer collateral–CA1 LTP is NMDA receptor

dependent and is based upon cooperativity of AMPA and NMDA ion

channels (Bliss and Collingridge 1993). AMPA channels are responsible

50

for early LTP (<20 min), while late-phase LTP is NMDA and AMPA

channel dependent (Lisman and Raghavachari 2006). Lower recruitment

and/or availability of silent NMDA synapses or partially silent

NMDA/AMPA synapses in SD may underlie the photoperiodic differences

in LTP (Poncer 2003; Lisman and Raghavachari 2006). Additionally,

AMPA-mediated transmission can be potentiated via multiple pathways

by the activated alpha subunit of CaM kinase II (Lisman and

Raghavachari 2006), and melatonin impairs calmodulin and CaMKII

activity (Fukunaga, Horikawa et al. 2002; Soto-Vega, Meza et al. 2004),

which is consistent with our current findings that both early- and late-

phase LTP are equally impaired in mice which had a longer duration of

melatonin exposure (Figure 2.3). Thus, we speculate that photoperiodic

differences in Ca2+ regulation, silent synapses, or expression of AMPA

and NMDA receptors may underlie photoperiodic differences in LTP.

However, the contribution of CA1-specific mRNA and protein expression

of ion channels, proteins involved in calcium regulation, and the

contribution of trophic factors such as Arc or Bdnf to SD impairment of

LTP and spatial learning and memory remain to be investigated.

Male P. leucopus maintain breeding territories in the spring and

early summer (King 1968), and the observed attenuation of spatial

memory and LTP may represent an adaptation to conserve energy during

the short days of winter when territorial maintenance is unnecessary. In

51

addition to energy conservation, the photoperiodic plasticity in P.

leucopus described in the current and previous studies also shares some

common adaptive non-reproductive photoperiodic traits with other

mammals, such as alterations in immune function, affective responses,

and social behaviors (reviewed in Chapter 1; Nelson, Denlinger et al.

2010). The extent to which using naturalistic mammalian models of

photoperiodic plasticity in brain structure and physiology, which show

robust responses to a single environmental signal (viz. day length), will

lead to insights into the mechanisms underlying seasonal human

pathologies remain unspecified.

52

Figure 2.1 Photoperiodic (A) body and (B) reproductive tissue responses in P.

leucopus to 11 week SD exposure. Long day (LD) n = 6, short day (SD) n = 8. *p

< 0.05. 3

53

Figure 2.2 Photoperiod induced deficits in spatial learning and memory in the

Barnes maze after 10 week SD exposure. A, Escape errors by day during

training. B, Latency to escape maze by day of training. C, Percent time in target

quadrant during 90 s probe trial. The dashed line indicates the expected time in

the target quadrant by chance (25%). D, Errors (incorrect escape hole choice)

during 90 s probe trial. Long day (LD) n = 6, short day (SD) n = 8. *p < 0.05. 4

54

Figure 2.3 Photoperiodic impairment of long-term potentiation of fEPSPs in the

Schaffer Collateral-CA1 pathway of hippocampal slices after 10 wk exposure to

SD. Upper left and middle; representative traces of fEPSPs (1) pre-tetanus and

(2) 50 min post-tetanus. Calibration bar: 0.2mV; 10 ms. Upper right; location of

stimulating (black arrow) and recording (white arrow) electrodes. Lower panel;

comparison of LTP between LD (open box) and SD (black box) mice. Tetanus

stimulation was delivered at the time indicated by two arrows. Long day (LD) n

= 9, short day (SD) n = 9. 5

55

Chapter 3: The Role of Melatonin in Photoperiodic Changes in

Hippocampal Plasticity.

1. Introduction

As discussed in Chapter 1, monitoring of environmental day length

in mammals living outside of the tropics is critical to coordinate

seasonally-appropriate adaptations in physiology and behavior to

promote survival and reproductive success. In mammals, nocturnal

melatonin synthesis and secretion from the pineal gland is the putative

endocrine signal of day length (Yellon, Tamarkin et al. 1982; Carter and

Goldman 1983; Bittman and Karsch 1984; Reiter, Tan et al. 2010).

However, it is the duration of elevated melatonin exposure, not peak

concentrations, that conveys environmental photic information (Bittman

and Karsch 1984; Bartness, Powers et al. 1993; Reiter 1993). Thus, short

and extended durations of melatonin exposure are physiologically

indicative of long and short days respectively. Extending the daily

duration of melatonin exposure via exogenous supplementation is

interpreted biologically as a short day, and can drive seasonally-

appropriate adaptive responses (Tamarkin, Hollister et al. 1977;

56

Johnston and Zucker 1980; Carter and Goldman 1983; Dark, Zucker et

al. 1983; Bartness, Powers et al. 1993).

The most striking and extensively studied of these day length

(photoperiodic) driven adaptive responses are seasonal rhythms of

reproduction (Malpaux, Migaud et al. 2001). Small mammals such as

rodents generally reproduce in long days. As autumnal day lengths

shorten, and the duration of nocturnal melatonin synthesis lengthens,

the reproductive axis is inhibited, presumably to shunt energy away from

reproduction to maximize survival across the harsh short days of winter

(Walton, Weil et al. 2011). However, melatonin can directly and indirectly

affect photoperiod-driven seasonal rhythms in non-reproductive

physiology, immune function, and behavior, which occur via both

gonadal steroid-dependent and steroid-independent mechanisms

(Walton, Weil et al. 2011).

As previously discussed, among photoperiodic rodents, white-

footed mice (Peromyscus leucopus) are particularly well-suited to

investigate photoperiod-induced changes in brain function and behavior.

Compared to counterparts held in long summer-like day lengths, white-

footed mice held in short winter-like days lengths have marked changes

in cognitive ability and in the function and connectivity of underlying

brain regions associated with these abilities, including olfactory memory

and neurogenesis (Walton, Pyter et al. 2012), fear memory and the

57

amygdala (Walton, Haim et al. 2012), and spatial memory and the

hippocampus (Pyter, Reader et al. 2005; Workman, Bowers et al. 2009).

Indeed, we have recently reported that short-day exposure impairs long

term potentiation (LTP), the putative mechanism for how memories are

formed and stored in the brain (Bliss and Collingridge 1993), in the

Schaffer collateral pathway of the hippocampus, and this impairment of

LTP is associated with impaired spatial learning and memory (Walton,

Chen et al. 2011).

Although melatonin has been implicated in driving photoperiodic

reproductive responses in white-footed mice (Johnston and Zucker 1980;

Petterborg and Reiter 1980; Dowell and Lynch 1987; Carlson,

Zimmermann et al. 1989), the role of melatonin in photoperiodic

hippocampal plasticity remains largely unknown. To determine the role

of melatonin in photoperiodic brain plasticity, adult male white-footed

mice were given subcutaneous implants of Silastic capsules filled with

melatonin or left empty, then exposed to long or short day lengths for ten

weeks to evoke the full short day phenotype in the blank implant group.

Chronic Silastic melatonin implants were used in the current study

because they are as effective as timed injections, without the

unnecessary repeated stress of those daily injections (Johnston and

Zucker 1980). After ten weeks in their respective day length and

melatonin treatments, one cohort of mice was assessed for Schaffer

58

collateral LTP, and a separate cohort of mice was assessed for spatial

learning and memory in the Barnes maze. At the conclusion of spatial

testing, brains were Golgi impregnated and the morphology of neurons in

the CA1, CA3, and dentate was assessed using computerized

morphometric analyses.

2. Materials and Methods

2.1. Animals

A total of sixty-four male white-footed mice (P. leucopus) were used

in this study. All mice were bred in our colony maintained at The Ohio

State University, which was derived from wild-caught stock obtained

through the Peromyscus Genetic Stock Center at the University of South

Carolina. Mice were group housed with same-sex littermates from

weaning until reaching adulthood (60-90 days of age). Mice were then

pseudo-randomly assigned to the four experimental groups and singly

housed thereafter. The pseudo-random assignment ensured that pups

from each breeding pair were distributed among experimental groups to

avoid litter-specific effects. Throughout the study, mice were housed in

cages (32 cm x 18 cm x 14 cm), maintained at constant temperature and

humidity (21±4 °C, 50±5%), and given ad libitum access to food (Harlan

Teklad 8640, Indianapolis, IN, USA) and filtered tap water. All husbandry

59

was provided by Ohio State University Laboratory Animal Resources

staff. All animal procedures were approved by the Ohio State University

Institutional Animal Care and Use Committee, and were in compliance

with guidelines established by the National Institutes of Health and the

United States Department of Agriculture (Institute of Laboratory Animal

Resources, U.S. 1996).

2.2. Melatonin implants and photoperiod treatment

Under dim lighting, implants were prepared by packing Silastic

tubing (1.47 mm ID x 1.96 mm OD x 15mm length; Dow Corning) 10mm

with melatonin powder (MEL; Sigma #, St Louis, MO, USA) or left empty,

and sealed with Silastic (Turek, Desjardins et al. 1976). Prior to use,

implants were rinsed twice with 70% EtOH, and soaked in sterile saline.

Mice were randomly assigned to treatment group and implanted

subcutaneously with either empty (BL) or MEL packed capsules, which

remained in place for the duration of the study. At the conclusion of the

study, MEL delivery was verified by visually inspecting the implants and

via radioimmunoassay of plasma collected in the middle of the light

phase. After surgery, mice were pseudo-randomly assigned to either

remain in long day lighting (LD; 16 h light: 8 h dark) or be transferred to

short day lighting (SD; 8 h light: 16 h dark) forming four experimental

groups: LD-BL, LD-MEL, SD-BL, SD-MEL. For both photoperiods lights

60

were extinguished at 15:00 EST. Mice remained in their respective

photoperiods for 10 weeks to induce the suite of adaptive responses to

day length (Chapter 2; Pyter, Reader et al. 2005; Walton, Chen et al.

2011).

2.3. Long-term potentiation (LTP)

After 10 weeks in photoperiod, mice (n = 8 from each group) were

processed for hippocampal LTP in the Schaffer collateral pathway as

described previously (Chapter 2; Walton, Chen et al. 2011). Briefly,

during the light phase, mice were rapidly decapitated, and 400 μm thick

hippocampal slices were prepared. Slices were held at 28 °C

carboxygenated (95% O2-5% CO2) in aCSF containing (in mM) 124 NaCl,

4 MgSO4, 4 KCl, 1.0 Na2HPO4, 4 CaCl2, 26 NaHCO3, and 10 D-glucose.

A bipolar stimulating electrode (67 µm, NiCr) was placed in the Schaffer

collateral pathway in the striatum radiatum. Following 90 min

incubation, a slice was placed in a recording chamber affixed to a

microscope (model E600FN, Nikon) with a custom stage (Syskiyou

Instruments, Grants Pass, OR). The chamber was continuously perfused

with carboxygenated ACSF at a rate of 2 ml/min. Field excitatory post-

synaptic potentials (fEPSPs) were recorded using patch clamp amplifier

(model 2400, AM Systems) with a glass pipette (1–2 MΩ) filled with aCSF.

After obtaining a stimulus-response curve, the intensity of a test

61

stimulus (0.033 Hz, 0.1 ms pulse duration) was selected to yield a 30-

40% of the maximal response. After 30 min of steady baseline recording,

LTP was induced using two trains of tetanizing stimulus (100 Hz for 1s, 5

min apart) and fEPSPs were recorded from the CA1 during baseline

stimulation and for 60 min post-tetanus.

2.3. Spatial learning and memory

After ten weeks in their respective photoperiods, forty mice (n = 10

from each group) were tested for spatial learning and memory ability in

the Barnes maze as previously described (Chapter 2; Walton, Chen et al.

2011). Briefly, mice were trained to find the location of the escape box

across five days, which consisted of three 120 s trials per day, with an

inter-trial interval of 5-7 min. At the end of each trial mice were allowed

to remain in the escape box for 45-60 s, then returned to their home

cage. For training trials, latency to escape, number of errors, and path

length were recorded using a video tracking system (HVS Image,

Buckingham, UK). After 5 days of training, on the sixth day of testing,

mice were given a single 90 s probe trial where they could not escape

from the maze. For the probe trial, the number of errors and time spent

in the vicinity of the former escape hole were recorded. To avoid transfer

of olfactory cues, the maze was wiped with 70% EtOH between trials, and

62

the surface was rotated 90° each day, without changing the orientation of

the escape box with the extramaze spatial cues.

2.4. Hippocampal neuronal morphology

Twenty-four hours after the completion of behavioral testing, in the

middle of the light phase, under deep isoflurane anesthesia, mice were

killed and the brains were rapidly removed and processed for Golgi-Cox

staining according to the manufacturer‟s instructions using a

commercially available kit as previously described (Pyter, Reader et al.

2005; Workman, Bowers et al. 2009; Walton, Haim et al. 2012). After

impregnation, 100 μm coronal brain sections were thaw mounted on

gelatin-coated slides, developed, and coverslipped. Neuronal morphology

was quantified on a using a Zeiss microscope (Axio Imager A2) and

commercially available software (Neurolucida, MBF Bioscience, Williston,

VT, USA). For dendritic complexity analysis pyramidal neurons in the

CA1 and CA3, and granule cells within the dentate gyrus (DG), were

selected if they met the following criteria: 1) were completely and evenly

stained, 2) did not have truncated dendrites due to sectioning, and 3)

were not overlapping with other stained cells. Neurons and their

processes (4-6 per region per brain) were traced at 200x and their digital

reconstructions were analyzed for dendritic complexity using Sholl

analysis. For dendritic spine density, from each of the neurons selected

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above, at 1000x spines were counted on four 20 μm unbranched

dendrite segments that were at least 80 μm away from the cell body. Any

protrusion from the dendrite shaft was counted as a spine, independent

of morphology (Pyter, Reader et al. 2005; Workman, Manny et al. 2011;

Walton, Haim et al. 2012).

2.5. Radioimmunoassay

To verify delivery of melatonin from the Silastic implants, 24 h

after the completion of behavioral testing, in the middle of the light

phase, terminal blood samples were collected through the retro-orbital

sinus. Protected from light and held at 4 °C throughout, blood was

centrifuged at 6000 RCF for 30 min; plasma was drawn off and frozen at

-80 °C until assay. Plasma was assayed for melatonin concentration

using a commercially available I125 RIA kit (#01-RK-MEL2; Alpco

Immunoassays, Salem, NH, USA) according to manufacturer‟s

instructions. Intra-assay coefficient of variation was <10%.

2.6. Statistics

Plasma melatonin concentrations were analyzed using a Student‟s t test.

Reproductive tissue masses, Barnes maze probe, neuronal cell body,

dendrite length, and spine density data were analyzed by ANOVA.

Significant differences were followed up by LSD post-hoc tests. Barnes

maze acquisition, LTP, and dendritic Sholl analysis data were analyzed

64

using repeated measures ANOVA. All analyses were performed using

SPSS software (v.19, IBM, Armonk, NY, USA). For all analyses α was set

at 0.05 and mean differences were considered statistically significant if p

≤ 0.05.

3. Results

3.1. Melatonin assay

Compared to mice receiving empty capsules, 12 weeks after

implantation, in the middle of the light phase, mice with melatonin

capsules had elevated plasma MEL concentrations (Fig 3.1A; t11 = -

14.378, p < 0.05).

3.2 Reproductive responses to MEL and SD

Compared to mice in LD with empty implants, extending the daily

duration of melatonin exposure via exposure to either short day lengths

or melatonin implants, reduced paired testes mass in all other groups

(F(3,32) = 3.460, p < 0.05 Fig 3.1B. t34 = 2.758, p < 0.05 Fig 3.1C).


There were no effects of photoperiod (F(1,24) = 3.209, p > 0.05),

melatonin implants (F(1,24) = 1.797, p > 0.05), or interaction of day length

and melatonin treatment (F(1,24) = 0.085, p > 0.05) on acquisition errors

65

across days in the Barnes maze (Fig 3.2A). However, compared to LDBL

mice, extending melatonin exposure via implants or SD exposure

impaired spatial memory in the probe trial (p < 0.05 all comparisons, Fig

3.2B). When extended melatonin exposure groups are collapsed, as

above, in comparison to the short duration of melatonin exposure

(LDBL), there were no differences due to extended MEL duration in

acquisition (F(1,26) = 0.029, p > 0.05, Fig 3.2C) and spatial memory was

impaired by extended melatonin exposure in the probe trial (t32 = -2.091,

p < 0.05, Fig 3.2D).

