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Estradiol-induced changes in the production and survival of granule neurons born in the dentate gyrus… Ormerod, Brandi Kirshane 2003

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ESTRADIOL-INDUCED CHANGES IN THE PRODUCTION AND SURVIVAL OF GRANULE NEURONS BORN IN THE DENTATE GYRUS OF ADULT RODENTS by Brandi Kirshane Ormerod B.Sc, Queen's University, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Graduate Programme in Neuroscience) We accept this thesis as conforming tg the required standard  THE fjNIVERSITY OF BRITISH COLUMBIA April 2003 © Brandi Kirshane Ormerod, 2003  U B C Rare Books and Special Collections - Thesis Authorisation F o r m  Page 1 o f 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my department o r by h i s o r her r e p r e s e n t a t i v e s . It is understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department  of  The U n i v e r s i t y o f B r i t i s h Vancouver, Canada  Columbia  file://C:\Documents%20and%20Settings\Guess\My%20Documents\PhD\Dissertation\Thes...  6/11/2003  ABSTRACT Controlling neurogenesis constitutive to the hippocampal dentate gyrus o f adult mammals could improve strategies geared toward replacing neurons lost i n the diseased or injured human C N S . This thesis resolves and expands upon conflicting reports about estradiol's influence over neurogenesis (progenitor proliferation and daughter cell differentiation and survival) in the adult rodent dentate gyrus. Chapter 2 showed that reproductive status regulates neurogenesis in the dentate gyri o f adult laboratory-reared female meadow voles. Specifically, reproductively inactive (low estradiol) females had more dividing cells than reproductively active (high estradiol) females or females exposed to estradiol for 48 h. However, females exposed to estradiol for 4 h had more dividing cells than reproductively inactive females, suggesting that estradiol dynamically regulates cell proliferation. Because the ratio o f new cells surviving 5 weeks versus 2 h was higher i n the dentate gyri o f reproductively active versus inactive females, estradiol appeared to enhance the survival o f young cells. Chapters 3 and 4 confirmed that estradiol dynamically regulates dentate cell proliferation robustly across rodent species. Cell proliferation i n the dentate gyri o f female rats (Chapter 3) and meadow voles (Chapter 4) increased 4 h after but decreased 48 h after estradiol- versus vehicle-treatment. In part, estradiol suppressed proliferation by stimulating adrenal activity because adrenalectomy eliminated the suppression in adult female rats. Consistent with the effects reported in other species, N M D A r activation decreased and N M D A r inactivation increased proliferation i n the dentate gyri o f adult female voles but estradiol did not stimulate N M D A r s to influence cell proliferation. Chapter 5 showed that estradiol potently enhances young granule neuron survival and that enhanced survival is related to improved hippocampus-dependent memory (but not learning). Specifically, estradiol doubled the number o f 16-day old neurons i n the dentate gyri o f adult male meadow voles when administered over Days 6-10 after the neurons are born. Estradiol-  Ill  treated voles (Days 6-10), exhibited similar hormone-free performance Morris water maze training trials but outperformed vehicle-treated voles on a probe trial. Chapter 6 discusses how estradiol-induced changes i n components o f neurogenesis may influence normal hippocampus function and discusses how the findings o f this thesis may relate to neuronal replacement strategies.  iv  T A B L E OF CONTENTS ABSTRACT.  i.  T A B L E OF CONTENTS LIST O F T A B L E S  II IV VIII  LIST OF FIGURES  IX  ABBREVIATIONS  XI  ACKNOWLEDGEMENTS  XII  CHAPTER 1  1  GENERAL INTRODUCTION  1  1.1 1.2 1.3 1.4 1.5 1.6 1.7  HISTORICAL SYNOPSIS OF A D U L T CNS NEUROGENESIS R E S E A R C H DEFINING S T E M C E L L S NEUROGENESIS IN T H E A D U L T M A M M A L I A N B R A I N A R E N E U R O N S P R O D U C E D IN THE D E N T A T E G Y R U S F U N C T I O N A L ? ESTRADIOL I N F L U E N C E S H I P P O C A M P A L NEUROGENESIS ESTRADIOL E N H A N C E S C E L L S U R V I V A L IN DIFFERENT SYSTEMS O V E R V I E W A N D OBJECTIVES  5 7 12 18 21 24 26  CHAPTER 2  32  REPRODUCTIVE STATUS INFLUENCES C E L L PROLIFERATION AND C E L L S U R V I V A L IN T H E D E N T A T E G Y R U S O F A D U L T F E M A L E M E A D O W V O L E S : A POSSIBLE R E G U L A T O R Y R O L E FOR ESTRADIOL  32  2.1 INTRODUCTION 32 2.2 METHODS 35 Animals 35 Procedure 35 Drug preparation 37 Histology 38 Peroxidase immunohistochemistry 38 Hormone assays 39 Data analyses 40 Statistical analyses 40 Experiment 1. Reproductive status, possibly via estradiol, influences cell proliferation in the dentate gyrus of adult female meadow voles 41 Experiment 2. The elevated labelled cell density observed in RI adult female meadow vo persists for 5 weeks 45 2.4 DISCUSSION 46 Reproductive status rapidly influences cell proliferation in the dentate gyrus of adult female meadow voles 46 Estrogen receptor localization and effects within the hippocampus 49 Neurogenesis in natural and laboratory populations 50 Reproductive status influences granule cell survival in the dentate gyrus 5 Reproductive status does not influence the density of pyknotic cells in the dentate gyrus adult female meadow voles 52  V  Reproductive status-related changes in cell proliferation and/or survival in the female meadow voles may be related to maternal behaviour in natural settings 2.5 I M P L I C A T I O N S  53 55  CHAPTER 3  56  ESTRADIOL I N I T I A L L Y E N H A N C E S BUT SUBSEQUENTLY SUPPRESSES (VIA A D R E N A L S T E R O I D S ) P R O G E N I T O R C E L L P R O L I F E R A T I O N WITHIN T H E DENTATE GYRUS OF ADULT F E M A L E RATS  56  3.1 I N T R O D U C T I O N 56 3.2 M E T H O D S 57 Animals 58 Procedure 58 Histology 61 Peroxidase immunohistochemistry 62 Fluorescence immunohistochemistry 63 Hormone assays 63 Data analyses '. 64 Statistical analyses 66 3.3 R E S U L T S 67 Experiment 3. Relative to vehicle, the number of BrdU-labelled cells observed in the dentate gyrus of adult female rats increases following exposure to EB for 4 h but decreases following exposure to EB for 48 h 67 Experiment 4. The Estradiol-induced Suppression in Cell Proliferation Within the Dentate Gyrus of Adult Female Rats is Reversed by Adrenalectomy 71 3.4 D I S C U S S I O N 72 Estradiol interacts with adrenal steroids to suppress cell proliferation 74 Estradiol could act time-dependently through numerous pathways 75 Estradiol time-dependently influences many forms ofplasticity within the hippocampus... 77 3.5 I M P L I C A T I O N S 78 CHAPTER 4  80  NMDA RECEPTOR ACTIVITY AND ESTRADIOL: INDEPENDENT R E G U L A T I O N O F C E L L P R O L I F E R A T I O N IN T H E D E N T A T E G Y R U S OF ADULT FEMALE MEADOW VOLES  80  4.1 I N T R O D U C T I O N 4.2 M E T H O D S Animals Procedure Drug preparation Histology Peroxidase immunohistochemistry Data Analyses Statistical analyses 4.3 R E S U L T S Experiment 5. Relative to vehicle, estradiol increased and NMDA decreased cell proliferation and a 4h estradiol exposure did not appear to stimulate NMDA receptor activity to influence cell proliferation  80 83 83 :. 84 85 88 88 89 88 90  90  vi Experiment 6. Relative to vehicle, estradiol significantly decreased and MK-801 significantly increased cell proliferation but estradiol did not interact with NMDA receptors to alter cell proliferation within 48 h : 92 4.4 DISCUSSION 94 Estradiol suppresses cell proliferation by stimulating adrenal activity but not NMDA receptor activity 95 Estrogen could time-dependently influence cell proliferation in the dentate gyrus of adult rodents through numerous pathways 97 Changes in cell proliferation within the dentate gyri of inctact females support that estradiol alters cell proliferation dynamically 99 New neurons appear functional and influence hippocampus-dependent behavior 100 4.5 IMPLICATIONS 102  CHAPTER 5  103  ESTRADIOL ENHANCES NEUROGENESIS BY INCREASING THE S U R V I V A L O F Y O U N G N E U R O N S A N D T H E I N C R E A S E IS R E L A T E D T O BETTER SPATIAL M E M O R Y IN ADULT M A L E RODENTS  103  5.1 INTRODUCTION 103 5.2 M E T H O D S 106 Animals 106 Surgery 107 Drug preparation 107 Procedure 108 Morris water maze training 110 Histological procedures Ill Peroxidase immunohistochemistry 112, Immunofluorescent labelling 112 Data analyses 114 Statistical analyses 115 5.3 RESULTS 116 Experiment 7. Estradiol promotes the survival of new neurons 116 Experiment 8. Estradiol-induced changes in young neuron number did not influence performance on water maze acquisition trials 118 Estradiol-induced changes in neuron number did influence performance on a water maze retention trial 119 5.4 DISCUSSION 121 Estradiol influences the survival of neurons in various systems 122 Estradiol regulates different components of neurogenesis that occurs in adulthood 125 New neurons have functional characteristics 126 Neurogenesis and hippocampus-dependent behaviour in adulthood 128 5.5 IMPLICATIONS 130  CHAPTER 6  132  G E N E R A L DISCUSSION  132  Reproductive status and estradiol influence cell proliferation  132  Estradiol partially suppresses cell proliferation by stimulating adrenal activity Estradiol did not infuence the differentiation of daughter cells Estradiol enhances the survival of young granule neurons  135 137 140  vii Net neurogenesis and behaviour in adult voles Estradiol-induced changes in components of neurogenesis and their implications for neuronal replacement Summary and overall implications  R E F E R E N C E LIST  142 145 147  151  LIST OF T A B L E S Table 1. Mean (+SEM) density o f BrdU-labelled and pyknotic cells in the granule cell layer and hilus o f adult female meadow voles measured 2h after B r d U was injected in Experiment 1  43  Table 2. Pearson product-moment correlations between dependent variables measured in adult female meadow voles i n Experiment 1  43  Table 3. Mean ( ± S E M ) adrenal mass, gonad mass, serum C O R T level and serum estradiol level measured i n adult female meadow voles in Experiment 1  44  Table 4. Mean ( ± S E M ) density o f [ H]thymidine-labelled and pyknotic cells measured in the granule cell layer and hilus 5 weeks after [ H]thymidine was injected i n Experiment 2  45  Table 5. Mean ( ± S E M ) % B r d U - i r cells expressing a neuronal (TUC-4-ir) or glial ( G F A P - i r ) phenotype measured 24 h after B r d U was injected did not significantly differ in adult female rats exposed to estradiol or vehicle for either 4 or 48 h i n Experiment 3  69  Table 6. Mean ( ± S E M ) serum hormone levels and adrenal masses i n adult female rats injected with B r d U either 4 or 48 h after estradiol administration i n Experiments 3 and 4  69  Table 7. Mean ( ± S E M ) serum hormone levels and adrenal masses i n samples taken from animals either 4 or 48 h after an estradiol injection in Experiment 3 (no B r d U injected)  70  Table 8. Mean ( ± S E M ) % B r d U - i r cells expressing a neuronal ( T U C - 4 - i r or doublecortin-ir) or glial ( G F A P - i r ) phenotype measured 4 days after B r d U was injected did not significantly differ in adult female rats exposed to estradiol for 4 h in Experiment 3 ,  71  Table 9. Mean (+SEM) % B r d U - i r cells expressing a neuronal (TUC-4-ir) or glial (GFAP-ir) phenotype i n adrenalectomized females following a 24 h survival time after B r d U injection i n Experiment 4 did not significantly differ between groups  71  3  3  Table 10. M e a n ( ± S E M ) dentate gyrus volume i n vehicle- and estradiol-treated adult male meadow voles i n Experiment 7  117  Table 11. Mean ( ± S E M ) % B r d U - i r cells expressing a neuronal ( N S E - or NeuN-ir) or glial ( G F A P - i r ) phenotype measured 16 d after B r d U was injected did not significantly differ i n adult male meadow voles treated with estradiol or vehicle i n Experiment 7  118  ix LIST OF FIGURES Figure 1. Representation of a coronal hippocampal section from the rat with inset depicting neurogenesis and a time course for the maturation of new neurons  14  Figure 2. Microphotographs of a bromodeoxyuridine (BrdU)-labeled cell, a [ H]thymidine-labeled cell and a pyknotic cell in Experiments 1 and 2  42  Figure 3. Photomicrographs of BrdU-labeled and pyknotic cells and confocal images of neurons or glia in the dentate gyri of adult female rats in Experiments 3 and 4  65  Figure 4. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female rats given BrdU either 4 h or 48 h after estradiol or vehicle and sacrificed 24 h later in Experiment 3  68  Figure 5. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adrenalectomized adult female rats when BrdU was administered 48 h after estradiol or vehicle in Experiment 4  72  Figure 6. Time line of Experiment 5  84  Figure 7. Time line of Experiment 6  85  Figure 8. Photomicrographs of a BrdU-labelled cells and a pyknotic cell  87  Figure 9. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female voles injected with BrdU 4 h after estradiol or oil and 1 h after N M D A or vehicle in Experiment 5  91  Figure 10. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female voles injected with BrdU was administered 48 h after estradiol or oil and 1 h after MK-801 or vehicle in Experiment 6  93  3  Figure 11. Timeline of Experiment 7  109  Figure 12. Timeline of Experiment 8  110  Figure 13. Microphotographs of cells analyzed in Experiment 7  113  Figure 14. Stereo logical estimates of BrdU-labelled and pyknotic cells in the dentate gyrus of adult male meadow voles following estradiol or vehicle treatment in Experiment 7  117  Figure 15. Performance of vehicle- versus estradiol-treated meadow voles in training trials of the Morris water maze in Experiment 8  120  Figure 16. Performance of vehicle- versus estradiol-treated meadow voles on a probe trial in the Morris water maze in Experiment 8  121  Figure 17. Stereo logical estimates o f BrdU-labelled cells i n the dentate gyrus o f adult female meadow voles following the administration o f estradiol or vehicle  xi  ABBREVIATIONS ACTH ADX AP-1 BrdU CAI CA3 cAMP  Ci CNS CORT CRE Cy3 Cy5 CRMP-4 d DCX CREB DNA E EB EB4 EB48 EGF ER„ ERp ERE ERK1/2 2  F344 FGF FGF-2 bFGF FITC g GABA GCL h Hil HP A  - adrenocorticotropic hormone - adrenalectomy - activator protein-1 - bromodeoxyuridine - cornu Amnion's regio 3 - cornu Amnion's regio 1 - cyclic adenosine monophosphate - curies - central nervous system - corticosterone - corticosterone response element - indocarbocyanine 3 - indodicarbocyanine 5 - collapsin response-mediated protein 4 - day - doublecortin - camp-responsive element bindingprotein - deoxyribonucleic acid - estradiol - 17-/7 estradiol benzoate - 4 h exposure to E B - 48 h exposure to E B - epidermal growth factor - estrogen receptor (a subtype) - estrogen receptor (P subtype) - estrogen response element - extracellular signal-regulated kinase 1 or 2 - Fischer 344 rats - fibroblast growth factor - fibroblast growth factor-2 - basic fibroblast growth factor - fluorescein - grams - gamma amino butyric acid - granule cell layer - hour - hilus - hypothalamic-pituitary-adrenal  i.p. IGF ir LTP MAPK N NDS NHS NeuN NFKB  NIH NMDA NMDAr NSE NURR1 OVX PB PBS RA RNA mRNA RI s.c. SD SFRE SGZ Shh SRE SVZ TBS TOAD-64kD TUC-4 Ulip V V4 V48  -  intraperitoneal insulin-like growth factor immunoreactive long-term potentiation mitogen activated protein kinase - normal - normal donkey serum - normal horse serum - neuronal nuclei - nuclear factor K B - National Institute of Health - /V-methyl-D-aspartate - 7V-methyl-D-aspartate-receptor - neuron specific enolase - nuclear orphan receptor 1 - ovariectomy - phosphate buffer - phosphate-buffered saline - reproductively active - ribonucleic acid - messenger R N A - reproductively inactive - subcutaneous - Sprague Dawley rats - steroidogenic factor binding element - subgranular zone - sonic hedgehog - steroid response element - subventricular zone - tris-buffered saline - turned on after division-64kD - Toad-64kD/Ulip/CRMP-4 - Unc 3 3-like phosphoprotein - vehicle - 4 h exposure to vehicle - 48 h exposure to vehicle  ACKNOWLEDGEMENTS This thesis was completed with the scholarly and emotional support o f several people whom I feel fortunate to have interacted with. First and foremost, m y sincerest thanks go to Dr. Liisa Galea who has been an exceptional mentor and good friend. Her wisdom in realms both scientific and mundane reflects that o f a seasoned veteran and is always appreciated. Furthermore, her enthusiasm for the research contained i n this thesis has been nothing short o f inspiring. I w i l l forever fondly recall m y days as Liisa's first graduate student! I express deepest thanks to m y supervisory committee comprised o f Cathy Rankin (double thanks to Cathy for adopting me this past year), Jane Roskams and Joanne Weinberg who not only offered excellent input regarding the studies described i n this thesis but who have been outstanding role models; they are exceptional people and exceptional scientists.  I thank Erin Falconer for endless hours o f thought provoking scientific discussion and for providing the collegial support (both i n and out o f the lab) that makes pursuing a degree enjoyable. Stan Floresco has been a great friend and colleague and I thank him for all o f his amazing advice.  I especially thank m y family for their undying motivation and undeserved awe and most o f all I thank Rob Helmer for being a pillar o f support thoughout the duration o f m y graduate career.  M a n y people, i n many different ways, enriched m y graduate student life. Thanks gang! Steve Barnes (for coffee breaks), Brian Christie (for daily motivation), Sue and Bruce Connup (for being great friends), Ariane Coury (for treating me like a peer), Brennan Eadie (for too much to name), Chris Fennell (Nach Fennel), Dave Froc (for keeping me humble), Jessica Grant (for great dinner parties), Darrin Hanneson (for his great legs), Hockey Pool participants not already listed, Melissa Holmes (for keeping it interesting), John Howland (.. .ah Howland), Kristin Laurens (for bringing Aussie slang to U B C ) , Tiffany Lee (for all her great help), Amanda L i (for asking advice), Del Paulhus (for really late night chats), Rebecca Pillai (for reducing my smoke intake at departmental parties), Jackie Rose (for too much to name), Gayle Smith (for all the encouragement), Starbucks, Elissa Strome (for being a great camping and party buddy), Chris Sturdy (for being Chris), Matt Tata (for some of the most memorable academic discussions), Victor Viau (for being inspirational), Athena Vouloumanous (for getting through neuroscience with me), Jennifer Wide (for her wit), Bob Kemp (for saving the day.. .often), Doug Wong-Wylie (for being my euchre buddy).  