3.4. Hippocampal long-term potentiation

Exposure to short day lengths impaired Schaffer collateral LTP in

the CA1 region of the hippocampus (F(1,17) = 7.351, p < 0.05 Fig 3.3A) as

previously shown (Walton, Chen et al. 2011). Extending melatonin

exposure duration similarly impairs hippocampal LTP (F(1,19) = 4.676, p <

0.05, Fig 3.3B).

3.5. Morphometry of hippocampal neurons

3.5.1. Cell soma

Neither photoperiod nor melatonin affected cell body size in CA1

and CA3 neurons (p > 0.05, data not shown). In the dentate gyrus,

extending the duration of melatonin exposure reduces cell soma size

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(short duration 221.5 ± 17.6 µm2, extended duration 163.5 ± 8.9 µm2; p <

0.05).

3.5.2. Dendritic material

Extending duration of melatonin exposure reduced the overall

amount of dendritic material in CA1 pyramidal neurons (t22 = -2.520, p <

0.05); this effect was significant in the apical dendrites (t22 = -2.896, p <

0.05, Fig 3.4D). Conversely, extending melatonin exposure increased

total dendritic material in CA3 neurons (t24 = 2.355, p < 0.05), with the

greatest effect in the basilar dendritic field (t24 = 2.452, p < 0.05, Fig

3.4E). Melatonin did not affect total amounts of dendritic material of the

granule cells within the dentate gyrus (t19 = 1.165, p > 0.05, Fig 3.4F).

3.5.3. Dendritic complexity

Extending the duration of melatonin exposure reduced total

dendritic complexity, as measured by the number of intersections with

outwardly concentric circles centered on the call soma (Sholl analysis), of

the CA1 neurons (F(1,22) = -6.198, p < 0.05 Fig 3.5C,F). Reduced

complexity was found in both apical (F(1,22) = -5.990, p < 0.05 Fig 5A,D)

and basilar (F(1,22) = -4.416, p < 0.05 Fig 3.5B,E) dendritic fields. As with

total dendritic material, extended melatonin duration increased overall

dendritic complexity in CA3 neurons (F(1,24) = 6.287, p < 0.05 Fig 3.6C,F),

and increased complexity was found in both apical (F(1,24) = 4.302, p <

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0.05 Fig 6A,D) and basilar (F(1,22) = 7.061, p < 0.05 Fig 3.6B,E) CA3

dendritic fields. No differences were observed in DG granule neurons

(F(1,19) = 1.065, p > 0.05 Fig 3.7).

3.5.4. Spine density

Extended melatonin exposure reduced spine density in the basilar

tips of CA1 dendrites (t21 = -2.265, p < 0.05, Fig 3.8D), without affecting

spine density in the CA1 apical dendrites (t21 = 1.685, p = 0.11, Fig

3.8D), CA3 neurons (apical: t20 = -2.265, p = 0.07; basilar: t21 = 1.568, p

= 0.13, Fig 3.8E), or DG neurons (t21 = -0.690, p > 0.05, Fig 3.8F).

4. Discussion

Extending the daily duration of melatonin exposure by exposure to

short days impairs hippocampal LTP, spatial learning and memory, and

alters neuronal morphology of hippocampal neurons (current study;

Pyter, Reader et al. 2005; Workman, Bowers et al. 2009; Walton, Chen et

al. 2011). The current findings demonstrate that extending the duration

of melatonin exposure via exogenous melatonin delivery through chronic

subcutaneous implants recapitulates the effects of short day exposure,

independent of environmental day length, on hippocampal function and

morphology in white-footed mice, providing further evidence for the role

of melatonin in seasonal brain plasticity in photoperiodic rodents.

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Melatonin has direct effects on spatial memory and neuronal

physiological plasticity in the hippocampus. In both rats and mice, acute

application of melatonin to the hippocampus impairs both LTP in the

CA1 region (El-Sherif, Hogan et al. 2002; Feng, Zhang et al. 2002; Talaei,

Sheibani et al. 2010) and hippocampal-mediated learning and memory

(Collins and Davies 1997; Feng, Zhang et al. 2002; Cao, Wang et al.

2009). Melatonin also alters hippocampal function in an indirect manner.

When tested during the light phase when endogenous melatonin levels

are at nadir (Figure 3.1A), SDBL mice have impaired performance in

spatial tasks (Figure 3.2) and impaired hippocampal physiological

plasticity in the form of LTP (Figure 3.3; Walton, Chen et al. 2011).

Independent of environmental day length, extending the daily duration of

melatonin exposure with chronic implants, which is interpreted

biologically as a short day, recapitulates the effects of SD exposure on

both spatial ability (Figure 3.2) and LTP (Figure 3.3). Although melatonin

potentially directly affected hippocampal function during behavioral

testing in mice with melatonin implants, that LDMEL and SDMEL mice

do not differ from SDBL mice (Figure 3.2) argues against this possibility

and argues for these effects being mediated via indirect mechanisms.

Additionally, that LTP experiments were performed in vitro in the absence

of both endogenous and exogenous melatonin, and that extended

melatonin duration groups did not differ significantly from each other

69

(Figure 3.3), further supports indirect mechanisms underlying the effects

of melatonin on hippocampal plasticity described herein.

The impairments resulting from the indirect effects of extended

melatonin duration also extend to hippocampal neuronal morphology

(Figures 3.4-3.8; Pyter, Reader et al. 2005). Short day lengths, and thus

extended melatonin exposure, reduce hippocampal volume in multiple

rodent species (reviewed in Yaskin 2011), including white-footed mice

(Pyter, Reader et al. 2005). Some of the reduction in hippocampal volume

may be accounted for by reduced dendritic complexity and dendritic

material in CA1 (Figures 3.4 & 3.5), and by reduced soma size in DG

neurons, thus reducing volume via tighter packing of the hippocampal

neurons in SD exposed brains. Although this hypothesis is appealing, it

remains to be determined empirically whether this is the case by careful

stereological quantification of hippocampal neurons in both extended

and short duration melatonin exposed brains, which is beyond the scope

of the current study. Arguing against neuronal morphologicial changes

contributing to smaller hippocampi in SD by tighter neuronal packing is

the increase in dendritic material and complexity in CA3 (Figures 3.4 &

3.6). The alteration of CA3 neuronal morphology may not arise as direct

result of melatonin exposure, but it could be driven indirectly by gonadal

regression induced by extended melatonin exposure (Figure 3.1), as

reduced testosterone concentrations enhance mossy fiber connectivity in

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the CA3 (Skuckas, Duffy et al. 2013). There are two main types of CA3

pyramidal neurons identified by differences in apical dendrite length and

branch morphology: long-shaft and short-shaft (Fitch, Juraska et al.

1989), and in at least one photoperiodic rodent species, Siberian ground

squirrels (Citellus undulates), CA3 pyramidal neurons change rapidly

between long- and short-shaft types dependent on torpid state (Popov,

Bocharova et al. 1992). Thus, the current findings provide evidence that

extended melatonin exposure in white-footed mice may either 1) shift

phenotype of the CA3 pyramidal neurons from long- to short-shaft, or 2)

make short-shaft CA3 neurons more susceptible to impregnation via the

Golgi-Cox method.

Photoperiodic rodents, such as white-footed mice, reproduce

seasonally and extended duration of melatonin exposure inhibits the

hypothalamic-pituitary-gonadal axis and drives regression of the

reproductive system (Figure 3.1C; Chapter 1; reviewed in Walton, Weil et

al. 2011). It is possible that the indirect effects of melatonin on spatial

memory and hippocampal LTP described herein may be modulated

through altered gonadal sex steroid signaling. Gonadal sex steroids

(estrogen and testosterone) can affect spatial memory (Galea, Kavaliers et

al. 1994; Galea, Kavaliers et al. 1996), hippocampal morphology (Cooke

and Woolley 2005), and hippocampal LTP (Gupta, Sen et al. 2001;

Leranth, Petnehazy et al. 2003). Consistent with the current findings,

71

testosterone rescued photoperiodic impairment of spatial navigation

ability in gonadectomized male white-footed mice, but only in short days

(Pyter, Trainor et al. 2006). However, in that study it was also reported

that day length (and thus melatonin duration) did not affect hippocampal

androgen or estrogen receptor expression in naïve mice in SD and LD,

and that testosterone had no effect on spatial performance in mice held

in long days, so the effects of testosterone on hippocampal mediated

behaviors were likely indirect effects, however the role of other gonadal

steroids in this behavior will be explored in Chapter 4. Similarly, the

impaired LTP of mice with extended melatonin exposure is not likely a

direct effect of reduced testosterone levels as androgens generally impair

LTP in the hippocampus (Harley, Malsbury et al. 2000; Hebbard, King et

al. 2003; Skuckas, Duffy et al. 2013).

In conclusion, the current results demonstrate that chronic

melatonin implants can recapitulate the effects of short days on the

hippocampus and implicate melatonin signaling as a critical factor in

day-length induced changes in the structure and function of the

hippocampus in a photoperiodic rodent. Although the exact mechanisms

underlying melatonin‟s effects on the hippocampus are largely not

described, hippocampal responses to extended melatonin exposure are

likely the result of multiple direct and indirect neuroendocrine responses

72

to changing day length which underlie the adaptive photoperiodic

responses to short day lengths.

73

Figure 3.1 Melatonin delivery from implants and reproductive responses to

extended melatonin exposure. A) Independent of photoperiod, 12 weeks after

implant surgery, plasma melatonin concentrations in the middle of the light

phase were elevated in mice with melatonin implants. B) Extending the

duration of daily melatonin exposure by short day lengths (SDBL) or by

melatonin implants (LDMEL, SDMEL) reduces paired testes mass. C) Extended

duration of daily melatonin exposure reduces paired testes mass. *p < 0.05. 6

74

Figure 3.2 Spatial learning and memory in the Barnes maze. Photoperiod and

melatonin treatment did not affect errors (incorrect escape choice) during

acquisition trials (A,C). Both SD exposure and melatonin impair spatial memory

during the probe trial (B,D). *p < 0.05. 7

75

Figure 3.3 Schaffer collateral long-term potentiation. Exposure to short day

lengths impairs hippocampal LTP (A), and extending melatonin exposure

duration with exposure to SD or melatonin implants impair CA1 LTP (B). *p <

0.05 repeated measures ANOVA. 8

76

Figure 3.4 Dendritic material of hippocampal neurons. Extending melatonin

exposure duration reduces total and apical dendritic material of pyramidal

neurons in CA1 (D). Extending melatonin increases total dendritic material of

the CA3 neurons, mainly in the basilar dendrites (E). Shared letters indicate no

significant differences LSD post-hoc test. *p < 0.05 Student‟s t test. 9

77

Figure 3.5 CA1 dendritic complexity. Extended duration of melatonin exposure

reduces complexity in CA1 apical (A,D), basilar (B,E), and total (C,F) dendritic

fields. Representative Neurolucida tracings of CA1 pyramidal neurons from

each experimental group (G). 10

78

Figure 3.6 CA3 dendritic complexity. Extended duration of melatonin exposure

increases complexity in CA3 apical (A,D), basilar (B,E), and total (C,F) dendritic

fields. Representative Neurolucida tracings of CA3 pyramidal neurons from

each experimental group (G). 11

79

Figure 3.7 Dentate dendritic complexity. Extended duration of melatonin

exposure did not affect dendritic complexity in dentate granule cells.

Representative Neurolucida tracings of DG granule cells from each experimental

group (C). 12

80

Figure 3.8 Neuronal spine density in hippocampal neurons. Extended duration

melatonin exposure reduces spine density in the basilar dendrites of CA1

without affecting neuronal spine density in other areas of the hippocampus.

Shared letters indicate no significant differences LSD post-hoc test. *p < 0.05

Student‟s t test. 13

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Chapter 4: The Role of Sex Steroids in Photoperiodic Changes in

Hippocampal Plasticity

1. Introduction

As discussed in Chapter 1, mammals living outside of the tropics

have day-length driven adaptations of physiology and behavior to survive

the energetic constraints of winter to promote fitness. Among the

photoperiodic adaptations to short day lengths in long-day breeders,

including white-footed mice, is the suppression of hypothalamic-

pituitary-gonadal (HPG) axis activity which is followed by the involution

of the gonads (see Figure 2.1; Pyter, Hotchkiss et al. 2005) with a

concurrent reduction of circulating gonadal steroids to basal levels. As

described in Chapter 2, exposure to short days is associated with altered

hippocampal function, size, and impaired spatial learning and memory in

white-footed mice (Pyter, Reader et al. 2005; Walton, Chen et al. 2011).

These photoperiodic responses in hippocampal physiology and behavior

are driven by melatonin signaling (Chapter 3), yet it is possible that the

effects of melatonin are indirect and mediated by altered gonadal steroids

downstream of altered melatonin rhythms.

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Males of photoperiodic species generally display altered patterns of

behavior in short days, some of which are hippocampal-mediated and

associated with altered reproductive physiology (Chapter 1; Nelson,

Denlinger et al. 2010). Short days impair hippocampal-mediated

behaviors in male deer mice (P. maniculatus) and male white-footed mice

(P. leucopus) (Chapter 2; Galea, Kavaliers et al. 1994; Perrot-Sinal,

Kavaliers et al. 1998; Pyter, Reader et al. 2005; Walton, Chen et al.

2011), which is consistent with the hypothesis that enhanced spatial

learning and memory in reproductively active males is advantageous in

navigating territories and finding mates (Jacobs 1996). However, these

studies have only indirectly assessed the role of gonadal steroids on

spatial behaviors in a photoperiodic context. One study that directly

assessed the role of testosterone in spatial memory in a photoperiodic

context in white-footed mice reported that, whereas testosterone rescued

short-day deficits in spatial ability in the water maze, testosterone did

not affect spatial navigation in long days (Pyter, Trainor et al. 2006). No

photoperiodic differences were reported in the hippocampus for androgen

receptor gene expression (AR), or gene expression for both main types of

estrogen receptor (ERα and β), however the hippocampus from naïve

mice were assessed, and not the hippocampus from the mice used in the

behavioral study that received testosterone and photoperiod

manipulations (Pyter, Trainor et al. 2006). Furthermore, in white-footed

83

mice it is not known whether testosterone directly affects spatial

behavior, or if the effects are indirect and mediated through the primary

metabolites of testosterone, as testosterone can be reduced to

dihydrotestosterone via 5α-reductase, or aromatized to estrogen via

aromatase (Clemens and Pomerantz 1982; Leonard and Winsauer 2011).

Thus, I designed the current study to directly assess the effects of

testosterone and its primary metabolites on photoperiod-mediated spatial

learning and memory in white-footed mice. Gonadal steroids have

organizational and activational effects on the hippocampus and spatial

memory (Dawson, Cheung et al. 1975; Isgor and Sengelaub 1998; Sisk

and Foster 2004). In the current study I wanted to specifically assess the

activational effects of testosterone and its metabolites; thus, prior to any

manipulation male mice were allowed to complete puberty and reach

adulthood (>60 d old). To clamp gonadal steroid levels, adult male white-

footed mice were gonadectomized and then implanted with Silastic

capsules containing one of the following: testosterone,

dihydrotestosterone, estradiol, or cholesterol, the metabolic precursor of

gonadal steroids. Mice were then placed in short or long days for 10

weeks, assessed for spatial learning and memory in the Barnes maze;

then hippocampal AR, ERα, and ERβ gene expression was assessed to

determine the potential mechanism by which testosterone interacts with

day length to affect spatial navigation.