This research was supported by a National Sciences and Engineering Research Council postgraduate scholarship and a K i l l a m predoctoral fellowship.  CHAPTER 1 GENERAL INTRODUCTION Over the last decade, evidence has been gathered to demonstrate that new neurons are added daily to the hippocampal dentate gyri and the olfactory bulbs o f mammals throughout adulthood (Eriksson et al., 1998; see Gage, 2000, Cameron and M c K a y , 2001; Gould and Gross, 2002 and Magavi and Mackliss, 2002 for review). Widespread acceptance that neurogenesis occurs within the adult mammalian central nervous system ( C N S ) has ignited research aimed toward identifying the mechanisms that control the process, largely with the agenda o f improving neuronal replacement strategies to restore the neuronal loss associated with neurodegenerative disease or neurotrauma. However, the field is young and the mechanisms regulating the proliferation o f progenitor cells and the differentiation and survival of daughter cells either in vivo or in vitro need to be more fully understood before such an approach is attempted. A n equally important research avenue being currently undertaken strives to understand the role that neurogenesis through adulthood plays i n normal hippocampus and olfactory bulb function. Evidence suggests that new neurons are functionally integrated into the hippocampus and olfactory bulbs and that depleting young neuron number i n either area can impair associated behaviours (Gheusi et a l , 2000; Shors et al., 2001, 2002; van Praag et al., 2002). These findings tempt speculation that adult-generated neurons could be used to replace neurons lost in the diseased or injured C N S to perhaps restore lost function. Several groups have proposed that the symptoms o f neurodegenerative diseases or neurotauma with relatively homogenous aetiologies w i l l be those most imminently improved by neuronal replacement strategies (see Shihabuddin et al., 1999; Bjorklund and Lindvall, 2000; Rossi and Cantaneo, 2002). For example, Parkinson's disease is associated with a progressive loss o f dopaminergic neurons in the substantia nigra that culminates i n the depleted  2 striatal dopamine levels thought to underlie the abnormal motor symptoms o f bradykinesia, rigidity and tremor (Duvoisin, 1992). Bjorkland and Lindvall (2000) suggest that neuronal replacement could alleviate the symptomology o f a disease, such as Parkinson's, in two ways. First, cells able to differentiate into the appropriate neuronal phenotype could be grafted into the affected C N S region to replace lost neurons by establishing appropriate efferent and afferent connections. Second, cells genetically engineered to secrete growth factors or neurotransmitters could be grafted into the affected region to promote the survival or regeneration o f existing neurons. In fact, fetal mesencephalic tissue grafted into the striatum o f Parkinson's disease patients appears to produce long-lasting (5-10 yr) improvements in symptomology both by becoming integrated into striatal circuitry and by increasing striatal dopamine (Bjorklund and Lindvall, 2000). Despite the apparent success o f fetal-tissue derived grafts in alleviating the symptoms o f some Parkinson's disease patients, using fetus-derived tissue for medical purposes is problematic for several reasons. Acquiring enough fetus-derived tissue is difficult (the tissue from several embryos is required to treat 1 Parkinson's patient), samples are seldom standardized and the use o f fetal tissue for medical purposes is ethically controversial. Clearly, sources o f cells that are abundant, standardizable and ethically acceptable that could be grafted into the diseased or damaged C N S could improve neuronal replacement strategies. Adult C N S progenitor cells are a potentially viable source o f cells for grafting either homotopically (into the donor C N S ) or heterotopically (into a non-donor C N S ) into the diseased or injured C N S . Progenitor cells can be isolated surgically from neurogenic (subventricular and subgranular zones) and non-neurogenic (spinal cord, septum and striatum) adult C N S regions (Morshead et al., 1994; Gage et a l , 1995; Palmer et al., 1995,1997; Weiss et al., 1996; Shihabuddin et al., 1997; Kukekov et al., 1999; R o y et al., 2000; Aresenijevic et al., 2001). Recently, progenitor cells have even been isolated from neurogenic and non-neurogenic  3 regions o f the human cadaver C N S (Palmer et al., 2001), further demonstrating their accessibility and potentially bypassing ethical controversy regarding their use. Adult C N S derived progenitor cells can be expanded long-term i n culture to produce many clones and can generate multiple C N S cell types both in vitro and when grafted into neurogenic C N S regions (Gage et al., 1995; Weiss et al., 1996; Palmer et al., 1995,1997), suggesting that the variety o f cell types required to treat different diseases could be generated. In fact, Gage and his colleagues (Sakurada et al., 1999) were able to direct the differentiation o f adult rat hippocampus-derived progenitor cells to a dopamine neuron phenotype in vitro. Adult rat hippocampus-derived progenitors grafted into various C N S regions survive for several months (Gage et al., 1995; Suhonen et al., 1996), indicating that trophic support for new cells is abundant throughout the adult C N S . However, these cells only adopt a neuronal fate when transplanted into neurogenic regions (Suhonen et al., 1996). Thus, neuronal replacement in non-neurogenic regions o f the adult C N S using this type o f approach may require the grafting of cells that have been partially differentiated in vitro or the co-transplantation o f cells engineered to secrete factors that direct the differentiation o f progenitors. Alternatively, by understanding the cues that regulate neurogenesis (progenitor cell differentiation, daughter cell differentiation and the survival o f young neurons) it may become possible to manipulate endogenous progenitor cells in situ to replace neurons lost in the diseased or injured C N S . In the normal rodent C N S , thousands o f neuroblasts are produced daily i n the subventricular zone and the hippocampal subgranular zone (Lois and AlvarezBuylla, 1994; Cameron and M c K a y , 2001; Seri et al., 2001; Chapters 3, 4, 5). Neuroblasts migrate several mms from the subventricular zone to the olfactory bulbs or several urns from the subgranular zone to the granule cell layer where they differentiate into neurons o f the appropriate phenotype (Cameron et al., 1993b; Lois and Alvarez-Buylla, 1994). Therefore, i f  4 the cues that regulate daughter cell differentiation and neuroblast migration in vivo were understood, neuroblasts o f the appropriate phenotype could be enticed to migrate into affected C N S areas. A s mentioned previously, progenitor cells are situated throughout the C N S . In fact, targeted cell death i n the cortex can induce resident progenitors to generate neurons that morphologically resemble cortical pyramidal neurons (Magavi et al., 2000). Therefore, the potential to stimulate neurogenesis could also be present throughout the adult mammalian C N S . Although the biochemical and behavioural regulators o f neurogenesis constitutive to the dentate gyrus are beginning to be understood, studies seldom investigate the cumulative effects of a single factor on the proliferation o f progenitor cells, the differentiation o f daughter cells and survival o f young neurons. Understanding the cumulative effects o f a single factor on these components o f neurogenesis is important because neuronal replacement strategies strive to increase neuron number i n an affected C N S area. A factor that increases progenitor cell proliferation but decreases the survival o f young neurons would produce no net change in new neuron number, and would therefore yield limited therapeutic benefit i f used in isolation to increase neuron number. The experiments described i n the present thesis adopt the approach o f investigating the influence o f estradiol upon multiple components o f neurogenesis in the dentate gyrus o f adult rodents. The findings o f this thesis are that estradiol first increases (within 4 h) but then stimulates adrenal activity to decrease (within 48) cell proliferation, does not affect the differentiation o f daughter cells and enhances the survival o f new granule neurons in the dentate gyrus o f adult rodent. Increased cell survival is related to improved hormone-free performance on a retention trial, but not on acquisition trials i n the hippocampus-dependent Morris water maze, suggesting that memory (but not learning) is improved by an estradiolinduced increase i n young neuron number. The findings are discussed i n the context o f how estradiol-induced changes in dentate neurogenesis could influence normal hippocampal function and neuronal replacement strategies.  5  1.1 H I S T O R I C A L SYNOPSIS O F A D U L T CNS N E U R O G E N E S I S R E S E A R C H  "...once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centres the nerve paths are something fixed, ended and immutable. Everything may die, nothing may be regenerated. It is for the future to change, if possible, this harsh decree." Ramon Y Cajal (1913-1914) translated by Raoul May  Forty years ago, Joseph Altman challenged the dogma that the adult mammalian C N S was 'fixed and immutable' by using the cell synthesis marker [ H]-thymidine to demonstrate 3  that new cells, which eventually become morphologically indistinguishable from neighbouring granule neurons, are added daily to the dentate gyrus and olfactory bulbs o f adult rats (Altman, 1962; Altman and Das, 1965; Altman, 1969). Few papers describing the topic o f adult neurogenesis were published i n years following Altman's very interesting discovery. In their 1984 paper, Kaplan and B e l l suggest that researchers resisted the idea that neurogenesis persisted throughout adulthood i n mammals because o f scepticism about whether adult neural "stem" or "blast" cells could exhibit mitotic potential in vivo and whether the apparently lowlevel [ H]-thymidine incorporation observed to occur i n the rodent hippocampus represented 3  D N A repair rather than cell division. They attempted to dispel the scepticism by identifying synapses on the soma and processes o f [ H]-thymidine-labelled cells under the electron 3  microscope. Nonetheless, scientists largely resisted the idea that new cells produced in the adult mammalian C N S differentiated into neurons based upon a purely morphological analysis, likely because glia also receive synaptic input (Ventura and Harris, 1999 for example). Nottebohm did manage to stimulate interest i n adult neurogenesis by showing that more new cells are added to the higher vocal centre ( H V C ) o f adult male canaries during the fall non-breeding season when new song is learned than during the spring breeding season when song is stable (Goldman and Nottebohm, 1983; Nottebohm et al., 1986; Nottebohm, 1989). However,  6 Nottebohm's discovery was touted as an interesting avian phenomenon after Rakic (1985a; 1985b) reported that although new cells were produced i n the hippocampus o f adult rhesus monkeys, he could not verify the phenotype o f the new cells using techniques available at that time. Fortunately, stereological and immunohistochemical methods that could be used to more accurately count and identify the phenotype o f new cells in the adult mammalian C N S were developed. Gould and her colleagues first used immunohistochemistry to show that many cells that incorporate the cell synthesis marker [ H]thymidine i n the adult rat hippocampus 3  eventually express mature neuronal protein (Cameron et al., 1993b). Coupled with the report made a year earlier by Reynolds and Weiss (1992) that cells derived from the adult rat forebrain generated both primary and secondary neurospheres in vitro, a property indicative o f stem cell potential, this evidence prompted some interest in discovering how neurogenesis constitutive to the adult rodent C N S is regulated. In the years following Gould's rediscovery, neurogenesis was identified i n the hippocampus o f adult tree shrews, marmosets and macaques (Gould et al., 1997a, 1999a,b,c; Kornack and Rakic, 1999). Then Eriksson and his colleagues (1998) found new neurons i n the post mortem dentate gyrus and olfactory bulbs o f 57 to 72 year-old patients who had received B r d U to monitor tumour growth up to 2 yrs prior to succumbing to cancer. Importantly, post-mortem examinations found no evidence o f tumour metastasis to cerebral tissue i n that study. This finding demonstrated that neurogenesis is not a phenomenon merely vestigial to the C N S o f species lower in phylogenetic rank to humans and that the human brain may i n fact retain some regenerative capacity during adulthood. Most important, this finding ignited research i n what appears to be the emerging field of'adult stem cell biology'. In fact, a report created by the National Institutes o f Health and Published by The Department o f Health and Human Services (2001, June 17) states " . . .the field o f stem cell biology is advancing at an incredible pace with new discoveries being reported i n the scientific  literature on a weekly basis". Indeed, the often-cited decree that within the adult mammalian C N S "everything may die and nothing may be regenerated" appears lifted.  1.2 D E F I N I N G S T E M C E L L S  Because the field o f adult neural stem cell biology is relatively new and advancing at such a rapid pace, the terminology used in publications is generally inconsistent and confusing. The Department o f Health and Human Services recently published a report (2001) developed by The National Institutes o f Health regarding the current state o f understanding about stem cells and their potential and pitfalls for therapeutic use. One o f the goals o f the report was to establish conventions about the terminology used for describing cells with mitotic potential found in neural or other tissue. According to the report, stem cells make copies o f themselves (or exhibit clonality) for the host organism's lifetime and produce all o f the cells that compose specialized organs and tissues derived from the three embryonic germ layers (mesoderm, ectoderm and endoderm; or exhibit pluripotentiality). Adult stem cells are derived from specialized tissue (i.e. neural tissue), exhibit clonality for the host organism's adult life and produce all the cell types unique to the specialized tissue they were derived from (or exhibit multipotentiality; adult neural stem cells produce neurons, astrocytes and oligodendrocytes). Precursor or progenitor cells divide symmetrically (or asymmetrically for a short time) to produce cells o f the specialized tissue they were derived from and typically are the mediaries between adult stem and specialized cells. Although the terms precursor and progenitor are often used interchangeably i n the literature, Fabel and colleagues (2003) suggest that the term 'progenitor cell' is becoming used more often to describe stem-like cells in which either the property o f clonality or multipotentiality has not been tested. The generally accepted view is that new neurons are only generated by cells resident to the hippocampal subgranular zone and the lateral ventricle subventricular zone (Temple and  8 Alvarez-Buylla, 1999; Gage, 2000; M a o and Wang, 2001; Gould and Gross, 2002; Rakic, 2002). Although reports that neurons are added to the normal feline visual cortex, non-human primate neocortex and amygdala and rodent cortex and amygdala exist (Kaplan, 1981; Gould et al., 1999c, 2001; Bernier et al., 2002; Bedard et al., 2002; Fowler et al, 2002), these results have not generally been replicable (Magavi et al., 2001; Kornack and Rakic, 2001; Koketsu et al., 2003). However, neurogenesis can be induced i n the rodent neocortex by targeted cell death (Magavi et a l , 2001). In vivo characterization o f proliferative cells within the subventricular and subgranular zones is difficult technically, primarily because stem and progenitor cells likely remain quiescent until they divide and at least do not express known proteins that could be used as specific stem cell markers (Gage, 2000; Temple 2001). Therefore, most information about adult neural stem or progenitor cells has been derived using a combination o f in vitro and transplant techniques. One question posed by adult neural stem cell biologists asks whether cells in the proliferative zones o f the adult C N S are neural stem cells (i.e. are they clonogenic and multipotential?). Studies have shown that adult human and rodent hippocampus-derived cells cultured on an adherent substrate i n media containing fibroblast growth factor (FGF-2) can generate progeny for at least one year, the majority o f which express markers found in stem cells, such as nestin, 0 2 - 4 and A 2 B 5 (Gage et al., 1995; Palmer et al., 1995,1997; Kukekov et al., 1999; R o y et al., 2000). M o r e convincing evidence that these cells are clonogenic has been shown by studies that genetically tagged cultured adult hippocampus-derived cells to track their lineage and then verified the presence o f clones after many divisions using Southern blot analysis (to verify cell genotypes; Palmer et al., 1997). Growth factor withdrawal, high-density cell cycle arrest or the addition o f factors such as retinoic acid, c A M P or neurotrophic factors, induces cultured cells to differentiate into multiple C N S cell lineages in vitro (Gage et al., 1995; Palmer et al., 1995,1997; R o y et al., 2000). For example, under these conditions cells  9 either tend to acquire neuronal- or glial-like processes, to express markers typical o f neurons, glia or oligodendrocytes and cells that express neuronal protein can generate sodium and potassium currents typical o f neurons. When cultured