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2. Materials and methods

2.1. Animals

Sixty male P. leucopus were used in this study. Mice were bred in

our colony maintained at The Ohio State University, which was derived

from wild-caught stock from the Peromyscus Genetic Stock Center at the

University of South Carolina. All mice were housed in standard mouse

cages (32 cm x 18 cm x 14 cm), maintained at constant temperature

(21±4°C), constant humidity (50±5%), and given ad libitum access to food

(Harlan Teklad 8640, Indianapolis, IN, USA) and filtered tap water. After

weaning, mice were group housed with same-sex littermates in long day

length conditions (LD; 16h light:8h dark; lights on 23:00h EST) until

reaching adulthood (60-90 days of age). After surgery (see below) and

photoperiod assignment, mice either remained in LD or were transferred

to short day lengths (SD; 8h light:16h dark; lights on 07:00 h EST) for 10

weeks prior to any behavioral testing. Throughout the experiment Ohio

State University Laboratory Animal Resources staff provided all

husbandry. All animal procedures were approved by the Ohio State

University Institutional Animal Care and Use Committee, and were in

compliance with guidelines established by the National Institutes of

Health and the United States Department of Agriculture (Institute of

Laboratory Animal Resources, U.S. 1996).

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2.2. Gonadectomy and steroid implants

To clamp gonadal steroids at constant concentrations, post-

pubertal (>60 d old) mice were first gonadectomized and then implanted

with Silastic steroid capsules as previously described (Pyter, Trainor et

al. 2006). Briefly, under isoflurane anesthesia using sterile surgical

technique, the testes were externalized through a small ventrum midline

incision and the testicular artery cauterized and then sectioned. While

still under anesthesia, mice were then implanted on the dorsum, through

an incision in the mid-scapular region, with a sterile Silastic implant.

Several days prior to surgery, steroid implants were prepared by packing,

and then sealing with Silastic glue, Silastic tubing (1.47 mm ID x 1.96

mm OD x 15mm length; Dow Corning) 10mm with one of the following

compounds: testosterone (T; Steraloids A6950), dihydrotestosterone

(DHT; Sigma A8380), estradiol (E: 2% in cholesterol; Sigma E1024), or

cholesterol (CH; Sigma C3045). Prior to use, implants were rinsed twice

with 70% EtOH, and soaked overnight in sterile saline at 37ºC to avoid

supraphysiological bolus delivery of steroid after implantation (Cohen

and Milligan 1993). After surgery, mice were pseudo randomly assigned

to either remain in LD, or were placed in SD forming 8 groups (Table

4.1).


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After 10 weeks in their respective photoperiods, mice were tested

for spatial learning and memory in the Barnes maze as previously

described (Chapter 2; Walton, Chen et al. 2011). Briefly, mice were

trained to find the location of the escape box across five days, which

consisted of three trials/day (120s max/trial). For training trials, latency

to escape, number of errors, and path length were recorded using a video

tracking system (HVS Image, Buckingham, UK). After 5 days of training,

on the sixth day of testing, mice were given a single 90 s probe trial

where they could not escape from the maze. For the probe trial, the

number of errors and time spent in the vicinity of the former escape hole

were recorded. To avoid transfer of olfactory cues, the maze was wiped

with 70% EtOH between trials. Mice were tested in 3 separate cohorts (n

= 20 each cohort), and each cohort was made up of mice representing all

treatment groups.

2.4. Tissue collection and gene expression

Twenty-four hours after the conclusion of behavioral testing, mice

were killed and the remaining reproductive tissues were collected to

verify expected responses to steroid treatment. To assess the expression

of the androgen receptor (AR) and estrogen receptors (ERα, ERβ) in the

hippocampus, brains were rapidly removed and placed into RNAlater

(Ambion) to preserve RNA integrity. After stabilization of RNA, the

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hippocampus was dissected out of each brain and total RNA was

extracted from each hippocampal homogenate using TRIzol (Invotrogen;

after Rio, Ares et al. 2010). MMLV Reverse Transcriptase (Invitrogen) was

used to convert ~2 μg of RNA to cDNA following manufacturer‟s

instructions. Gene expression levels for the genes of interest were

assessed using quantitative real time PCR on an ABI 7500 FAST

Sequence Detection System under standard conditions with P. leucopus

gene-specific probes and primers as previously described (Pyter, Trainor

et al. 2006). Prior to analysis, gene expression levels for each gene of

interest were normalized to 18s rRNA gene expression.

2.5. Statistics

Barnes maze probe, gene expression, and remaining reproductive

tissue mass data were analyzed by MANOVA with photoperiod and

steroid treatment as factors. Significant differences were followed up by

LSD post-hoc tests. Barnes maze acquisition data were analyzed using

repeated measures MANOVA. Based on previous studies, we predicted E

and T would have opposite effects on cognitive behavior dependent upon

photoperiod, (Pyter, Trainor et al. 2006; Trainor, Lin et al. 2007; Trainor,

Rowland et al. 2007). Thus, planned comparisons among photoperiod, E,

and T for the above measures were also performed. All analyses were

performed using SPSS software (v.19, IBM, Armonk, NY, USA). For all

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analyses α was set at 0.05 and mean differences were considered

statistically significant if p ≤ 0.05.

3. Results

3.1. Reproductive tissues

Tissue mass of the seminal vesicles and epididymides is supported

by both androgens and estrogens (Higgins, Burchell et al. 1976; Belis,

Adlestein et al. 1983; Pelletier, Labrie et al. 2000), thus the masses of the

remaining reproductive tissues were used as a biological marker to verify

steroid delivery across the experiment. Testosterone increased body mass

(F(1,3) = 6.266, p ≤ 0.05), but photoperiod did not affect body mass (F(1,3) =

0.042, p > 0.05)(Figure 4.1A). Steroid treatment supported seminal

vesicle mass (F(1,3) = 157.8, p ≤ 0.05) in the following order: T > DHT > E

> CH (Figure 4.1B). Photoperiod did not affect this measure (F(1,3) =

1.173, p > 0.05); Figure 4.1B). Steroid treatment also supported mass of

paired epididymides (F(1,3) = 23.803, p ≤ 0.05) in the following order: T >

DHT > E, CH (Figure 4.1C). Photoperiod did not affect epididymides mass

(F(1,3) = 1.689, p > 0.05; Figure 4.1C).

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During acquisition trials, inclusive of all four steroids (T, DHT, E,

CH), neither photoperiod (F(3,51) = 0.011, p > 0.05) nor steroid treatment

(F(3,51) = 2.492, p > 0.05) affected latency to escape, and there was no

interaction of photoperiod and steroids on escape latency across days

(F(3,51) = 0.821, p > 0.05) (Data not shown). Similarly, across training days

the number of errors prior to escape was not affected by photoperiod

(F(3,51) = 0.003, p > 0.05) or steroid treatment (F(3,51) = 0.678, p > 0.05),

and the two variables did not interact to affect errors (F(3,51) = 1.725, p >

0.05)(Data not shown). In the memory probe trial, inclusive of all steroid

treatments (T, DHT, E, CH), neither photoperiod (F(3,49) = 0.308, p > 0.05)

nor steroids (F(3,49) = 1.742, p > 0.05) affected the number of errors, nor

did photoperiod and steroids interact to affect errors (F(3,49) = 1.563, p >

0.05) (Data not shown).

A priori planned comparisons of the effects of estradiol and

testosterone in long and short days on Barnes maze acquisition were

then performed. Neither photoperiod (F(1,25) = 0.054, p > 0.05) nor steroid

treatment (F(1,25) = 0.020, p > 0.05) affected escape latency across days,

and there was no interaction of steroid treatment with photoperiod on

escape latency (F(1,25) = 2.235, p > 0.05) (Figure 4.2A). Whereas neither

photoperiod (F(1,25) = 0.078, p > 0.05) nor steroid treatment (F(1,25) =

0.419, p > 0.05) affected the number of errors across days, there was an

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interaction of T and E with photoperiod in training errors prior to escape

across days (F(1,25) = 4.825, p ≤ 0.05); testosterone improved performance

in short days and estrogen improved performance in long days (Figure

4.2B). This pattern was also present in the probe memory trial, where

estrogen and testosterone interacted to affect the number of errors

dependent on photoperiod (F(1,24) = 4.176, p ≤ 0.05), without any main

effects of photoperiod (F(1,24) = 0.008, p > 0.05) or steroid treatment (F(1,24)

= 0.043, p > 0.05) on probe errors (Figure 4.2C). Mice exposed to

estrogen in long days and those exposed to testosterone in short days

had improved performance in the probe trial of spatial memory.

3.3 Sex steroid receptor gene expression

NOTE: the gene expression data presented here are preliminary due

to low sample size in each of the groups (n=3), and thus are interpreted

with extreme caution. Inclusive of all steroid treatment groups (T, DHT,

E, CH), neither photoperiod nor steroid treatment affected expression of

ERα (photoperiod: F(1,3) = 2.221, p > 0.05; steroid treatment: F(1,3) =

1.915, p > 0.05; Figure 4.3A), ERβ (photoperiod: F(1,3) = 1.295, p > 0.05;

steroid treatment: F(1,3) = 1.388, p > 0.05; Figure 4.3B), or AR

(photoperiod: F(1,3) = 1.023, p > 0.05; steroid treatment: F(1,3) = 0.823, p >

0.05; Figure 4.3C). Given that testosterone and estrogen exert their

effects through androgen and estrogen receptors, respectively (Kerr, Beck

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et al. 1996; McEwen 2001), planned comparisons were performed

between photoperiod in the ERα and ERβ in the estrogen treatment

group and of AR between photoperiods in the testosterone treatment

group. Photoperiod had no effect on ERα or ERβ receptor expression in

the estrogen treated mice, nor did it affect AR expression in mice

receiving testosterone implants (p > 0.05 all measures; Figure 4.3D).

4. Discussion

This study provides further evidence on how gonadal steroids

interact with photoperiod to affect hippocampal function and behavior.

Consistent with previous reports in male white-footed mice (Pyter,

Trainor et al. 2006), testosterone improved spatial learning and memory

in short days (Figure 4.2). Additionally, photoperiod and gonadal steroids

interacted to affect spatial learning and memory. Whereas testosterone

supported spatial navigation in short days, estrogen supported spatial

navigation in long days (Figure 4.2B, C). Preliminary data on gene

expression levels of the receptors for testosterone (AR) and estrogen (ERα

and β) from the hippocampus of the mice tested for spatial navigation,

although not reaching significance, appear to mechanistically support

this interaction. In short days, AR expression is elevated when

testosterone enhanced spatial navigation, whereas ERα and ERβ

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expression is elevated in long days when estrogen enhanced spatial

navigation (Figure 4.3D). However, this association regarding steroid

receptors is made with extreme caution, as more data are necessary to

confirm or refute these preliminary findings.

Testosterone and its metabolites, DHT and E, have been reported

to facilitate hippocampal-mediated behaviors in male rodents (Edinger,

Lee et al. 2004). However, some hippocampal morphological measures,

such as dendritic spine density and neurogenesis, are only affected by

androgens, and not estrogen, in males (Leranth, Petnehazy et al. 2003;

Spritzer and Galea 2007). In males, testosterone is secreted into

circulation from the testes and acts hormonally in the brain as an

androgen, or T can be metabolized locally in the hippocampus and affect

behavior in a paracrine manner as either an androgen (DHT) or as an

estrogen (Edinger and Frye 2005; Edinger and Frye 2007). Independent

of gonadal sex steroids, de novo neurosteroidogenesis, which has been

implicated in maintenance and support of synaptic plasticity, occurs

within the hippocampus (Hojo, Hattori et al. 2004; Rune and Frotscher

2005; Schmidt, Pradhan et al. 2008). Thus, a complex interaction exists

among gonadal steroids, brain neurosteroidogenesis, and behavior.

Compounding this interaction is the role of photoperiod, which can alter

steroid receptor expression in the brains of several closely related

Peromyscus species (Trainor, Rowland et al. 2007), and affect the signal

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transduction mechanism of steroid hormone receptors in the brain

(Trainor, Lin et al. 2007).

In their natural environment, male white-footed mice during the

short days of winter form communal nests and do not maintain

territories. However, in the longer days of spring and summer,

reproductively active mice maintain and defend breeding territories (King

1968). Reproductive status-dependent enhancement of hippocampal

function has been reported in white-footed mice and other photoperiodic

rodent species, and naturalistic photoperiodic changes in range size

correspond with altered spatial ability in laboratory experiments (Chapter

2; Galea, Kavaliers et al. 1994; Galea, Kavaliers et al. 1996; Pyter, Reader

et al. 2005; Pyter, Trainor et al. 2006; Walton, Chen et al. 2011),

reviewed in Chapter 1). The increase in hippocampal demands (for

greater spatial navigation and memory) during the long days of the

breeding season to maintain a breeding territory and to support

reproductive behaviors is energetically expensive. White-footed mice have

evolved adaptations in the regulation of hippocampal function, such as

decreased hippocampal volume (Pyter, Reader et al. 2005), reduced

synaptic connectivity (Chapter 3), and impaired physiology (Walton, Chen

et al. 2011). All of these changes are driven by photoperiod and result in

impaired hippocampal function in short days, potentially to conserve

energy (Chapter 1; Jacobs 1996; Walton, Weil et al. 2011).

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Taken together along with previous studies, male white-footed mice

held in short day lengths have increased sensitivity in the hippocampus

and hippocampus-mediated behavior to testosterone, whereas male mice

held in long days have increased sensitivity to estrogen. These changes in

sensitivity may result in part from photoperiod-mediated changes in

steroid receptors in the hippocampus, however this must be probed

deeper as previous reports on photoperiod mediated steroid receptor

expression in several Peromyscus species have yielded inconsistent

results (Pyter, Trainor et al. 2006; Trainor, Lin et al. 2007; Trainor,

Rowland et al. 2007). Independent of the mechanism, after 10 weeks in

short days, the hippocampus of white-footed mice is sensitive to the

activational effects of testosterone, which, in a naturalistic context, may

then initially support increased hippocampal function and organization

in preparation for maintaining breeding territories and seeking mates in

the lengthening days of spring. Concurrent with lengthening days of

spring is the activation of the HPG axis and recrudescence of the gonads

when testosterone levels are elevated. Once the hippocampus has

responded to the organizational effects of elevated circulating

testosterone levels resulting from gonadal recrudescence, the

hippocampus may then switch in long days to become insensitive to

testosterone and increase sensitivity to the activational effects of

estrogen.

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Cholesterol Testosterone Estradiol Dihydrotestosterone

LD LDCH LDT LDE LDDHT

SD SDCH SDT SDE SDDHT

Table 4.1 Experimental groups formed by photoperiod and steroid treatment. 1

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Figure 4.1 Body and reproductive tissue mass responses to steroid and

photoperiod treatment. Independent of photoperiod, mice receiving testosterone

had the largest body mass (A). Verifying the delivery of steroids from the

implants across the experiment, remaining reproductive tissues (B, seminal

vesicles; C, epididymides) varied in mass according to steroid (T > DHT > E >

CH). Shared letters indicate differences (p ≤ 0.05) from other groups due to

steroid treatment. 14

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Figure 4.2 Photoperiod and steroid treatment interact to affect spatial learning

and memory. Photoperiod and steroids had no affect on latency to escape (A).

Photoperiod interacted with steroid treatment to alter errors during acquisition

(B) and retention (C) of the spatial task. Mice receiving testosterone (T) in short

days (SD) and mice receiving estrogen (E) in long days (LD) made fewer errors

prior to escape than their opposite-photoperiod counterparts. 15

98

Figure 4.3 Effects of photoperiod and steroids on estrogen and androgen

receptor expression in the hippocampus. LD – long days, SD – short days, T –

testosterone, DHT – dihydrotestosterone, E – estrogen, CH – cholesterol. 16

99

Chapter 5: Photoperiod, Fear Behavior, and the Basolateral

Amygdala.

1. Introduction

As noted in Chapter 1, photoperiodism is the biological ability to

measure environmental day length (photoperiod) to ascertain the time of

year and engage in seasonally appropriate adaptations of physiology and

behavior. These seasonal changes in physiology and behavior can be

induced in a laboratory setting by simply exposing animals to different

static day lengths (Chapter 1; Walton, Weil et al. 2011). White-footed

mice (P. leucopus) display a suite of changes in physiology and behavior

induced by exposure to short days, including changes in behavior, brain

volume and functional connectivity, and enhanced hypothalamic-

pituitary-adrenal (HPA) axis reactivity (Chapter 2; Pyter, Reader et al.