adult hippocampus-derived cells are grafted heterotopically into either the hippocampus or olfactory bulbs, they generate phenotypically appropriate glia and neurons (Gage et al., 1995; Suhonen et al., 1996), further demonstrating that adult hippocampus-derived cells are capable o f producing multiple C N S cell phenotypes. Cells derived from the adult mammalian subventricular zone also appear able to self renew and generate multiple C N S cell lineages. Adult mouse and human subventricular zonederived cells aggregate into spheres o f proliferating cells when plated on a non-adherent substrate in media containing either epidermal growth factor ( E G F ) or F G F - 2 , and individual cells derived from theses spheres can produce secondary spheres, suggesting that these cells are capable o f self-renewal. W h e n these sphere-producing cells are plated on an adherent substrate in either E G F or F G F - 2 many daughter cells acquire processes and express neuronal or glial markers (Reynolds and Weiss, 1992; Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Reynolds and Weiss, 1996; Gritti et al., 1996; Chiasson et al., 1999; Kukekov et al., 1999; Arsenjivec et al., 2001), suggesting that these cells are also multipotential. Palmer and his colleagues (1995) have confirmed that adult rat subventricular zone-derived cells, like cells derived from the hippocampus, are clonogenic and multipotential by identifying genetically tagged clones that generate multiple cell lineages after many divisions i n culture using Southern blot analysis. Clearly, cells located within the proliferative adult dentate gyrus and subventricular zone exhibit the properties o f self-renewal and multipotentiality and, therefore, could be adult neural stem cells. Because gliogenesis persists throughout the adult mammalian C N S (Ichikawa and Hirata, 1982; K o r r et al., 1983), the possibility that stem cells exist i n non-proliferative C N S  10 regions exists. In fact, the first adult CNS-derived cells shown to exhibit stem cell-like properties in culture were isolated from the mouse striatum (Reynolds and Weiss, 1992). Since then, adult neural stem-like cells have been isolated and cultured from the adult rat septum and striatum (Palmer et al., 1995), the rat and human cortex (Palmer et al., 1999; Arsenijevic et al., 2001), the mouse and human rostral migratory stream and olfactory bulbs (Pagano et al., 2000; Gritti et al., 2002) and the mouse and rat spinal cord (Weiss et al., 1996; Shihabuddin et al., 1997). Interestingly, more cells that exhibit stem-cell properties i n culture can be isolated from neurogenic versus quiescent C N S regions (Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Palmer et al., 1995; Seaberg and van der Kooy, 2002). Because stem-like cells can be isolated from normally quiescent regions o f the adult C N S , the potential for neurogenesis in these areas must exist. However, the finding that adult hippocampal progenitors only adopt a glial phenotype when grafted into these quiescent C N S regions (Gage et al., 1995; Suhonen et al., 1996) suggests that these areas do not express the cues that induce neuronal differentiation and guide the migration o f neuroblasts. There is debate about whether the stem-like cells i n different adult mammalian C N S regions share the same phenotype and about how primitive these cells i n each region may be. In fact, at least five different cell classes have been purported to be the source o f new olfactory bulb neurons. For example, Johansson and his colleagues (1999) reported that a sub-population of ependymal cells lining the third ventricle generates progenitor cells that then generate neurons and glia, in vitro. However, van der K o o y and his colleagues (Chiasson et al., 1999) argue that subependymal cells (but not ependymal cells) self-renew and generate multiple C N S cell types i n their culture system. Doetsch and her colleagues (1997) suggest that undifferentiated cells found i n the rostral migratory stream are self-renewing and multipotential while Alvarez-Buylla and his colleagues (Garcia-Verduga et al., 1998) have reported that rostral migratory stream astrocytes exhibit stem cell properties in vitro. Finally, Gritti and her  11 colleagues (2002) suggest that cells in the rostral extension o f the olfactory peduncle and bulb exhibit stem cell-like characteristics i n culture. Recently, debate about the phenotype o f cells that produce new dentate granule neurons has begun. Although Gage and his colleagues (Palmer et al., 1997) have described adult dentate gyrus-derived cells as clonogenic and multipotential i n their culture system, van der K o o y and his colleagues (Seaberg and van der Kooy, 2002) argue the same cells exhibit limited self-renewal i n their culture system and that subependymal zone cells are the only true stem cell located i n the hippocampus. Seri and her colleagues (2001) have argued that astrocytes within the subgranular zone exhibit self-renewal in culture and generate multiple C N S cell types. Reconciling the findings o f these studies is difficult because the host strain and species, dissection methods, culture substrate and media are rarely controlled between studies (see Gage, 2000 for review). In fact, Palmer and his colleagues (1997 but see Palmer et al., 1999) have shown that cultures initiated from hippocampus-derived tissue containing mixed cell types retain a normal diploid karyotype for approximately 35 population doublings when cultured in F G F - 2 but then spontaneously transform genetically to become increasingly aneuploid, demonstrating that culture conditions can transform CNS-derived cells. Understanding the behaviour o f adult C N S progenitor/stem cells in vitro would benefit from studies that control all o f these variables. Taken together, these findings demonstrate that adult mammalian CNS-derived cells exhibit long-term self-renewal and can generate both neurons and glia in vivo and when transplanted back into the C N S and could be stem cells (Weiss et al., 1996; Fisher et al., 1995; Palmer et al., 1997). In fact, the proliferative cells found within the adult C N S are often referred to as adult neural stem cells (Shihabuddin et al., 1995; Gage 2000 for example). However, the property o f clonality has not been demonstrated in vivo and therefore, cells with proliferative capacity i n the subventricular and subgranular zones should remain classified as progenitor or precursor cells. In fact, according to Fred Gage (2002) " . . .no individual neural  12 stem cell has been identified and isolated adequately to separate it unambiguously from other, more committed [progenitor or precursor] cells in vitro or in vivo". Thus i n the experiments described in this thesis, cells that incorporate B r d U upon dividing and subsequently produce daughter cells that express either neuronal or glial protein w i l l be referred to as progenitor cells.  1.3 N E U R O G E N E S I S I N T H E A D U L T M A M M A L I A N B R A I N  Another line o f research undertaken by adult stem cell biologists is aimed toward understanding how neurogenesis i n the adult mammalian C N S is controlled. T o visualize dividing progenitor cells or their progeny in situ, animals are commonly injected with [ H]thymidine or the thymidine analogue bromodeoxyuridine (BrdU). A n y cell in the synthesis 3  phase o f its cell cycle or undergoing D N A repair w i l l preferentially incorporate either nucleotide into its D N A instead o f endogenous thymidine (Packard et al., 1973; M i l l e r and Nowakowski, 1988; Cameron and M c K a y , 2001). Then, the tissue o f interest can be processed autoradiographically or immunohistochemically to reveal progenitor cells that divided in the approximately 2 h that [ H]thymidine or B r d U , respectively, was bioavailable or their progeny 3  depending upon the amount o f time that elapses before perfusion (see Figure 1). B r d U and [ H]-thymidine labelling studies have demonstrated that cells located i n the subgranular zone divide producing daughter cells that migrate several urns, possibly along radial glia, into the granule cell layer (Altman and Das, 1965; Cameron et al., 1993b; Palmer et al., 2000; Figure 1). These markers have also demonstrated cells located i n the subventricular zone divide producing daughter cells that chain migrate several mms through the rostral migratory stream and then along radial glia to the olfactory bulbs (Lois and Alvarez-Buylla, 1993; Rousselot et al., 1995).  