2005; Pyter, Adelson et al. 2007; Walton, Chen et al. 2011). In recent

years, photoperiodic rodent models are finding more utility as models of

human pathologies involving alterations in brain structure and function

(Shekhar, McCann et al. 2001; Workman and Nelson 2010).

Among human post-traumatic stress disorder (PTSD) patients,

reduced hippocampal volume is associated with susceptibility to PTSD,

100

but not severity of symptoms (Yehuda and LeDoux 2007), amygdala

activity is associated with symptom severity (Dickie, Brunet et al. 2011),

and PTSD patients have enhanced HPA axis negative feedback (Yehuda

and LeDoux 2007). Additionally, the medial prefrontal cortex (mPFC) is

hyporesponsive to fear-related stimuli in patients suffering from PTSD

(Liberzon and Sripada 2008; Gold, Shin et al. 2011). Translational rodent

studies have identified these three brain regions as critical components

of the neural circuit underlying associative fear memory. The basolateral

amygdala (BLA), hippocampus, and mPFC all play critical roles in fear

memory: complex modulation of reciprocal connections among the

mPFC, hippocampus, and basolateral amygdala (BLA) are integral to

associative fear memory (Maren and Quirk 2004; Yehuda and LeDoux

2007; Guimarais, Gregorio et al. 2011; Vouimba and Maroun 2011).

Although standard laboratory rodents (Rattus norvegicus and Mus

musculus) have been widely used to model PTSD and other human

psychiatric disorders, to fully understand combined factors underlying

the development, maintenance, and treatment of these diseases, more

diverse animal models are needed (Shekhar, McCann et al. 2001).

Toward this end, photoperiod-induced changes in the brain of white-

footed mice mirror several components of the etiology of PTSD. For

example, in common with the human PTSD condition, short day exposed

white-footed mice have reduced hippocampal volume (Pyter, Reader et al.

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2005) and increased HPA axis feedback (Pyter, Adelson et al. 2007). To

my knowledge, photoperiod–mediated changes in fear memory and

responses in this, and other, photoperiodic rodent species remain largely

undescribed. A preliminary study in our lab indicated that, unlike short-

day induced impairments in hippocampal-mediated spatial learning and

memory, white-footed mice exposed to short days may have enhanced

non-spatial fear memory (Pyter, Reader et al. 2005).

Based on the commonality of photoperiodic changes in white

footed mice with the etiology of PTSD described above, and on

preliminary data demonstrating short day enhancement of fear memory,

I hypothesized that exposure to short days would enhance fear memory

and alter neuronal morphology in brain regions implicated in associative

fear memory. To test my hypothesis, I exposed male white-footed mice to

either short or long day lengths for ten weeks to induce maximal

photoperiodic responses, tested them in two separate behavioral fear

paradigms (passive avoidance and auditory fear conditioning), and

examined the neuronal morphology in the BLA and the infralimbic region

of the mPFC (IL) using Golgi-Cox staining.

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2.1. Animals

Twenty-eight adult (>55 d of age) male P. leucopus, from our breeding

colony maintained at the Ohio State University, were randomly assigned

to either long (LD; 16L:8D, n = 14) or short day lengths (SD; 8L:16D, n =

14) for 10 weeks to establish photoperiod-induced changes prior to

behavioral testing (Chapter 2, (Pyter, Reader et al. 2005; Walton, Chen et

al. 2011). Mice were housed in standard polycarbonate cages (32 x 18 x

14 cm), maintained at constant temperature (21 ± 4 °C) and relative

humidity (50 ± 5%), provided ad libitum access to filtered tap water and

food (Harlan Teklad 8640, Indianapolis, IN, USA), and received care from

the Ohio State University Laboratory Animal Resource staff for the

duration of the study. All procedures proposed were approved by the

Ohio State University Institutional Animal Care and Use Committee and

are in compliance with guidelines established by the National Institutes

of Health (Institute of Laboratory Animal Resources, U.S. 1996).

2.2. Behavioral Tests

2.2.1. Passive avoidance

To assess non spatial contextual and cued fear memory, 9 mice (n = 4

LD; n = 5 SD) were tested in the passive avoidance task as previously

described (Pyter, Reader et al. 2005). Briefly, early in the dark phase

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mice were placed in a closed side of a dark divided conditioning chamber

(Gemini, San Diego Instruments, San Diego, CA, USA) and allowed to

habituate for 30 s. After habituation, the side of the chamber containing

the mouse was illuminated, the guillotine-like door to the dark chamber

was raised, and latency for mice to step through to the dark chamber

was recorded. Upon avoiding the aversive stimulus (light) by stepping

through into the dark chamber, the door closed, mice received a 1 s 0.6

mA foot shock, and were then returned to their home cages. Twenty-four

hours after training, mice were returned to the chamber and tested as

above; latency to step through into the dark chamber was recorded for a

maximum of 300 s. Longer latencies to step through 24 h after training

reflect enhanced fear memory. To avoid carry-over effects from using the

same aversive stimulus, these mice were not used for auditory fear

conditioning.

2.2.2. Auditory fear conditioning

To assess tone-conditioned fear acquisition and retention, 19 (n = 10

LD; n = 9 SD) mice were assessed using the Near-IR Video Fear

Conditioning System (Med Associates Inc., St. Albans, VT, USA). For

acquisition of the tone-conditioned fear, during the light phase mice were

brought directly from their vivarium rooms and were placed in the test

chamber illuminated with white light for a 2 min habituation period with

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68dB white noise. Mice were then exposed to a series of 8 conditional

stimuli (80dB tone, CS) for 6 s with the last 2 s paired with a 0.75 mA

foot shock (unconditioned stimulus, US). Mice remained in the chamber

for an additional 60 s after the last CS/US pairing before being returned

to their home cages. Freezing behavior was recorded by the software for

the 2 minute baseline, during the first 4 s of each tone, during the 30 s

interval between CS presentations, and for the 60 s after the final CS. To

assess contextual fear retention, 24 h after the acquisition session, mice

were placed in the original unmodified chamber and freezing behavior to

the unmodified chamber was recorded for 180 s, and mice were returned

to their home cages. Four hours after the contextual fear retention test,

mice were tested for retention of the CS-US pairing with the following

modifications to alter context. Mice were transported from their vivarium

rooms via sound- and light-attenuating boxes to a staging area. From

there, mice were brought into the testing lit with dim red light and placed

into the chambers. To avoid context-dependent freezing, the chamber

was modified via the addition of a smooth plastic floor, a semi-circular

unlit testing chamber, lights were extinguished, and a gauze pad with a

drop of vanilla extract was placed in the chamber to present the CS in a

novel environment. Mice were then tested for retention of the CS/US

pairing by using the procedure described above for the acquisition trial

above without receiving the foot shock (US).

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2.3. Sample collection and histology

Twenty hours after the completion of auditory fear conditioning,

under deep isoflurane anesthesia, mice were exsanguinated via the retro-

orbital sinus, plasma was collected from the blood samples as previously

described (Pyter, Reader et al. 2005), and samples were stored at -80°C

for corticosterone assay. Immediately after blood collection, mice were

rapidly decapitated and brains were processed to study neuronal

morphology (after (Pyter, Reader et al. 2005; Workman, Bowers et al.

2009)) using a commercially available Golgi-Cox impregnation kit (FD

NeuroTechnologies, Ellicott City, MD, USA) according to the

manufacturer‟s instructions. Briefly, after impregnation, brains were cut

into 100 µm coronal sections and thaw mounted on to gelatin-coated

slides. Slides were then developed, counterstained with cresyl violet

acetate, dehydrated, cleared with xylenes, and coverslipped with

Permount (Fisher).

2.3.1. Dendritic arborization analysis

Pyramidal neurons (n = 4 - 6 for each mouse) in the infralimbic

cortex (IL), identified by its cytoarchitecture and neuroanatomical

position medial to the forceps minor and cingulum between 1.3 to 1.9

mm anterior to bregma (Paxinos and Franklin 2004), were traced at 400x

and quantified using neuronal tracing software (Neurolucida,

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Microbrightfield, VT, USA). Neurons were traced only if they met the

following criteria: 1) completely and uniformly impregnated with Golgi

stain, 2) all dendrites were intact and visible, and 3) not obscured by

other stained neurons (after Chapter 3; Pyter, Reader et al. 2005). The

basolateral amygdala (BLA), identified by its location bounded by the

branched arms of the external capsule between 0.8 and 2.0 mm posterior

to bregma (Paxinos and Franklin 2004), did not contain sufficient

numbers of neurons that met the above criteria for analysis.

Representative values for each parameter measured by the software (see

results) from each animal were calculated by averaging values from all

neurons traced. Representative values calculated for each animal were

then used for further analysis.

2.3.2. Dendritic spine density analysis

Dendritic spines of the neurons were traced at 1000x using

Neurolucida software (Microbrightfield, VT, USA). Within the BLA, for

each animal average spine density was calculated by selecting six

neurons, and an unbranched, unbroken, and consistently stained

dendritic segment at least 50 µm away from the soma from each neuron

was quantified. Any protrusion originating from the dendritic shaft was

classified as a spine, and all spines along a continuous 80 µm segment

were counted for spine density analysis (after Chapter 3; Vyas, Jadhav et

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al. 2006). For the IL, average spine densities were quantified for each

animal from both basilar and apical dendrites, selected as above. For

each neuron a total of 80 µm of basilar and 80 µm apical dendrite were

quantified for spine density. For each brain region, the average spine

density calculated from each animal was then used for further

comparative analysis.

2.3.3. Corticosterone assay

Frozen plasma samples were thawed on ice and assayed for

corticosterone using a commercially available double antibody RIA kit

according to manufacturer‟s instructions (Cat# 07120102; MP

Biomedicals, Costa Mesa, CA, USA). The intra-assay coefficient of

variation was 7.9%.

2.3.4. Statistics

Repeated measures ANOVA were used to compare auditory fear

responses over time and dendritic arborization measures (Sholl analysis).

Student‟s t tests were used for comparisons between photoperiods for

physiological data, spine density analysis, within trial passive avoidance,

and for specific time points within trials for auditory fear testing. Paired t

tests were used for within photoperiod comparisons across trials in

passive avoidance testing. Data with unequal variance were log

transformed prior to analysis. All analyses were performed using SPSS

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software (v19; IBM, NY, USA) and differences were considered

statistically significant at p ≤ 0.05.

3. Results

3.1. Physiological measures

Exposure to short day lengths did not affect body mass (t(17) = 0.185,

p > 0.05) or inguinal fat pad mass (t(17) = 0.036, p > 0.05). Compared to

LD-exposed counterparts, exposure to SD reduced masses of all

reproductive tissues assessed (paired testes, t(15) = 4.934, p < 0.001;

epididymides, t(17) = 2.872, p < 0.05; seminal vesicles, t(17)= 2.655, p <

0.05 Figure 5.1 left). Short day exposure did not affect basal

corticosterone concentrations at the terminal bleed (t(16) = 0.904, p >

0.05; Figure 5.1 right).

3.2. Behavioral measures

3.2.1. Passive avoidance

As previously reported in an earlier study in this species [4],

comparing groups there were no photoperiodic differences between

groups in step through latency during training (t(7) = 0.275, p > 0.05) or

testing (t(7) = -0.737, p > 0.05). LD mice did not differ in latency to step

through 24 h after training (t(3) = 0.-0.751, p > 0.05), whereas SD mice

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displayed enhanced fear memory by taking significantly longer to step

through 24 h after training (t(4) = -0.4875, p < 0.01)(Figure 5.2A).

3.2.2. Auditory-cued fear conditioning

LD and SD mice did not differ in their freezing responses during

acquisition of the CS-US pairing across trials (repeated measures

ANOVA: F(1,15) = 0.056, p > 0.05: Figure 5.2B). Comparing baseline to

post CS-US presentation, both SD and LD mice increased freezing

behavior across acquisition (LD, t(8) = -2.714, p < 0.05; SD t(7) = -3.457, p

< 0.05), however photoperiod did not affect increases in freezing between

pre and post-stimulus (Figure 5.2B). There were no differences due to

photoperiod in freezing to context 24 h after acquisition (t(17) = 0.777, p >

0.05; not shown). Because of individual variance in inter-trial interval

freezing behavior, freezing responses during retention tone trials were

corrected by subtracting freezing during the 30s immediately prior to

tone presentation from freezing during the tone. Compared to mice

exposed to LD, SD exposure increased freezing to tone across retention

trials (repeated measures ANOVA: F(1,17) = 8.160, p < 0.05). Follow up,

within-trial, Student‟s t test comparisons show that SD mice froze

significantly more than LD counterparts during the initial (t(17) = -2.668,

p < 0.05) and the fifth (t(17) = -2.839, p < 0.05) tone presentation during

retention trials (Figure 5.2C).

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3.3. Neuronal morphology

3.3.1. Dendritic spine density

Compared to LD mice, mice exposed to SD had increased spine

density on dendrites in the BLA (t(10) = -4.196, p < 0.01; Figure 5.3). In

the IL, there were no differences in spine density due to photoperiod in

either basilar (t(13) = -0.332, p > 0.05) or apical dendrites (t(13) = 0.753, p >

0.05; Figure 5.4A).

3.3.2. IL pyramidal neuronal morphology

No differences due to photoperiod were found in IL pyramidal neurons

for cell body perimeter (t(14) = 1.002, p > 0.05), cell body area (t(14) =

1.332, p > 0.05), basilar dendrite length (t(14) = -0.066, p > 0.05), apical

dendrite length (t(14) = -0.061, p > 0.05), or total dendrite length (t(14) = -

0.074, p > 0.05)(data not shown). No differences were observed due to

photoperiod in number of intersections (repeated measures ANOVA:

F(1,14) = 410.144, p > 0.05; Figure 5.4C) or dendrite length (repeated

measures ANOVA: F(1,14) = 761.671, p > 0.05; not shown) via Sholl

analysis of dendritic arborization complexity.

4. Discussion

The present study in white-footed mice demonstrates three novel

findings. 1) Exposure to short day lengths enhances associative fear

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memory in the auditory-cued fear conditioning. 2) Photoperiod and fear

conditioning interact to increase spine density of the neurons in the BLA.

However, 3) neither photoperiod nor the interaction of photoperiod and

auditory-cued fear conditioning alter the morphology of pyramidal

neurons within the IL.

Exposure to short days in white-footed mice reduces hippocampal

volume, alters hippocampal dendritic spine density, and impairs LTP

within the hippocampus (Chapter 2; Pyter, Reader et al. 2005; Walton,

Chen et al. 2011). Pursuant to these day length induced morphological

and physiological changes, SD mice have impaired hippocampal-

dependent spatial learning and memory (Chapter 2; Pyter, Reader et al.

2005; Workman, Bowers et al. 2009; Walton, Chen et al. 2011). Non-

hippocampal learning and memory, assessed by several behavioral tests,

was previously reported to be unaffected by photoperiod (Pyter, Reader et

al. 2005). However, in a passive avoidance test SD mice had enhanced

fear memory compared to LD mice, indicating photoperiod may affect fear

memory in an opposite manner than spatial memory (Pyter, Reader et al.

2005). I have replicated these results in the current study (Figure 5.2A)

and have extended them to show SD-induced enhancement of fear

memory in the auditory-cued fear test (Figure 5.2C). These results

support my hypothesis that SD exposure enhances fear memory.

Additionally, increased spine density in the dendrites of the BLA neurons

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is associated with increased fear memory in SD mice (Figure 5.3). The

BLA is critical for encoding fear memory and imparting emotional valence

on memories (Maren 1999; McGaugh 2002; Maren and Quirk 2004;

Vyas, Jadhav et al. 2006). Furthermore, enhanced spine density within

the BLA is associated with enhanced auditory fear memory (Koseki,

Matsumoto et al. 2009). Thus, SD exposure causes hippocampal atrophy

and functional impairments in the hippocampus, whereas the amygdala

becomes hypertrophic and functionally enhanced by exposure to short

day lengths. While circadian differences have been reported in fear

learning (Rudy and Pugh 1998; Chaudhury and Colwell 2002; Eckel-

Mahan, Phan et al. 2008), the differences are generally confined to

contextual fear and not tone-cued fear (Rudy and Pugh 1998; Eckel-

Mahan, Phan et al. 2008). The current findings are not likely due to

differences in circadian patterns of fear learning and memory between

SD and LD mice as we conducted our acquisition trials during the light

phase when fear learning is strongest (Rudy and Pugh 1998; Eckel-

Mahan, Phan et al. 2008), and all of our testing occurred at the same

circadian time for both groups, thus limiting any circadian effects.

What is the role of the IL area of the mPFC in photoperiodic alteration

in fear memory? The IL/mPFC is critical for orchestrating the balance of

the BLA and the hippocampus in the formation and extinction of

emotionally charged memories, such as fear conditioning. Reciprocal

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connections between the mPFC and the BLA regulate encoding and

extinction of fear memories (Vouimba and Maroun 2011). The mPFC-

hippocampus pathway also coordinates extinction of fear memories

(Takita, Izaki et al. 1999; Milad, Wright et al. 2007; Koseki, Matsumoto et

al. 2009), and the BLA-hippocampus pathway is critical for modulating

fear memory encoding and recall (Seidenbecher, Laxmi et al. 2003;

Phelps 2004; Huff, Frank et al. 2006). Well-orchestrated coordination of

all the three brain regions is critical for encoding, recall, and extinction of

fear memories (Maren and Quirk 2004; Quirk and Mueller 2008;

Guimarais, Gregorio et al. 2011). Although SD mice do show cognitive

inflexibility in spatial reversal learning (Pyter, Reader et al. 2005),

presumably mediated by the IL (Li and Shao 1998), the current

neuroanatomical findings (Figure 5.4) do not support a specific role for

altered IL function in the SD enhancement of fear memory. However, the

role of photoperiodic impairment in the mPFC-hippocampus pathway in

cognitive flexibility in both spatial fear learning remains undescribed.

One of the hallmarks of photoperiodic responses in rodents is the

involution of the gonads by exposure to short day lengths (Figure 5.1) via

inhibition of the hypothalamic-pituitary-gonadal (HPG) axis, which

results in low gonadal sex steroid concentrations (reviewed in Chapter 1;

Walton, Weil et al. 2011). Gonadal sex steroids can influence neuronal

morphology in the regions above implicated in fear memory (Cooke and

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Woolley 2005; McEwen 2010). Although gonadectomy can recapitulate

some of the appropriate photoperiodic responses, gonadal steroids are

not the sole contributing factor to the suite of behavioral and

physiological responses in photoperiodic rodents (reviewed in Walton,

Weil et al. 2011). Indeed, gonadal steroids enhance fear memory;

estrogens facilitate fear conditioning by upregulating CRH expression in

the amygdala (Jasnow, Schulkin et al. 2006), and androgens

(testosterone and dihydrotestosterone) also facilitate conditioned fear, but

the enhanced effects are mediated in the hippocampus, and not the

amygdala (Edinger, Lee et al. 2004). Thus, reduced sex steroid

concentrations in SD mice should lead to impaired fear memory, which

argues strongly against a significant role of gonadal steroids in SD

enhancement of fear memory. However, we currently have ongoing

experiments to study the effects of sex steroids on hippocampal- and

amygdala-mediated memory.

In addition to inhibiting the HPG axis, short days enhance HPA

axis responsiveness and negative feedback in white footed mice (Pyter,

Adelson et al. 2007). Although photoperiod does not affect baseline

corticosterone concentrations (Figure 5.1; Pyter, Adelson et al. 2007), SD

mice display increased HPA axis responsiveness to stressors and

increased negative HPA axis feedback, potentially regulated at the level of

the hippocampus by SD elevation of hippocampal glucocorticoid and

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mineralocortocoid receptors (Pyter, Adelson et al. 2007). Photoperiod

alterations in the HPG and HPA axis have adaptive significance for this

species. To survive the energetic bottleneck of reduced food availability

and increased thermogenic demands during the short days of winter,

energy is conserved by reduction of reproductive tissue mass and

reproduction-associated behaviors (reviewed in Chapter 1; Walton, Weil

et al. 2011). Additionally, activation of the HPG axis (glucocorticoid

response to stressors) to mobilize energy for the fight-or-flight response is

energetically expensive, thus increased regulation (efficiency) of the HPA

axis in short days may be an adaptive response to conserve energy

(Pyter, Adelson et al. 2007).

In the North American temperate zone home range of white-footed

mice, photoperiodic changes in habit alter behavior and distribution

(King 1968). Short day mice inhabit an environment that is both

energetically demanding and devoid of dense understory cover, thus the

alterations in endocrine responses to stress (HPA axis) and behavioral

responses to fearful stimuli (fear memory) described above may provide

an adaptive advantage to survive in their winter habitat. During the short

days of winter forest understory is greatly reduced and leaf cover is

minimal (Bratton 1976), and small photoperiodic mammals, including

white-footed mice, alter their habitat use and movement to areas within

their home range with maximum leaf understory density for predator

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avoidance (Kaufman, Peterson et al. 1983; Litvaitis, Sherburne et al.

1985; Barnum, Manville et al. 1992; Bowers and Dooley 1993). To our

knowledge, the effect of photoperiod on predator avoidance has never

been directly tested on white-footed mice, however short days increase

predator avoidance behavior in other rodent species (Herman and Valone

2000; Borowski 2002). In short days, enhanced fear memory may be

advantageous for survival by offsetting the interaction of reduced

available ground cover for predator avoidance with impaired

hippocampal-mediated spatial navigation (Chapter 2; Pyter, Reader et al.

2005; Walton, Chen et al. 2011). Thus, it is possible that the

photoperiodic differences in fear responses reported here are ecologically

relevant and are an integral part of a suite of adaptive responses to short

days, which include changes in physiology and hippocampal function.

In summary, short day exposed white-footed mice have reduced

hippocampal volume (Pyter, Reader et al. 2005), increased HPA axis

negative feedback (Pyter, Adelson et al. 2007), increased fear memory

(Figure 5.2), and increased connectivity within the BLA (Figure 5.3).

Among human PTSD patients, reduced hippocampal volume is

associated with susceptibility to PTSD, but not severity of symptoms

(Yehuda and LeDoux 2007), amygdala activity is positively correlated

with symptom severity (Dickie, Brunet et al. 2011), and PTSD patients

have enhanced HPA axis negative feedback (Yehuda and LeDoux 2007).

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One of the greatest research challenges of mechanisms involved in

psychiatric disorders is the difficulty in replicating the symptoms of the

disease in animal models (Miller and McEwen 2006), and it is important

to develop new ethologically-relevant models for translational research of

psychiatric disorders (Shekhar, McCann et al. 2001; Clinchy, Schulkin et

al. 2010). In the laboratory setting, a simple day length manipulation in

white-footed mice, exposure to short days, concurrently replicates three

main features of PTSD and enhances fear memory responses. Taken

together, the photoperiodic modulation of brain fear circuits and fear

memory in white-footed mice described in this, and previous studies,

may argue for the potential utility of this species as an additional, yet

unique, animal model to research how a single environmental factor (day

length) can interact with genes to alter phenotype to resemble a human

psychiatric disorder, such as PTSD.

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Figure 5.1 SD exposure reduced the mass of the reproductive tissues (left), but

did not alter basal plasma corticosterone concentrations (right). * p < 0.05

Student‟s t test. 17

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Figure 5.2 SD exposure enhances fear memory in P. leucopus. A) LD mice did

not differ in step through latency in the passive avoidance test 24 h after

training, whereas SD mice hesitated longer to step through. B) LD and SD mice

did not differ in freezing responses during acquisition in the auditory-cued fear

conditioning test. Both LD and SD mice increased freezing across acquisition.

C) Freezing responses to tone 24 h after acquisition are increased in SD mice

compared to LD mice. Follow up within-trial comparisons show that SD mice

froze significantly more than LD counterparts during the initial tone

presentation and during tone 5. *p < 0.05: A) paired t test, C) Student‟s t test.

18

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Figure 5.3 Effects of SD exposure and fear conditioning on dendritic spine

density in the basolateral amygdala. Upper panel: SD mice have increased

dendritic spine density in the BLA. Lower panel: 1000x photomicrographs of

representative dendritic segments of BLA neurons from LD (left) and SD (right)

mice. Scale bar = 10 µm. * p < 0.05 Student‟s t test. 19

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Figure 5.4 Effects of SD exposure and fear conditioning on pyramidal neuron

morphology in the infralimbic cortex. A) SD exposure did not alter spine density

on either apical or basilar dendrites. B) 200x photomicrograph of a

representative IL pyramidal neuron (upper) and its Nuerolucida reconstruction

upon which Sholl analysis was performed (lower). C) Exposure to SD did not

alter complexity of pyramidal cell dendritic arborization (Sholl analysis) of

infralimbic cortical pyramidal neurons. 20

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Chapter 6: Photoperiod and Olfactory Bulb Neurogenesis.

1. Introduction

In the previous chapters, I explored the effects of photoperiodic

alterations in brain structure and function, demonstrating behavioral,

structural, and functional outcomes of smaller brains and hippocampi in

short days. In the next two chapters, I will assess a factor which may

contribute the reduction in brain volume in short day mice: the role of

photoperiod on altered recruitment of new neurons in the brain.

Neurogenesis in discrete areas of the mammalian brain is an

ongoing process which continues throughout adulthood. Nascent granule

neurons arise in the subgranular zone (SGZ) of the hippocampus and are

incorporated into the dentate gyrus as they mature. Neural progenitor

cells arising from the subventricular zone (SVZ) of the lateral ventricles

migrate via the rostral migratory stream (RMS) to the olfactory bulbs.

Once in the olfactory bulb (OB), they migrate radially outward and

mature; mainly becoming interneurons (reviewed in Lazarini and Lledo

2011). Although the function of adult neurogenesis in the hippocampus

has been well described (Deng, Aimone et al. 2010), the behavioral and

functional outcomes of adult neurogenesis within the olfactory bulb

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remain unspecified (reviewed in Lazarini and Lledo 2011; Kageyama,

Imayoshi et al. 2012).

Olfactory information enters the central nervous system through

the sensory olfactory epithelium in the nasal cavities (and the

vomeronasal organ in many species), and is then relayed through the

olfactory nerve to the glomerular layer of the olfactory bulb, synapsing on

the tufted and mitral cells, which are the projection neurons of the

olfactory bulb. In addition to forming synaptic connections with the

tufted and mitral cells of the olfactory bulb, the inhibitory interneurons

of the granule cell layer and periglomerular cells also receive input from

sensory neurons in the olfactory epithelium (Whitman and Greer 2009).

In adult mammals, the granule and periglomerular cells, which modulate

the projection cells from the olfactory bulb, are continuously replaced via

neurogenesis (Lledo and Saghatelyan 2005). Continuous adult

neurogenesis is necessary to maintain innate olfactory responses

(Sakamoto, Imayoshi et al. 2011) and it has been recently hypothesized

that the continuous turnover of the inhibitory granule cells in adulthood

is responsible for optimizing pattern separation, thus optimizing

encoding of olfactory information (Sahay, Wilson et al. 2011).

In a naturalistic context, optimization of olfactory information is

critical for fitness, especially in animals that rely primarily on olfactory

information for social communication and predator avoidance, such as

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small rodents. These social and avoidance cues can vary seasonally

(Ferkin and Gorman 1992; Heth, Nevo et al. 1996; Borowski and

Owadowska 2010). Photoperiodism is the biological ability of animals to

track day length and to make seasonally appropriate adaptive responses

to survive differing seasonal energetic demands in non-tropical latitudes

(reviewed in Chapter 1; Walton, Weil et al. 2011). Photoperiodic changes

in olfaction and odor responsiveness, which are generally associated with

reproduction, have been identified in many species across vertebrate and

invertebrate taxa (reviewed in Nelson, Denlinger et al. 2010). Seasonal

changes in hippocampal neurogenesis have been documented in several

photoperiodic rodent species (Huang, DeVries et al. 1998; Galea and

McEwen 1999; Smith, Pencea et al. 2001; Ormerod and Galea 2003;

Bartkowska, Djavadian et al. 2008); however, few studies have

investigated the role of photoperiod on SVZ/OB neurogenesis in these

species (Huang, DeVries et al. 1998; Bartkowska, Djavadian et al. 2008),

and to my knowledge, no studies have demonstrated a functional

difference in olfaction associated with photoperiodic changes in olfactory

bulb neurogenesis in rodents.

As previously described, white-footed mice (P. leucopus) are small

photoperiodic rodents, indigenous to central and northern regions of the

United States east of the Rocky Mountains (King 1968). As with other

well-studied photoperiodic small rodents, the reproductive,

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immunological, and behavioral responses to photoperiod have been well

described in this species (Chapter 2; Chapter 5; Lynch 1973; Pyter, Neigh

et al. 2005; Pyter, Reader et al. 2005; Walton, Chen et al. 2011; Walton,

Haim et al. 2012). However, the effects of photoperiod on olfactory bulb

neurogenesis and associated olfactory behavior remain undescribed.

Toward this end, I assessed the role of photoperiod on olfaction, olfactory

bulb neuronal morphology, and olfactory bulb neurogenesis in male

white-footed mice by asking the following questions: 1) does photoperiod

alter cell proliferation and neurogenesis in the olfactory bulbs of this

species, 2) does photoperiod alter olfactory bulb neuronal morphology,

and 3) are photoperiod-mediated changes in olfactory bulb neurogenesis

or morphology associated with changes in an olfactory behavior?


2.1. Animals

Sixty-five adult (>55 days of age) male white-footed mice

(Peromyscus leucopus) from our breeding colony were used in this study.

Animals were housed individually in polypropylene cages (27.8 x 7.5 x 13

cm) with a constant temperature and humidity of 21 ± 5°C and 50 ±

10%, respectively, and ad libitum access to food (Harlan Teklad 8640

rodent diet, Indianapolis, IN) and filtered tap water. Mice were either

126

housed in reversed long days (LD; 16 h light/day), or in short days (SD; 8

h light/day) for 10-15 weeks, depending on experiment. Photoperiodic

responsiveness (SD-induced reduction of reproductive tissue mass) was

verified for mice in all experiments. All procedures were approved by the

Ohio State University Institutional Animal Care and Use Committee and

comply with guidelines established by the National Institutes of Health

(Institute of Laboratory Animal Resources, U.S. 1996).

2.2. Experiment 6.1: Effects of photoperiod on olfactory bulb cell

proliferation and neurogenesis

2.2.1. BrdU injections

To estimate neurogenesis, after 10 weeks of photoperiod exposure,

17 mice (n = 11 LD, n = 6 SD) were given daily IP BrdU injections (50

mg/kg in 0.1 ml saline; Sigma-Aldrich, St. Louis, MO, USA) for 6

consecutive days. Prior to perfusions, mice remained undisturbed for 4

weeks following the conclusion of injections. The time of injections was

randomized each day to control for circadian differences in cell division

among the treatment groups. To estimate olfactory bulb cell proliferation,

after 14 weeks in photoperiod, 17 mice (n = 9 LD, n = 8 SD) were given a

single intraperitoneal (IP) injection of the cell division marker,

bromodeoxyuridine (BrdU 50 mg/kg in 0.1 ml saline) 1.5-2.5 h prior to

perfusion.

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2.2.2. Tissue collection and histology

Following BrdU treatment (proliferation, 1.5-2.5 h; neurogenesis, 4

wk), mice were deeply anesthetized with sodium pentobarbital (Abbott

Laboratories, North Chicago, IL, USA) and transcardially perfused with

50 ml of ice-cold saline followed by 75 ml of 4% paraformaldehyde in

0.1M PBS. Paired testes were removed and weighed to determine

reproductive responsiveness to photoperiod treatment. Brains were

removed, post-fixed in paraformaldehyde for 3 h at room temperature,

transferred into 0.2 M phosphate buffer overnight at 4°C. The next day,

brains were transferred to 30% sucrose in 0.1 M PBS until permeated,

then frozen and stored at -70°C. Using a cryostat, brains were cut into 25

µm sections and thaw mounted onto positively charged slides (Superfrost

Plus, Fisher Scientific, Pittsburgh, PA, USA). For immunohistochemistry,

one series of every eighth section from all mice were fluorescently triple-

labeled for BrdU, glial fibrillary acidic protein (GFAP), and neuronal

nuclei (NeuN) to detect newly-born, glial and neuronal cells, respectively.