13 M a n y daughter cells produced in the subgranular zone eventually differentiate into granule neurons upon migrating into the granule cell layer. Morphologically, they become indistinguishable from mature dentate granule neurons (Altman and Das, 1965; Cameron et al., 1993b), acquire synapses on their dendrites and soma (Kaplan and B e l l , 1983; Kaplan and Hinds, 1977; Markakis and Gage, 1999) and extend an axon to the C A 3 layer o f the hippocampus within 4-10 days after birth (Stanfield and Trice, 1987; Hastings and Gould, 1999; Markakis and Gage, 1999). Three-dimensional confocal laser microscopy has been used to confirm the neuronal or glial phenotype o f new cells by colocalizing fluorescent probe tagged anti-BrdU with fluorescent probe-tagged antibodies that recognize either neuronal or glial protein i n single cells. This technique has revealed that approximately 60-70% o f new cells found in the granule cell layer express immature neuronal markers such as doublecortin ( D C X ) and tubulin-P (Tuj 1P) and then mature neuronal markers such neuron specific enolase (NSE) and calbindin and approximately 15-20% express glial protein such as glial acidic fibrillary protein ( G F A P ; see Figure 2 for examples). These percentages are reasonable considering that most labelled cells migrate into the granule cell layer and the granule cell layer contains few glia (Kosaka and Hama, 1986; Cameron et al., 1993b; Palmer et al., 2000). The remainder o f labelled cells do not express known proteins (Cameron et al., 1993b; Kempermarm et al., 1997a; Eriksson et al., 1998; Tanapat et al., 1999; Palmer et al., 2000; Ormerod et al., 2002) and therefore could be daughter cells that retain a parent progenitor cell phenotype. Today, B r d U is chosen more often than [ H]-thymidine to label dividing cells in adult 3  mammals primarily because immunohistochemical processing is faster than autoradiography, working with radioactivity is labour-intensive and confocal laser microscopy can be used to phenotype BrdU-labelled cells. However, both markers are advantageous for labelling  Cells  in  14  I n j e c t BrdU  2h  4-1 Od  24h  2-3wks  Netjroblas^igration?  BrdU-labelled progenitor cells  neuroblasts, glioblasts and possibly progenitor cell copies; neuroblasts begin expressing immature neuronal protein  Neuroblasts extend axons and express Immature neuronal protein  new neurons express mature neuronal protein  Figure 1. Representation of a coronal hippocampal section from the rat with inset depicting neurogenesis and a time course for the maturation of new neurons. Mature neurons situated in the granule cell layer (GCL) extend dendrites into the molecular layer and an axon through the hilus that synapses with pyramidal cells in the CA3 region of the hippocampus (Amaral and Witter, 1995). Cells located in the subgranular zone (the w50um band between the hilus and GCL) divide producing daughter cells. Many daughter cell migrate into the GCL, and extend and axon into the CA3 region and dendrites into the polymorphic layer. Experimentally, changes in cell proliferation are assessed 2 h after a cell synthesis marker, such as BrdU, is injected as this amount of time is not sufficient for progenitor cells to complete mitosis. Twenty-four h after BrdU is injected, the number of daughter cells can be counted, but very few daughter cells express known proteins by this time making identification of their phenotypes difficult. Presumably neuroblasts migrate deeper into the granule cell layer between 1-4 days after birth and begin to express immature neuronal proteins (Chapter 5). Neuroblasts extend axons between 4 and 10 d after birth (Hastings and Gould, 1999) and 2-3 week old neurons begin to express mature neuronal proteins (Cameron et al., 1993b; Palmer et al., 2000).  15 different situations. In processed tissue, each [ H]-thymidine substitution stoichiometrically appears as a silver grain, permitting the quantification o f labelling degree (heavily labelled cells are progenitor cells or their progeny) and the number o f divisions among daughter cells (heavily labelled cells are first generation and lightly labelled cells are subsequent generation; Packard et al., 1973; Nottebohm, 2002). Immunohistochemistry involves a series o f steps that amplify the B r d U signal and therefore, is more sensitive but the amplification makes B r d U non-stoichiometric (Nowakowski and Hayes, 2000). Therefore, the hypothesis that B r d U labelled cells could represent a population o f cells that repaired rather than synthesized D N A has been forwarded. However, no evidence o f B r d U labelling D N A repair in vivo has ever been published and several lines o f evidence dismiss the D N A repair hypothesis. First, labelled cell number approximately doubles between 2 and 24 h after B r d U is injected (Cameron and M c K a y , 2001). Second, studies using B r d U immunohistochemistry to detect D N A damage in cultured cells deliver insults that induce the replacement o f approximately 100 nucleotides at each damaged site whereas in vivo, only 1-2 nucleotides are replaced and current immunohistochemical techniques are not sensitive enough to detect this low-level repair (Schmitz et al., 1999 for review). Third, many BrdU-labelled cells come to exhibit morphology and express proteins similar to mature granule neurons in the granule cell layer (see previous paragraph). Finally, Palmer and his colleagues (2000) demonstrated that the B r d U incorporated by cultured fibroblasts following gamma irradiation, which produces relatively similar damage to what is observed in vivo, could not be detected immunohistochemically. Taken together, the evidence available suggests that B r d U can only be detected i n cells that are synthesizing rather than repairing D N A . Another advantage associated with B r d U is that anti-BrdU can penetrate the relatively thick tissue sections required to maintain the integrity o f fixed tissue whereas emulsion only reveals [ H]thymidine i n the top 3 um o f a section (Feinendagen, 1971; M i l l e r and 3  Nowakowski, 1988). Therefore, stereological estimates o f total labelled cell number can be determined on BrdU-labelled tissue (Gunderson et al., 1988; West et al., 1991; Cameron and M c K a y , 2001). Stereology was designed to estimate the total number o f cells i n a structure that  16 would be observed i f the entire structure could be visualized under the microscope (Gunderson et al., 1988; West et al., 1991). For example, i f BrdU-labelled cells were counted on every section through the dentate gyrus, then the total number o f labelled cells would be underestimated because only those observed in the focal plane o f the microscope (i.e. typically the top 0.005 m m o f each section using a lOOx light objective) can be counted. Stereology, as applied to BrdU-labelled tissue, projects the number o f labelled cells counted in the focal plane of the objective through the section thickness and i f cells are counted on sections o f equal intervals (i.e. every 10 section) through the dentate gyrus then cells are projected through its th  estimated volume (see Chapter 4 for more detail). The application o f stereological techniques to estimate BrdU-labelled cell number through the dentate gyrus o f adult mammals appears partially responsible for the relatively recent surge in interest regarding the phenomenon. In fact, Kaplan and B e l l (1984) suggested that few researchers were interested i n investigating adult neurogenesis partially because the phenomenon appeared to be quite low-level based upon the densities o f [ H]thymidine-labelled cells reported (see Altman and Das, 1965; 3  Cameron et al., 1993b). Using stereology, Cameron and M c K a y (2001) have determined that up to 9,000 new cells are produced daily i n the dentate gyri o f adult rats (Cameron and M c K a y , 2001). Other methods o f visualizing dividing cells or their progeny have also been used and can be advantageous i n certain situations. Retroviral vectors carrying a reporter gene such as Lac-Z or green fluorescent protein can be used to visualize entire dividing cells and their progeny (see Sanes et al., 1986; van Praag et al., 2002). Because these vectors are expressed in the soma, dendrites and axon o f new cells, they can be useful for localizing new neurons in tissue slice preparations used for electrophysiological recording, for example. However, because infection rates can be low-level and retroviral expression can be downregulated once the progeny o f dividing cells differentiate (Gage, 2000) their usefulness for stereologically estimating the total number o f new cells may be limited. Endogenous markers o f mitosis such as K i 6 7 and P C N A appear as effective as B r d U or [ H]-thymidine for detecting proliferating 3  cells (Nacher et al., 1996; Tanapat et al., 1999; Wojtowicz) and do not require an injection.  