Briefly, slides were rinsed in 0.1 M TBS for 30 min, incubated in 2N HCl

at 37°C for 15 min to denature DNA, and then were immediately

transferred to 0.1M borate buffer for 10 min at room temperature.

Following 3 rinses in TRIS buffered saline (TBS), slides were blocked (3%

donkey serum in TBS + 0.5% Triton-X + 0.2% sodium azide) for 4 h at

room temperature with constant agitation. A primary antibody mixture

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[1:200 rat anti-BrdU (Accurate Chemical & Scientific Corporation,

Westbury, NY, USA), 1:200 mouse anti-NeuN (Chemicon International,

Temecula, CA, USA), 1:500 rabbit anti-GFAP (Sigma-Aldrich, St. Louis,

MO, USA)] was applied to all slides for 24 h at room temperature with

constant agitation. Slides were thrice rinsed in TBS and a secondary

antibody mixture (1:200 anti-rat Alexa594, 1:200 anti-mouse Alex488,

1:500 anti-rabbit Alexa647, all raised in donkey; Molecular Probes,

Carlsbad, CA, USA) was applied to all slides for 3 h at room temperature

with constant agitation. Slides were rinsed in TBS and then coverslipped

with Fluoromount (Fisher Scientific, Pittsburgh, PA, USA). A mean

(±SEM) of 13.9 ± 0.4 sections were analyzed per mouse. BrdU+ cells were

manually counted in each section within the periglomerular and

plexiform regions of the olfactory bulbs at 400X magnification with a

fluorescent microscope. Sections were designated as being rostral (~300

µm), central (~500 µm), or caudal (~750 µm) from the tip of the olfactory

bulbs. The number of BrdU+ cells within the granule cell layer was

estimated by manually counting the number of cells within 3 (dorsal,

medial, ventral) 300 µm x 225 µm grid boxes of the granule cell layer at

400X magnification. To determine the cellular phenotypes of BrdU+ cells

in the granule cell layer of the neurogenesis brains (i.e., 6 injections of

BrdU + 4 weeks), between 35 - 60 cells (mean 46.7 ± 9.3) from 8 - 15

sections (mean 12 ± 2.7) per animal were analyzed at 400X magnification

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using a confocal laser scanning microscope (Zeiss 510 META,

Thornwood, NY, USA) with excitation wavelengths of 488, 543, and 633

nm at the Microscope and Imaging Facility at Ohio State University.

2.3. Experiment 6.2: Effects of photoperiod on dendritic morphology in

the olfactory bulb granule cell layer.

2.3.1. Olfactory bulb dendritic morphology

After 10 weeks in respective photoperiods, a separate cohort of

mice (n = 5 LD, n = 8 SD) were rapidly decapitated. Brains with complete

and intact olfactory bulbs were removed and processed for Golgi staining

according to the manufacturer‟s protocol (FD Rapid GolgiStain Kit, FD

Neurotechnologies, Ellicott City, MD, USA) as previously described

(Chapter 3; Chapter 5; Pyter, Reader et al. 2005). Briefly, olfactory bulbs

were cut coronally in 80 µm sections on a cryostat and mounted on 3%

gelatin-coated slides, dried for 7-10 days, then counterstained with

cresyl violet before dehydration and coverslipping. Granule cells in the

olfactory bulbs (n = 5/mouse) were traced using a camera lucida at 400X

magnification (Neurolucida, MicroBrightField, Williston, VT, USA).

Dendritic spines were traced on five 10 µm distal segments of each

neuron at 1000X magnification on the terminal tips of randomly chosen

granule cell dendrites that had at least one branch point. Using the

accompanying software (NeuroExplorer, MicroBrightField) dendritic

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complexity (via Sholl analysis), dendritic length, and spine density were

calculated. Throughout analyses, all samples were number-coded so the

experimenter was unaware of the treatments.

2.4. Experiment 6.3: Effects of photoperiod on investigation of conspecific

male urine.

2.4.1. Habituation-dishabituation test

After 10 weeks of exposure to photoperiod, mice (n = 9 LD, n = 9

SD) were tested under dim red light at the beginning of the dark phase in

an olfactory habituation-dishabituation assay. On the first day of the

testing paradigm, mice were transferred in their home cages to the

behavioral testing room at the onset of the dark phase and allowed to

habituate for 1 h, then returned to their respective vivarium rooms. On

day 2, mice were transferred to the testing room and allowed to habituate

for a minimum of 30 min prior to olfactory testing. For odorants, fresh

urine from six experimentally naive male P. leucopus (3 LD, 3 SD), that

were unassociated with the behavioral testing, was collected immediately

prior to olfactory testing, pooled in a sterile 1.5 ml micro centrifuge tube,

and held on ice along with a fresh aliquot of ddH2O for the duration of

testing. All olfactory testing was conducted in one single session using

the same odorants for all mice. For odor exposure, the wire food hoppers

were removed from their home cages and replaced with an identical

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sterile empty hopper. Mice were allowed to habituate to the new hopper

for 5 minutes, and then a 1000 µl pipette tip containing the scented filter

paper was presented 6 times for 3 min with a 1 min interval between

presentations. The first 3 presentations were ddH2O and the final 3 were

male urine. Odorants were presented in the following manner:

immediately prior to odor presentation for each mouse, 25 µl of the

odorant was placed on a 1 cm2 piece of filter paper and inserted into the

wide end of a sterile 1000 µl pipette tip 5 mm below the edge, and then

the pipette tip was inserted 3 cm, open end down, through the wire cage

lid at the front of the cage. The location of the odorant source within the

cage was consistent for all mice tested. All behavior was recorded and

videos were scored for time spent directly investigating the pipette tip

(rearing up and placing snout within 1 cm of the open end of the pipette

tip), using The Observer software package (v8.0, Noldus, Leesburg, VA,

USA), by an observer unaware of both animal groups and odor

treatment.

2.5. Statistical analyses

Olfactory habituation-dishabituation data were analyzed by

repeated measures ANOVA, with investigation time as the repeated

measure and photoperiod as the between subjects factor. Significant

results were followed up by within trial two-tailed Student‟s t-tests. Two-

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tailed Student‟s t-tests were used for comparisons between photoperiods

for reproductive responsiveness, dendritic length and spine density, and

BrdU measures of cell proliferation and neurogenesis. Repeated

measures ANOVAs were used to compare photoperiod effects on dendritic

complexity (Sholl analysis). Data with unequal variance were log

transformed prior to comparisons. SPSS software (v.19, IBM, Armonk,

NY, USA) was used for all analyses, and all comparisons were considered

statistically significant if p ≤ 0.05 as calculated by SPSS.

3. Results

3.1. Reproductive responses to photoperiod

For all mice, exposure to short day lengths for 10-15 weeks

reduced paired testes mass (t55 = 4.508, p ≤ 0.05) and paired testes

mass corrected for body mass (t53 = 4.390, p ≤ 0.05) (Figure 6.1).

3.2. Experiment 6.1

Mice treated to examine neurogenesis (i.e., 6 BrdU injections + 4

weeks; Figure 6.2) displayed more BrdU+ cells in the glomerular layer

(Gl), plexiform layers (IPl/EPl), and granular cell layer (GrO) across the

rostro-caudal extent of the olfactory bulbs compared with mice treated to

examine cell proliferation (i.e., 1 BrdU injection + 2 h) (Figure 6.3). There

were no photoperiodic differences in progenitor cell (BrdU+) proliferation

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or 4 week survival (neurogenesis) in the anterior (p > 0.05; Figure 6.3A,B)

or the medial olfactory bulb (p > 0.05; Figure 6.3C,D). Although there

were no photoperiodic differences in proliferation in the posterior

olfactory bulbs (p > 0.05; Figure 6.3E), exposure to short days increased

4 week progenitor survival in the posterior plexiform (t15 = -2.256, p ≤

0.05) and posterior granule cell layers (t15 = -2.524, p ≤ 0.05)(Figure

6.3F). Including all layers across the entire rostro-caudal extent of the

olfactory bulbs, there were neither differences due to photoperiod in cell

proliferation (p > 0.05; Figure 6.3G), nor differences in progenitor

survival after 4 weeks in the anterior or medial olfactory bulbs (p > 0.05;

Figure 6.3F). However, within the posterior olfactory bulb, short days

increased 4 week progenitor survival (t15 = -2.102, p ≤ 0.05; Figure 6.3H).

Within the granule cell layer, the majority of BrdU+ cells were co-

labeled with the neuronal marker NeuN, with very sparse GFAP+ or cells

labeled with BrdU+ alone (Table 6.1). No differences in the percentages of

different BrdU+ cell phenotypes were observed between photoperiods

(Table 6.1).

3.3. Experiment 6.2

3.3.1. Olfactory bulb granule cell morphology

Exposure to short day lengths did not alter granule cell dendritic

complexity in the olfactory bulbs measured by Sholl analysis (F1,62 =

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0.018; p = 0.89; Figure 6.4A) and branch order analysis (F1,9 = 0.000; p =

0.99; Figure 6.4B). Short days did not alter dendritic length (t11 = 0.033,

p = 0.97) or dendritic spine density (t11 = -0.932, p = 0.37) (Figure 6.4C).

3.4. Experiment 6.3

3.4.1. Habituation-dishabituation

Compared to long day mice, exposure to short days did not affect

time spent investigating water odor across trials (F1,16 = 0.146; p = 0.70);

however, SD exposure reduced time spent investigating male urine (F1,16

= 4.358; p ≤ 0.05: Figure 6.5A). Follow up within-trial t-tests revealed

that SD mice did not differ from LD mice during the initial exposure to

male urine (t16 = 1.053; p = 0.31). SD mice did not spend less time

investigating male urine during trial 2 (t16 = 1.892, p = 0.08), but SD mice

spent significantly less time investigating during trial 3 (t16 = 2.452, p ≤

0.05: Figure 6.5A). There were no differences due to photoperiod in

latency to approach the odor source (F1,16 = 1.347; p = 0.26) (Figure

6.5B).

4. Discussion

In the current study, adult male white-footed mice exposed to

short days did not alter measurements of neuronal morphology of the

granule cells in the olfactory bulb (Figure 6.4), or alter proliferation of

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BrdU+ progenitor cells in the olfactory bulb (Figure 6.3), which are

mostly comprised of neurons (Table 6.1). However, SD exposure

increased BrdU+/NeuN+ cells 4 weeks after injections in the anterior

plexiform and granule layers of the olfactory bulbs (Figure 6.3F,H).

Concurrent with an increase in neurogenesis in the caudal olfactory

bulbs, SD mice also habituate faster (reduced investigation time) to

conspecific male urine odors (Figure 6.5). These findings demonstrate

that, in a photoperiodic rodent, day length can affect both olfactory-

mediated behavior and olfactory bulb neurogenesis, which may underlie

olfactory learning.

Neural progenitor cells arising from the subventricular zone (SVZ)

of the lateral ventricles migrate via the rostral migratory stream (RMS) to

the olfactory bulbs, and the caudal olfactory bulbs are closest to the SVZ

origin of the proliferating cells, which may account for the differences

found in the caudal olfactory bulb in the current study. However, BrdU+

cell numbers 4 weeks after injection did not differ across the rostro-

caudal extent of the olfactory bulb (Figure 6.3), which argues against this

possibility. It is also possible that subtle SD increases in SVZ

proliferation not detected in this study, altered survival during the

migratory process along the RMS from the SVZ to the OB (Peretto,

Merighi et al. 1999), altered programmed cell death of OB progenitor cells

(Winner, Cooper-Kuhn et al. 2002) in LD mice, or some combination of

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these factors, contributes to the current findings. Subtle photoperiodic

differences in olfactory bulb cell death in Sorex shrews, identified by

TUNEL labeling, have been reported (Bartkowska, Djavadian et al. 2008),

although overall rates were very low. The influence of photoperiod on

these factors in this species remains uninvestigated, thus, we cannot

rule out the possibility that subtle alterations in proliferation or

progenitor migratory survival, not detected in the current study,

contribute to the increase in olfactory bulb neuron survival found in SD

mice.

Neuronal precursors arising from the SVZ, or from a pool of

quiescent progenitor cells in the central OB (Liu and Martin 2003;

Mandairon, Jourdan et al. 2003), continuously replace a population of

the inhibitory interneurons (granular and periglomerular) of the adult

olfactory bulb. Two recent studies ablating SVZ neurogenesis in mice

have shown opposing roles for newly born neurons in the olfactory bulb:

they may be preferentially involved in formation of odor memories

(Breton-Provencher, Lemasson et al. 2009), or neurogenesis is uncoupled

from olfactory bulb function (Valley, Mullen et al. 2009). Although more

research is necessary to parse out the specific role of neurogenesis in

olfaction, it is possible that turnover and overproduction of new neurons

in the olfactory bulb is critical for optimally encoding new olfactory

memories (Sahay, Wilson et al. 2011), whereas old neurons (arising in

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perinatal development) are responsible for general olfaction (Winner,

Cooper-Kuhn et al. 2002; reviewed in Nissant and Pallotto 2011).

However, continuous adult neurogenesis is necessary to support innate

olfactory-dependent behavioral responses (Sakamoto, Imayoshi et al.

2011) and olfactory experience modulates the turnover of OB olfactory

bulb new neurons in a spatial and temporal manner (Sawada, Kaneko et

al. 2011). Although the role of olfactory bulb neurogenesis remains

unclear, taken together, a growing body of evidence, including the

current study, supports the necessity of neurogenesis for behavioral and

physiological plasticity within the olfactory bulb.

Within the olfactory bulbs, odors are topographically represented

by glomeruli (Pinching and Doving 1974; Vassar, Chao et al. 1994), and

odor-specific activation of patterns of glomeruli allow for differential

threshold sensitivity (Wachowiak and Cohen 2001) and encoding of

olfactory memories by the cortex (Buck 2004). Additionally, water-soluble

odorants preferentially activate glomeruli in the posterior olfactory bulb

(Johnson, Arguello et al. 2007). In the current study, SD exposure

increased neurogenesis in the posterior olfactory bulb (Figure 6.3H), and

in accordance with the topography of water-soluble odors, SD mice had

altered responsiveness to conspecific male urine (Figure 6.5A). Reduced

investigation time of urine may not be a function of short-term

habituation, but could potentially reflect altered motivation not related to

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the odor (Wilson 2009). However, the current data argue against this

possibility because SD mice did not differ from their LD counterparts in

latency to investigate all odor presentations (Figure 6.5B) or investigation

behavior of a socially neutral water-soluble odorant (water; Figure 6.5A).

One of the hallmarks of photoperiodic rodents is day length-

dependent plasticity of neural systems, including neuroendocrine and

behavioral circuits (reviewed in Chapter 1; Walton, Weil et al. 2011).

Photoperiodic responses in some rodent species are coupled to the

olfactory system. In nonphotoperiodic rodents, olfactory bulbectomy

(OBX) can unmask photoperiodic responsiveness, as demonstrated in

both lab rats (Rattus norvegicus; Nelson, Moffatt et al. 1994) and house

mice (Mus musculus; Nelson 1990). Olfactory input is necessary for

normal photoperiodic responses in gray mouse lemurs (Seguy and Perret

2005) and Syrian hamsters (Pieper, Tang et al. 1984; Clancy, Goldman et

al. 1986), potentially due to OBX-dependent increases in gonadotropins.

However, OBX in Siberian hamsters has no affect on photoperiodic

responses (Prendergast, Pyter et al. 2009). Thus, the exact role of

olfaction and the olfactory bulbs in photoperiodic responses is species-

specific, and in white-footed mice remains uninvestigated.