17 However, the transient expression o f endogenous mitosis markers makes phenotyping progeny difficult because cells are i n s-phase only for approximately 9.5 h (Cameron and M c K a y , 2001). Clearly, the choice o f a proliferation marker depends upon the question being asked i n an experiment. To understand the mechanisms and function o f adult neurogenesis, the factors affecting progenitor cell proliferation (mitosis i n progenitor cells) must be delineated from the factors that affect the survival (differentiation and maturation) o f young neurons. Altering the rate o f cell proliferation, the differentiation o f daughter cells or the survival o f young neurons could increase or decrease net neurogenesis (literally, the creation o f neurons) i n the dentate gyrus. A n experimental manipulation administered just before or during the time that B r d U is bioactive affects cell proliferation whereas an experimental manipulation administered after B r d U is bioactive can affect the survival o f labelled cells in the process o f differentiating, migrating or maturing. In both cases, the effect is reflected in the number o f labelled cells observed hours, days or weeks after B r d U is injected. Alternatively, a manipulation could be administered just prior to or during B r d U uptake and then labelled cell number could be assessed weeks later to verify the number o f new neurons surviving. However, in this case BrdU-labelled cells should also be assessed 2-24 h after B r d U is administered (the time required for 1 mitotic division; Cameron and M c K a y , 2001) to disentangle effects on proliferation versus survival. Although the number o f labelled cells has been assessed after multiple daily B r d U injections i n some studies to investigate the effect o f housing condition, physical activity or genetic strain on neurogenesis (Kempermann et al., 1997a, 1997b; van Praag et al., 1999a, 1999b), interpreting the results o f these studies is difficult. In the case that no difference between groups i n BrdU-labelled cell number emerges following multiple daily injections, it could be that proliferation was increased and the survival o f young neurons decreased or vice versa. Generally, administering a single injection o f cell synthesis marker either after or before a treatment facilitates the determination o f whether neurogenesis has been affected and whether the manipulation has influenced progenitor cell proliferation or the survival o f young neurons, respectively.  18  1.4 A R E N E U R O N S P R O D U C E D I N T H E D E N T A T E G Y R U S F U N C T I O N A L ?  Currently, theories about the functional role o f neurogenesis constitutive to the adult C N S are speculative. However, granule neurons are produced in the dentate gyrus o f every eutherian mammalian group studied including rodents (Altman, 1962; Kempermann 1997a; Galea and M c E w e n , 1999; Lavenex et al., 2000; Ormerod et al., 2002), lagomorphs (Gueneau et al., 1982), carnivores (Wyss and Sripanidkulchai, 1985), tupaiids (Gould et al., 1997a) and human/non-human primates (Gould et al., 1998; 1999a; 1999b; Eriksson et al., 1998; Kornack and Rakic 1999) throughout adulthood. The conservation o f this phenomenon across species certainly suggests that new granule neurons serve some functional role. In fact, the daily addition o f thousands o f new neurons to the adult dentate gyrus (Cameron and M c K a y , 2001) surely impacts the flow o f information through the hippocampus. Information prominently travels through the trisynaptic circuit o f the hippocampus (entorhinal cortex to the dentate gyrus to area C A 3 and to area C A I ) and to a lesser extent through disynaptic (entorhinal cortex to C A 3 to C A I ) and monosynaptic circuits (entorhinal cortex to C A I ; for review see Amaral and Witter, 1995). The neurons that are added to the dentate gyrus appear to be electrophysiologically plastic (Wang et al., 2000; Snyder et al., 2002; van Praag et al., 2002) and could alter how information is transferred through the hippocampus, v i a the trisynaptic circuit. For example, Wang and colleagues (2000) found that long-term potentiation (LTP) could be induced in presumably young granule neurons located near the subgranular zone i n presence o f intact G A B A  A  receptor-induced inhibition whereas  L T P can only be induced i n mature granule neurons when G A B A  A  receptor-induced inhibition  is antagonized. Recent work using a GFP-tagged retrovirus to birth-date granule neurons produced in the dentate gyrus o f mice found electrophysiological responses typical o f mature granule neurons 4 weeks after infection and a mature granule neuron morphology (dendritic  19 complexity and similar spine counts) 4 months after infection (van Praag et al., 2002). In addition, evidence suggests that i f young neurons (a few days to 3 weeks old) are reduced by approximately Vi by gamma irradiation, artificial cerebrospinal fluid-induced L T P that is normally observed cannot be generated, suggesting that young neurons do influence activity within the dentate gyrus (Snyder et al., 2001). Because new dentate granule neurons rapidly extend axons to the C A 3 region (Hastings and Gould, 1999), they could rapidly influence hippocampus-dependent function. In addition, because new neurons are extremely plastic, electrophysiologically, they may be primed to participate in learning and/or memory. In fact, hippocampus-dependent learning enhances the number o f young neurons that survive in the dentate gyri adult rats. The acquisition o f a trace-conditioned eyeblink response and the Morris water maze (localization o f a hidden platform) are dependent upon the integrity of the hippocampus whereas neither the acquisition o f delay-conditioned response nor the localization o f a visible water maze platform require an intact hippocampus (Morris et al., 1982; Morris et al., 1990; M c E c h r o n et al., 1998; Weiss et al., 1999). Gould and her colleagues (1999) found more 12 day-old neurons in the dentate gyri (but not subventricular zone) o f rats that were trained on a trace eyeblink conditioning task or in the Morris water maze versus a delay eyeblink conditioning task or a visible platform water maze task, beginning one-week post-BrdU injection. N o difference i n neuron number was found between groups o f rats trained on either trace eyeblink conditioning trials or delay eyeblink conditioning trials prior to B r d U administration (Gould et al., 1999), indicating that cell proliferation is not influenced differentially by these tasks. Although hippocampus-dependent learning can enhance the survival o f younger (4 to 7 day-old) neurons, the effect on older (1-2 week-old) neurons is much more robust (Ambrogini et al., 2000 versus Gould et al., 1999). These findings suggest that hippocampus-dependent learning enhances the survival o f young granule neurons in the dentate gyrus o f adult rodents.  20 Because thousands o f granule neurons are added daily to the dentate gyrus o f adult rodents (Cameron and M c K a y , 2001), preventing the integration o f young neurons could produce deficits in performance on hippocampus-dependent tasks. In fact, Shors and her colleagues (2001, 2002) found that rats' abilities to acquire either a trace-conditioned eyeblink response or a trace-conditioned fear response are impaired i f cell proliferation is prevented for 14 d (but not for 6 d) using a cytostatic agent just prior to training. The acquisition o f the traceconditioned fear response has been shown to rely upon the integrity o f the hippocampus (McEchron et al., 1998; Weiss et al., 1998). Animals given 21 d to recover from the effects o f the cytostatic agent readily acquire the trace-conditioned eyeblink response (Shors et al., 2001). Surprisingly, Shors and colleagues (2002) found that performance across training trials in the Morris water maze was not impaired following treatment with a cytostatic agent over 14 days just prior to training. Taken together, this evidence suggests that 1-2 week old neurons are critical for some, but not all forms o f hippocampus-dependent learning. Therefore, new neurons appear to participate i n or are influenced b y hippocampusdependent learning most significantly just after they begin extending their axons (4-10 after birth) and are presumably forming synapses with C A 3 region pyramidal neurons because they begin to express the mature neuronal protein N S E by 14 d after birth (Cameron et al., 1993b; Hastings and Gould, 1999). Less is known about the relationship between new neurons and hippocampus-dependent memory. However, relatively specific and complete destruction o f the granule cell layer produces severe deficits in performance on retention trials, as well as across training trials i n a large Morris water maze (Xavier et al., 1999), suggesting that young granule neurons could influence this aspect o f hippocampus-dependent function. Shors and her colleagues (2002) found that rats treated with cytostatic agent for 14 days prior to the onset o f Morris water maze training performed as well as control rats on a retention trial in the task, suggesting that young neurons may not influence retention on this hippocampus-dependent  21 task. However, rats with hippocampal damage are capable o f solving the Morris water maze task using non-spatial strategies (Whishaw, 1985; Long and Kesner, 1996; Pouzet et al., 2002), and rats treated with antimitotic agent prior to training could have also learned to solve the Morris water maze task using a non-spatial strategy that would permit good performance on a retention trial. Administering an antiproliferative agent after training trials have been administered (and prior to a retention trial) could provide better insight as to how young granule neurons influence memory for a platform position acquired using spatial information. In addition, increasing young neuron number prior to training trials could reveal their influence over spatial learning and/or memory. Chapter 5 describes the effect o f increasing young granule neuron number on hippocampus-dependent learning and memory.  