In common with the current study, seasonal differences in odor

responsiveness have been reported for many mammalian species,

ranging from blind mole rats (Heth, Nevo et al. 1996), to meadow voles

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(Ferkin and Gorman 1992; Perrot-Sinal, Ossenkopp et al. 2000), to

humans (Goel and Grasso 2004). Altered responses to presentation of

male urine discovered in the current study may be related to

photoperiodic changes in social structure in white-footed mice. During

the breeding season, male white-footed mice are territorial and aggressive

toward intruding males, whereas exposure to short days facilitates

prosocial behaviors as mice form communal groups (Wolff and Durr

1986), most likely for thermal energetic conservation. Thus, the

facilitation of habituation to conspecific odors we report here in SD mice

may support these prosocial behaviors.

In addition to altering behavioral circuits, exposure to short days

in photoperiodic rodents, including white-footed mice, alters

neuroendocrine and reproductive circuits; reducing paired testes mass

(Figure 6.1) and concentrations of sex steroids and gonadotropins

(reviewed in Chapter 1; Walton, Weil et al. 2011). Although adult

neurogenesis has been shown to be modulated by sex steroids (Shingo,

Gregg et al. 2003; Galea, Spritzer et al. 2006), the effects are temporally-,

sex-, and species-specific (reviewed in Galea 2008; but see Lavenex,

Steele et al. 2000; Ormerod and Galea 2003). Additionally, neurogenesis

in the SGZ and the SVZ are regulated by different mechanisms (Brown,

Cooper-Kuhn et al. 2003), and regulation of neurogenesis in some

photoperiodic rodents is mechanistically different between SD and LD

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animals (Huang, DeVries et al. 1998; Bartkowska, Djavadian et al. 2008).

In common with Syrian hamsters (Huang, DeVries et al. 1998), SD white-

footed mice in the current study displayed increased neurogenesis in the

olfactory bulb (Figure 3H). In contrast, photoperiod does not affect SGZ

neurogenesis in Eastern Gray squirrels (Lavenex, Steele et al. 2000), and

SD decreases SGZ and SVZ proliferation and neurogenesis in two species

of photoperiodic shrews (Bartkowska, Djavadian et al. 2008). Thus, we

cannot make generalizations about the interaction of steroids and

photoperiod in the regulation of adult neurogenesis, as this needs to be

addressed in species-specific manner.

In summary, the current study demonstrates that in white-footed

mice, photoperiod alters neurogenesis in the olfactory bulb, without

affecting proliferation or granule cell neuronal morphology. The short-day

increase in neurogenesis within the caudal olfactory bulb, the region

known to respond to water-soluble odorants, is associated with altered

behavioral responses to conspecific male urine. These changes in

behavior associated with altered olfactory bulb neurogenesis may

represent a neural substrate responsible, in part, for photoperiodic

changes in social structure in this species. In addition, the current study

adds to the growing body of literature describing the role of olfactory bulb

neurogenesis in olfactory behaviors, and provides novel insight into the

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role of photoperiod in the regulation of olfactory bulb-dependent

plasticity.

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Figure 6.1 Reproductive responses to photoperiod. Exposure to short day

lengths for 10-15 weeks reduced absolute paired testes mass (left) and paired

testes mass controlling for body mass differences (right). * p ≤ 0.05 21

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Figure 6.2 Olfactory bulb photomicrographs from LD (left panel) and SD (right

panel) white-footed mice. Within each panel, clockwise from lower left: GFAP

(blue), NeuN (green), BrdU (red), and merged images. Scale bar = 50 μm. 22

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Figure 6.3 Progenitor cell proliferation and survival in the olfactory bulb. There

were no effects due to photoperiod in progenitor cell proliferation 2 h after BrdU

injection (A,C,E,G). Exposure to short days increased progenitor survival in the

posterior olfactory bulb (H). The majority of these differences were in the

posterior plexiform and granule cell layers (F), whereas there were no

differences due to photoperiod the medial (D) or anterior (B) olfactory bulb. All

abbreviations after Paxinos & Franklin (2004): Gl, glomerular layer of the

olfactory bulb; IPl, internal plexiform layer of the olfactory bulb; EPl, external

plexiform layer of the olfactory bulb; GrO, granule cell layer of the olfactory

bulb. * p ≤ 0.05 23

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Figure 6.4 Olfactory bulb neuron morphology. Short day exposure did not alter

dendritic intersections (A; Sholl analysis) or branch order of the dendrites (B).

There were no differences due to photoperiod in dendrite length (C, left) or spine

density (C, right). Representative Neurolucida tracings of olfactory bulb granule

cells (D). p > 0.05 in all cases. 24

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Figure 6.5 Olfactory habituation-dishabituation test. A) Photoperiod had no

effect on habituation to water (H2O), whereas mice exposed to short days spent

significantly less time investigating male urine (U, p ≤ 0.05 repeated measures

ANOVA). SD mice did not differ from LD mice in initial investigation time of

novel male urine; however they habituated to the odor faster than their LD

counterparts. B) Photoperiod did not alter latency to investigate the presented

odor within or across trials. # p ≤ 0.05 repeated measures ANOVA, * p ≤ 0.05

Tukey‟s HSD. 25

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Table6.1

% (±SEM) of BrdU+ cells

NeuN+ GFAP+ Unlabeled

ALL MICE 99.75 0.25 0.17

LD 99.54 ± 0.46 0.46 ± 1.22 0.86 ± 0.32

SD 100 ± 0.00 0 0

Table 6.1 Phenotype of progenitor cells 4 weeks after BrdU injections in the

granule cell layer of the olfactory bulb of LD and SD exposed male P. leucopus. 2

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Chapter 7: Photoperiod and Hippocampal Neurogenesis.

1. Introduction

As previously noted in Chapter 6, neurogenesis in discrete areas of

the mammalian brain is an ongoing process that continues throughout

adulthood. Neurogenic niches have been identified in multiple areas of

adult mammalian brains (Gould 2007), however the subgranular zone

(SGZ) of the dentate gyrus (DG) represents one of the most active

neurogenic niches. Nascent progenitor cells arise in the SGZ of the DG of

the hippocampus, and generally are subsequently incorporated into the

dentate gyrus as granule cells (reviewed in (Zhao, Deng et al. 2008).

However, proliferating cells in the SGZ have several fates: they can die

off, become neurons (neurogenesis), become glia (gliogenesis), or remain

undifferentiated and quiescent (reviewed in Gage 2000). It is widely

accepted that neurogenesis in the hippocampus supports the formation

of new memories and can affect cognition, affective behaviors, and spatial

learning and memory, and there are a multitude of factors which

regulate proliferation, differentiation, and maturation of the progenitor

cells (reviewed in Zhao, Deng et al. 2008).

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Neurogenesis in adult vertebrate brains was initially identified by

Joseph Altman (Altman 1962). The definitive functional characterization

of neurogenesis in adult vertebrate brains in a naturalistic context has

been credited to Fernando Nottebohm when, while searching for an

explanation in changes in the brain volume of canaries (Serinus canarius)

in relation to seasonal breeding (Nottebohm 1981), he injected birds with

[3H]thymidine to label dividing cells by being incorporated into DNA in

the S-phase and discovered that new neurons were being produced and

integrated into functional song circuits (Goldman and Nottebohm 1983;

Paton and Nottebohm 1984). In addition to identifying neurogenesis in

adult vertebrates was the finding that altered neurogenesis was

associated with the observed changes in brain volume that were

dependent upon breeding status (Nottebohm 1981; Paton and Nottebohm

1984). This pattern has been reported in other bird species (Sherry and

Hoshooley 2010), and as discussed in Chapter 1, changes in breeding

status in birds are driven by changes in photoperiod, and thus

photoperiod can regulate neurogenesis in adult vertebrate brains.

Photoperiodic changes in brain volume have also been documented in

some adults of rodent species such as voles (Clethrionomys glareolus),

shrews (Sorex araneus, Sorex minutus), and white-footed mice (P.

leucopus) (Yaskin 1994; Pyter, Reader et al. 2005; Bartkowska,

Djavadian et al. 2008; Yaskin 2009). However, the role of neurogenesis

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in photoperiodic mammalian hippocampal volume fluctuation remains

unclear as some rodent species, such as meadow voles (Microtus

pennsylvanicus), have seasonally altered neurogenesis independent of

hippocampal volume changes (Galea and McEwen 1999; Ormerod and

Galea 2003), whereas other species, such as Eastern grey squirrels

(Sciurus carolinensis), Sorex shrews (S. araneus, S. minutus), and

Richardson‟s ground squirrels (Urocitellus richardsonii), have seasonally

altered brain or hippocampal volume changes which are not associated

with any changes in hippocampal neurogenesis (Lavenex, Steele et al.

2000; Bartkowska, Djavadian et al. 2008; Burger, Saucier et al. 2013), or

the association remains uninvestigated, as is the case with white-footed

mice (Pyter, Reader et al. 2005). Thus, the uncoupling or coupling of

neurogenesis to photoperiodic changes in hippocampal volume and

function are likely species-specific and dependent upon specific

photoperiodic adaptations.

As indicated above, one such species that undergoes photoperiodic

changes in hippocampal morphology and function is white-footed mice

(Chapter 2; Pyter, Reader et al. 2005; Walton, Chen et al. 2011), yet the

role of hippocampal neurogenesis in these photoperiodic brain changes

remains undescribed. As previously reported (chapter 6; Walton, Pyter et

al. 2012), when compared to long day counterparts neurogenesis in the

olfactory bulbs (the other main neurogenic niche in adult mammals) is

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increased after 10 weeks of exposure to short days, a time point at which

hippocampal structure is already altered (Pyter, Reader et al. 2005). A

shortcoming of this study was that it only assessed a single time point

during short day exposure; thus, it remains possible that the

contributions of altered neurogenesis to short day alterations in the

brain structure and function may occur earlier during exposure to short

days. To answer this specific question, we designed the following

experiment to assess hippocampal neurogenesis longitudinally at

multiple time points across ten weeks of short day exposure.


2.1 Animals

A total of 62 male white-footed mice (P. leucopus) were used in this

study. Mice were bred in our colony maintained at The Ohio State

University, which was derived from wild-caught stock obtained through

the Peromyscus Genetic Stock Center at the University of South Carolina.

All mice were housed in cages (32 cm x 18 cm x 14 cm), maintained at

constant temperature and humidity (21±4 °C, 50±5%), and given ad

libitum access to food (Harlan Teklad 8640, Indianapolis, IN, USA) and

filtered tap water. After weaning, mice were group housed with same-sex

littermates until reaching adulthood (60-90 days of age), and thereafter

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housed singly once assigned to experimental groups. All husbandry was

provided by Ohio State University Laboratory Animal Resources staff. All

animal procedures were approved by the Ohio State University

Institutional Animal Care and Use Committee, and were in compliance

with guidelines established by the National Institutes of Health and the

United States Department of Agriculture (Institute of Laboratory Animal

Resources, U.S. 1996).

2.2 Longitudinal assessment of neurogenesis using BrdU

To label cells undergoing mitosis the SGZ of the DG, mice were

injected IP with 100 mg/kg BrdU once a day for five days during the

middle of the light phase. Immediately prior to injection, BrdU (Sigma-

Aldrich) was dissolved in 0.9% sterile saline to a final concentration of 10

mg/ml, 0.2 μm filtered, and protected from light until injection.

All mice were housed in long day conditions (LD; 16 h light: 8 h

dark) and then pseudo-randomly assigned to either remain in long day

lighting or be transferred to short day lighting (SD; 8 h light: 16 h dark).

For both photoperiods lights were extinguished at 15:00 EST. To assess

neurogenesis longitudinally across 10 weeks of exposure to SD, mice

were pulsed with BrdU to label mitotic cells at the following time points

in relation to SD exposure: 0, 2, 4, 8, and 10 weeks (Figure 7.1). Mice

were maintained in their respective photoperiods for 4 weeks and then

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were killed to assess progenitor cell survival (BrdU+ cells) in the

hippocampus. To control for the potential effects of altered vivarium

environment across different durations of photoperiod treatment, each

time point group consisted of a SD cohort and a separate LD

counterpart.

2.3 Brain histology

Four weeks after the conclusion of BrdU injections, mice were

transcardially perfused with 4% PFA in 0.1M PBS, and brains were post-

fixed at 4°C overnight in the same solution. Brains were cryoprotected in

30% sucrose:0.1M PB solution, then frozen on dry ice and held at -80°C.

Every sixth coronal section (40 μm) throughout the hippocampus was

collected onto positively charged slides and processed

immunohistochemically for BrdU. For assessment of progenitor cell

survival and progenitor cell phenotype, tissues were processed using a

modified protocol adapted from (Leuner, Glasper et al. 2009). For all

staining, antigen retrieval was performed by microwaving tissues at

medium power for 5 minutes in boiling 0.1M citric acid (pH 6.0). Tissues

were then allowed to cool to RT in citric acid, rinsed 3x in 0.1M PBS,

denatured in 2N HCl for 30 min at 37°C, and then immediately placed in

0.1M sodium borate decahydrate (pH 8.5) for 10 min. After 3x PBS rinse,

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tissues were blocked for 30 min in a solution of 1% Tween, 0.1M PBS,

and 10% normal donkey serum.

For BrdU+ cell quantification, tissues were then incubated

overnight at 4°C in primary antibody (rat anti BrdU, Accurate Chemical

#OBT0030) at 1:200 in 1% Tween in 0.1M PBS, then in biotinylated

donkey anti rat secondary (1:200), and developed with ABC/DAB (Vector

Labs) following the manufacturer‟s instructions. To quantify progenitor

cell survival, all BrdU+ cells from three areas (sub-granular zone, granule

cell layer, hilus) of the two-blade dentate gyrus in the dorsal

hippocampus were then counted at 20x, the average number of cells per

section for each brain was calculated, and then each area was analyzed

separately. No differences were found in progenitor cell survival among

LD cohorts, so these data were combined for comparative analysis

against the short day exposed groups.

To assess the phenotype of the BrdU+ cells, tissues were processed

to label the mitotic marker BrdU, the neuronal marker NeuN, and the

glial marker GFAP. Following the antigen retrieval and blocking steps

described above, tissues were then incubated in the following primary

antibodies: rat anti BrdU (Accurate Chemical OBT0030) 1:200, mouse

anti NeuN (Chemicon MAB377) 1:1000, and rabbit anti GFAP (Abcam

AB7260) 1:2500 dissolved in the blocking solution described above.

Tissues were incubated in primary antibodies at RT for 24 h, rinsed 3x in

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PBS, re-blocked in blocking solution, and then incubated in the following

secondary antibodies at 1:500 dilution for 2 h at RT in 1% tween/0.1M

PBS: donkey anti rat 488 (712-485-150), donkey anti mouse (715-515-

150), and donkey anti rabbit (711-605-152, all from Jackson

ImmunoResearch). Following staining, twenty-five randomly selected

BrdU+ cells from the dorsal two-blade DG of each brain were assessed

for co-labeling with neuronal and glial markers using confocal

fluorescent microscopy (Nikon Eclipse C1 Plus). Due to a technical issue,

the group that spent 10 weeks in short days could not be analyzed for

progenitor cell phenotype.

2.4 Statistics

All data were analyzed using one-way ANOVA, with time point as

the main factor, and DG layer as the dependent variables for progenitor

cell survival data or co-label of BrdU+ cells as the dependent variables for

cell phenotype data. Significant differences were followed up by LSD

post-hoc tests. Statistical analyses were performed using SPSS (v.19,

IBM), and measures were considered significant if p ≤ 0.05.

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3. Results

3.1. Progenitor cell 4 week survival

In the subventricular zone (SVZ), there was a main effect of time in

photoperiod on progenitor cell survival (F(1,5) = 2.543, p < 0.05; Figure

7.2B). Follow up analyses showed that, compared to all other groups, the

group that spent 10 weeks in short days had the highest numbers of

BrdU+ cells (Figure 7.2B). Additionally, both the LD and 10 week groups

had significantly greater numbers of BrdU+ cells in the SVZ than all

other groups exposed to short days (Figure 7.2B). There were no effects of

photoperiod on progenitor cell 4 week survival in the granule cell layer

(GCL: F(1,5) = 0.942, p > 0.05; Figure 7.2C) or in the hilus (F(1,5) = 1.986, p

> 0.05; Figure 7.2D).

3.2 BrdU+ cell phenotype

Four weeks after being pulsed with BrdU, there were no differences

among photoperiod groups in dentate neurogenesis (F(1,4) = 0.532; p >

0.05), gliiogenesis (F(1,4) = 0.490; p > 0.05), or in undifferentiated BrdU+

progenitor cells (F(1,4) = 0.550; p > 0.05, Figure 7.3C & Table 7.1).

4. Discussion

In adult male P. leucopus exposed to both short and long days,

independent of photoperiod, progenitor cells arising from the SGZ of the

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dentate gyrus, which have survived for 4 weeks, mainly differentiate into

neurons (Figure 7.3, Table 7.1). However, the number of surviving

progenitor cells is impaired across the first 8 weeks of short day exposure

(Figure 7.2). Whereas progenitor survival is impaired early in SD

exposure, by 10 weeks in SD progenitor survival levels recover, and

exceed those of mice held in long days (Figure 7.2). The current data

provide further evidence that a single environmental signal, day length,

can alter neurogenesis in adult mammals. Although I did not directly

assess proliferation in this study, I have previously reported that

photoperiod alters progenitor survival without affecting proliferation rates

in the SVZ and olfactory bulb (Chapter 6; Walton, Pyter et al. 2012).

Additionally, my experimental design incorporated a control for

proliferation rates in the 0 WK group, as they were all pulsed with BrdU

in long days, and then randomly assigned to photoperiod (Figure 7.1).

Therefore, although this group had equal proliferation rates to its long

day counterpart, short day exposure for 4 weeks decreased survival rates

of those progenitor cells arising from pools proliferating at the same rate

(Figure 7.2B). This pattern has also been observed in male meadow voles

where photoperiod impaired progenitor cell survival without altering

proliferation rate (Ormerod and Galea 2003).

Among photoperiodic rodents in which neurogenesis has been

assessed, the regulation of neurogenesis in a photoperiodic context is

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mechanistically different dependent upon species and sex. Photoperiod

did not affect SGZ neurogenesis in male Eastern grey squirrels (Lavenex,

Steele et al. 2000), whereas, in common with male meadow voles

(Ormerod and Galea 2003), and two species of photoperiodic shrews

(Bartkowska, Djavadian et al. 2008), male white-footed mice in the

current study displayed decreased neurogenesis in the DG (Figure 7.3).

However, in the case of meadow voles there is an interaction of sex and

day length on neurogenesis; male voles have decreased neurogenesis in

short days (Ormerod and Galea 2003), whereas female meadow voles

have increased rates in short days (Galea and McEwen 1999).

Additionally, the longitudinal change in neurogenesis across short days

reported in the current study highlights an issue with drawing

conclusions from the previously mentioned studies regarding

neurogenesis: are the reported differences, or lack thereof, an artifact of

sampling time point in the short day group? Thus, I cannot make

generalizations about the role of photoperiod or the interaction of

steroids and photoperiod in the regulation of adult neurogenesis, as this

needs to be addressed in species- and sex-specific manner.

Functionally, hippocampal neurogenesis has documented roles in

supporting the formation of new memories, and can affect cognition,

affective behaviors, and spatial learning and memory (reviewed in Zhao,

Deng et al. 2008). Nascent dentate neurons can begin participate in

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learning and memory formation at 4-6 weeks of age (reviewed in Zhao,

Deng et al. 2008; Deng, Aimone et al. 2010), but they don‟t fully mature

for 2-4 months (van Praag, Schinder et al. 2002). I have previously

reported that mice exposed to short days for 10 weeks have impaired

hippocampal-dependent spatial learning and memory and impaired

hippocampal LTP (Chapter 2; Walton, Chen et al. 2011). The current

findings, that hippocampal neurogenesis is maximally impaired between

2-4 weeks in short days (Figure 7.2), provide support for a role of altered

neurogenesis in the short day impairment of hippocampal function in

white-footed mice, as new neurons born within this time frame would be

the ones most likely to contribute to the formation of new spatial

memories (Zhao, Deng et al. 2008; Deng, Aimone et al. 2010). Although

neurogenesis is increased at 10 weeks in short days, the neurons would

not contribute to formation of new memories for another 4-8 weeks.

Thus, neurons born at 10 weeks in short days would not be functional at

the time when hippocampal function has been tested in a photoperiodic

context in this species (Chapter 2; Chapter 3; Pyter, Reader et al. 2005;

Walton, Chen et al. 2011).

Why, after 10 weeks in short days, is neurogenesis is increased

above the rate found in long day mice? The natural history of white-

footed mice may provide some insight. Male P. leucopus maintain

breeding territories in the spring and early summer (King 1968), and the

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observed attenuation of neurogenesis upon exposure to short day lengths

may represent an adaptation to conserve energy during the short days of

winter (see Chapter 1) when territorial maintenance, and thus, elevated

spatial memory is unnecessary. As described in Chapter 1, upon

exposure to short days, the HPG axis in inhibited and the mice become

reproductively inactive. Maximal reproductive responses to short days

occur around 12 weeks, and then mice become refractory to short day

lengths and the reproductive system spontaneously recrudesces

(Chapter1; Pyter, Hotchkiss et al. 2005). Preceding gonadal

recrudescence, the neuroendocrine axis becomes activated and there is a

gonadotropin (LH and FSH) surge, which rises above levels found in long

days, prior to gonadal increases in volume and increased secretion of

testosterone (Berndtson and Desjardins 1974). Presumably, changes in

the brain precede measurable endocrine and gonadal changes. In

common with the gonadotropin surge described above, the enhancement

of neurogenesis in the hippocampus at 10 weeks in short days may

represent „recrudescence‟ of the hippocampus to prepare it for the

enhanced spatial memory necessary to maintain breeding territories, as

these neurons would need to born at minimum 4-8 weeks prior to

breeding season and territory establishment to be functional in the

hippocampus. In captive male meadow voles, reproductive status is

positively associated with neurogenesis (Ormerod and Galea 2003) and

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testosterone is positively correlated with neurogenesis in rats (Spritzer

and Galea 2007). Although we did not directly measure androgens or

gonadal responses in the current study, androgens did not likely

contribute to the enhanced neurogenesis found in mice at 10 weeks in

short days. At the time that the mice were killed and brains collected (14

weeks in short days, Figure 7.1), the photoperiodic cycle of gonadal

volume in the mice was at nadir (Pyter, Hotchkiss et al. 2005), and thus

gonadal androgen levels were also at nadir.

In conclusion, neurogenic rates in the dentate gyrus of male white-

footed mice vary across exposure to short days. Upon exposure to short

days, neurogenesis is attenuated for 8 weeks. However, by 10 weeks in

short day lengths, neurogenesis is enhanced above levels of long day

mice, potentially as a mechanism to recover the spatial memory function

of the hippocampus in preparation for breeding season. The current

findings are consistent with the natural photoperiodic cycle of

hippocampal function in male white-footed mice, and may help to inform

research on photoperiodic plasticity in neurogenesis and provide insight

into how the complex interplay between the environment, genes, and

adaptive responses to changing day lengths affects brain function and

behavior at multiple levels.

162

Figure 7.1 Schematic of the longitudinal assessment of neurogenesis

experimental design. Mice were pulsed with BrdU for 5 days (syringe) and

survived 4 weeks prior to being killed to assess neurogenesis. To label mitotic

cells at specific times across photoperiod exposure, BrdU was given at five time

points: 0 weeks (i.e. mice were all injected in LD conditions, then placed into SD

or remained in LD for 4 weeks), 2 weeks (i.e. mice were placed into SD or

remained in LD for 2 weeks, then were pulsed with BrdU and allowed to survive

for 4 weeks), 4 weeks, 8 weeks, and 10 weeks. 26

163

Figure 7.2 Progenitor cell survival in the dentate gyrus across exposure to short

day lengths. Representative photomicrograph of BrdU+ cells in the DG (A). In

the subgranular zone where the majority of BrdU+ cells were found, BrdU+

progenitor cell survival was impaired across the first 8 weeks of short day

exposure, however by 10 weeks in short days progenitor cell survival rebounded

and was increased compared to long day mice (B). Day length did not affect

dentate progenitor cell survival in the granule cell layer (C) or in the hilus (D). *

p ≤ 0.05 compared to 0, 2, 4, & 8 week groups; # p ≤ 0.05 compared to LD mice;

LSD post hoc test. LD = long day control group, other labels on the abscissa

indicate time in photoperiod when mice were pulsed with BrdU (see Figure 7.1).

27

164

Figure 7.3 Phenotype of dentate progenitor cells after 4 weeks survival.

Representative photomicrographs of the dentate gyrus at low magnification (A)

and high magnification of the granule cell layer (B). Photoperiod did not affect

the phenotype of progenitor cells (C). The majority of BrdU+ cells were NeuN+, a

small percentage of undifferentiated progenitor cells were present, and GFAP+

BrdU+ cells were very sparse (C, see Table 7.1). 28

165

% (±SEM) of BrdU+ cells

% NeuN % GFAP % Unlabeled

LD 92.3 ± 1.5 1.1 ± 0.7 6.6 ± 1.5

0 WK 93.7 ± 2.1 0.6 ± 0.6 5.8 ± 2.3

2 WK 95.8 ± 1.3 1.2 ± 0.8 3.1 ± 1.5

4 WK 95.2 ± 2.5 0.7 ± 0.7 4.1 ± 2.6

8 WK 93.4 ± 2.7 0.0 ± 0.0 6.6 ± 2.7

Table 7.1 Phenotype of progenitor cells, 4 weeks after BrdU injections, in the

dentate gyrus of LD and SD exposed male P. leucopus. 3

166

Chapter 8: Conclusions and Future Directions.

As described in Chapter 1, outside of the tropics, distinctive sets of

adaptations have evolved to cope with the unique demands of winter and

summer on survival and reproduction. Because these seasonal

adaptations often require significant time to develop, individuals rely

upon an environmental signal (day length) to alter gene expression in

order to produce the suite of season-specific adaptations. Photoperiodism

is the ability of plants and animals to measure environmental day length

(photoperiod) to ascertain the time of year and engage in seasonally

appropriate adaptations, and the annual cycle of changing photoperiod

provides the environmental switch between seasonal phenotypes. The

aim of this dissertation was to describe the influence of photoperiod on

phenotype, the distribution of energetically expensive processes across

the year, to maximize survival and fitness in males of a small

photoperiodic rodent species, white-footed mice (Peromyscus leucopus).

Among the adaptive responses to short days in male white-footed

mice is reduced in brain size, and a marked reduction in hippocampal

volume (Pyter, Reader et al. 2005). Adult mammalian brains are limited

167

in structural and volumetric plasticity; however altered recruitment and

retention of new neurons (neurogenesis) in adult brains across

photoperiod may contribute to the volumetric and mass changes

reported in white-footed mice. I explored the effects of photoperiod on

neurogenesis in the two main neurogenic niches of adult brains; the

olfactory bulb and the hippocampus. When assessed after 10 weeks of

short day exposure, both olfactory and hippocampal neurogenesis levels

are elevated above long day mice (Chapter 6, Chapter 7). A retrospective

longitudinal study revealed that across the first eight weeks of exposure

to short days, neurogenesis is impaired in the hippocampus, which may

contribute in part to the smaller hippocampus observed in short day

mice. Neurogenesis levels across the first eight weeks of short day

exposure in the olfactory bulbs remain to be investigated.

Given that the hippocampus is critical for spatial learning and

memory, in Chapter 2 I explored the functional outcomes of having a

smaller hippocampus and impaired hippocampal neurogenesis, utilizing

a rodent-specific spatial learning and memory task; the Barnes maze. I

also assessed neuronal physiology in the form of long-term potentiation

in the hippocampus, the form of neuronal plasticity that is the putative

synaptic mechanism of how memories are formed in the brain (Bliss and

Collingridge 1993). The functional outcomes of having a smaller

hippocampus in short days are: 1) impaired spatial learning and

168

memory, and 2) impaired synaptic plasticity (LTP) in the CA1 neurons of

the hippocampus. These findings provide a behavioral and physiological

correlate to naturalistic changes in hippocampal volume, which parallel

seasonally altered hippocampal demands (territoriality; King 1968) and

the adaptive photoperiodic reorganization of physiological systems to

survive the harsh short days of winter (Jacobs 1996; Walton, Weil et al.

2011).

Because mammals attend to the nightly duration of pineal

melatonin secretion to monitor day length in order to engage in

seasonally-appropriate adaptations (Walton, Weil et al. 2011), in Chapter

3 I explored the role that melatonin plays in the functional, structural,

and physiological changes of the hippocampus driven by short days

described in Chapter 1. An extended duration of melatonin exposure is

interpreted biologically as a long night (or a short day). Using either short

day lengths or an exogenous melatonin implant, extending the duration

of melatonin exposure for 10 weeks compared to the duration of

melatonin secretions in long days, independent of environmental day

length, recapitulates the short day hippocampal phenotype, including

altered neuronal morphology, impaired spatial navigation ability, and

LTP. Thus, photoperiodic plasticity in the hippocampus is driven by

changes in melatonin rhythms. However, melatonin has both indirect

169

and direct effects, and the effects of melatonin on hippocampal function

may be indirect. I explored this possibility in Chapter 4.

Downstream of the extended duration of pineal melatonin secretion

in short days, is the suppression of the HPG axis and the resultant

reduction in circulating gonadal steroids (discussed in Chapter 1).

Testosterone, which reaches the hippocampus hormonally, can act

directly to affect hippocampal function via androgen receptors, or it can

be enzymatically converted to either a non-aromatizable androgen

(dihydrotestosterone) or to estradiol, and then affect hippocampal

function. When assessed in a photoperiodic context, testosterone

enhances spatial navigation in short days, whereas estradiol enhances

spatial navigation in long days. These photoperiod-mediated changes in

steroid sensitivity may be, in part, based on altered receptor expression

for these specific steroids; however more data are needed to support this

possibility.

Given that the hippocampus is heavily interconnected with other

brain structures, and that photoperiodic plasticity in the hippocampus

drives behavioral and physiological changes, it follows that the other

brain structures, and their associated behaviors, are altered in short

days as well. In Chapter 5 I explored how photoperiod affects the

structure and function of the amygdala, and alters amygdala-dependent

fear behaviors. Indeed, short days enhance fear memory and synaptic

170

connectivity (spine density) in the basolateral amygdala, the region where

fear memories are encoded (Maren 1999). Thus, in white footed mice,

short days enhance amygdala function while impairing hippocampal

function.

In addition to the photoperiod-mediated changes in brain and

behavior described by my experiments in this dissertation, short day

white-footed mice have reduced hippocampal volume (Pyter, Reader et al.

2005) and increased HPA axis negative feedback (Pyter, Adelson et al.

2007). When taken together, this pattern of behaviors in short day mice

resembles a specific human anxiety disorder, namely, post-traumatic

stress disorder (PTSD). Among human PTSD patients, reduced

hippocampal volume is associated with susceptibility to PTSD, but not

severity of symptoms (Yehuda and LeDoux 2007), amygdala activity is

positively correlated with symptom severity (Dickie, Brunet et al. 2011),

and PTSD patients have enhanced HPA axis negative feedback (Yehuda

and LeDoux 2007). One of the greatest research challenges of

mechanisms involved in psychiatric disorders is the difficulty in

replicating the symptoms of the disease in animal models (Miller and

McEwen 2006), and it is important to develop new ethologically-relevant

models for translational research of psychiatric disorders (Shekhar,

McCann et al. 2001; Clinchy, Schulkin et al. 2010). In the laboratory

setting, a simple day length manipulation in white-footed mice, exposure

171

to short days, concurrently replicates three main features of PTSD and

enhances fear memory responses.

In conclusion, the photoperiodic modulation of brain fear circuits

and fear memory in white-footed mice described in this, and previous

studies, may argue for the potential utility of this species as an

additional, yet unique, animal model to research how a single

environmental factor (day length) can interact with genes to alter

phenotype to resemble a human psychiatric disorder, such as PTSD. I

also believe that studying photoperiodic phenotypic plasticity in white-

footed mice, and how light interacts with genes via melatonin hormonal

signaling, can provide novel and important insights into conserved

molecular mechanistic themes underlying the relationship between

phenotype and genotype.

172

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