1.5 E S T R A D I O L I N F L U E N C E S H I P P O C A M P A L N E U R O G E N E S I S  Estradiol is one factor that has been shown to influence neurogenesis in the dentate gyrus o f adult rodents, however the effects reported in rats and voles differ. Galea and M c E w e n (1999) reported that adult female meadow voles trapped during the non-breeding season (when estradiol levels are low) had more proliferating cells in their dentate gyri (measured 24 h after a single injection o f [ H]thymidine) than females trapped during the breeding season (when 3  estradiol levels are high) or males trapped during either season. In fact, Galea and M c E w e n (1999) found that serum estradiol was correlated negatively with labelled cell density in the dentate gyri o f adult female meadow voles. Then, Tanapat and her colleagues (1999) found more labelled cells i n the dentate gyri o f gonadally intact adult female rats than i n the dentate gyri o f males (measured 2 h after B r d U was injected). The rate o f cell proliferation was related to estrous cycle phase because more new neurons were produced during the afternoon o f the proestrus phase (when estradiol levels are high) relative to either the estrus or diestrus phase (when estradiol levels are low). In rats, ovariectomy reduces dentate cell proliferation and this  22 reduction is reversed 2 h after an estradiol injection (Tanapat et al., 1999; Banasr et al., 2001). Taken together evidence suggests that estradiol decreases cell proliferation i n the dentate gyri of adult female meadow voles but increases cell proliferation i n the dentate gyri o f adult female rats. One reason w h y the effects o f estradiol on cell proliferation i n the dentate gyri o f adult female meadow voles versus rats could differ is because the meadow voles that Galea and M c E w e n (1999) trapped during the breeding or nonbreeding season were feral and several factors that have been shown to influence cell proliferation cannot be controlled i n a feral sample. For example, all o f the females that Galea and M c E w e n (1999) captured during the breeding season were pregnant and evidence has shown that pregnancy can affect neurogenesis in the dentate gyrus o f adult rats (Madonia et al., 2000; Banasr et al., 2001). C e l l proliferation declines i n the dentate gyri o f aged rodents (Kuhn et al., 1996; Seki and A r a i , 1995; Montaron et al., 1998; Cameron and M c K a y , 1999). Because reproductively successful adult female meadow voles establish and defend territories while inhibiting the reproductive status o f subadults within their territories (Madison, 1980; Madison et al., 1985), the pregnant females that Galea and M c E w e n (1999) trapped during the breeding could have been older females. Finally, access to a running wheel increases cell proliferation i n the dentate gyrus o f adult mice (van Praag 1999a; 1999b). Female meadow voles reduce their space use and territory size during the breeding season (Madison, 1985; Sheridan and Tamarin, 1988), and therefore presumably run less during the breeding season relative to during the non-breeding season and relative to males in either season. Thus, replicating the effect o f season on cell proliferation in the dentate gyri o f a sample o f laboratory-reared female meadow voles would permit control over these potentially confounding variables to shed light upon the relationship between reproductive status, estradiol and cell proliferation in the dentate gyri o f adult voles.  23 Another reason why the effects o f estradiol on cell proliferation in the dentate gyri o f adult female meadow voles versus rats could differ is that the duration that each species was exposed to high-level circulating estradiol was different. The reproductive physiologies o f adult female rats and meadow voles are very different. Whereas female rats experience elevated estradiol levels only on the afternoon o f proestrus (Buckingham et al., 1978), female meadow voles experience elevated circulating estradiol levels that persist for up to 45 d only after ovulation is induced by male contact (Lee et al., 1970; Seabloom et al., 1985; Nubbemeyer, 1999). Tanapat and colleagues (1999) injected rats with B r d U at 2 pm, and therefore proestrus females would have likely been exposed to high circulating estradiol for a few hours (see Buckingham et al., 1978). Galea and M c E w e n (1999) injected females that were trapped in either the breeding or nonbreeding with [ H]thymidine and, therefore, females in the breeding 3  season would likely have been exposed to high circulating estradiol levels for a longer time period (Lee et al., 1970; Seabloom et al., 1985; Nubbermeyer, 1999). Therefore, following short duration exposure to estradiol cell proliferation could have been elevated in proestrus rats (Tanapat et al., 1999) and following longer exposure to high circulating estradiol cell proliferation could have decreased in the dentate gyri o f adult female meadow voles (Galea and M c E w e n , 2001). High-level estradiol could affect cell proliferation differentially i n a durationdependent manner. This hypothesis is tested i n Chapters 3 and 4. Banasr and her colleagues (2001) demonstrated that estradiol stimulates serotonin synthesis to increase cell proliferation within 2 h o f its administration and there is evidence to suggest that estradiol could suppress cell proliferation following longer exposures by stimulating adrenal activity. High-level adrenal steroids (corticosterone injection, stress) suppress and low-level adrenal steroids (low dose corticosterone replacement in adrenalectomized rats) enhance cell proliferation in the dentate gyri o f adult male rats (Cameron and Gould, 1994; Cameron et al., 1995; Cameron and M c K a y , 1999; Tanapat et al.,  24 2001). Dividing progenitor cells express neither Type I nor Type JJ glucocorticoid receptors (Cameron et al., 1993a), suggesting that adrenal steroids regulate cell proliferation indirectly. In fact, Cameron and her colleagues (1998) demonstrated that N M D A receptor activation works downstream o f adrenal steroids to suppress cell proliferation in the dentate gyri o f adult rats. N M D A receptor activation increases and N M D A receptor blockade decreases cell proliferation in the dentate gyri o f adult rats and tree shrews (Cameron et al, 1994; Cameron et al., 1995; Gould et al., 1997; Bernabau and Sharp, 2000; Nacher et al., 2001; Nacher et al., 2003 but see Bernabau and Sharp, 2000 and Arvidsson et al., 2001) and N M D A receptor activation can prevent an adrenalectomy-induced increase and N M D A receptor blockade can block an adrenal steroid-induced decrease i n cell proliferation in the dentate gyri o f adult male rats (Cameron et al., 1998). Interestingly, estradiol stimulates the rat and meadow vole hypothalamic-pituitaryadrenal axis (Coyne and Kitay, 1969; Christian, 1969; Burgess and Handa, 1992; Handa et al., 1994) and has been shown to increase both the sensitivity and number o f N M D A receptors in the rat hippocampus (Weiland et al., 1992; Gazzaley et al., 1996). Therefore, estradiol could eventually suppress cell proliferation by stimulating adrenal activity and/or by stimulating N M D A receptor activity. Chapter 3 describes an experiment that tests whether estradiol stimulates adrenal activity and Chapter 4 describes experiments that test whether estradiol interacts with N M D A receptors to suppress cell proliferation.  1.6 E S T R A D I O L E N H A N C E S C E L L S U R V I V A L I N D I F F E R E N T S Y S T E M S  Although thousands o f new neurons are added daily to the dentate gyri o f adult rodents, many o f the neurons appear to die between 2 and 4 weeks after birth (Gould et al., 1999; Cameron and M c K a y , 2001). Studies using [ H]thymidine or relatively high doses o f B r d U 3  (200-600 mg/kg) have demonstrated that the number o f labelled cells approximately doubles between 24 h and 1 week post-label, but that only about A o f the labelled cells observed at 24 h l  25 survive longer than 2-4 weeks (Cameron et al., 1993b; Gould et al., 1999; Cameron and M c K a y , 2001). Although some labelled cells appear to continue dividing to the point that B r d U and [ H]thymidine are too diluted to be detected immunohistochemically or 3  autoradiographically (Cameron et al., 1993b; Nowakowski and Hayes, 2002), the average number o f silver grains i n [ H]thymidine-labelled cells does not diminish until 3 weeks post3  label suggesting many cells die between 2 and 3 weeks (Cameron and M c K a y , 2001). Therefore, by increasing the number o f new granule neurons that survive 2 weeks or more, net neurogenesis could be increased. Estradiol enhances the survival o f numerous cell types (see Garcia-Segura et al. 2001 for review) including hippocampal neurons in various models o f injury both in vivo and in vitro. Estradiol increases the viability, differentiation and survival o f cultured hippocampal neurons (Sudo et al., 1998). In addition, estradiol rescues cultured hippocampal neurons from excitotoxicity-, oxidative injury- and P-amyloid toxicity-induced death (Goodman et al., 1996; Weaver et al., 1997). In vivo, estradiol reduces the death o f C A I region hippocampal neurons and decreases infarct size following experimental ischemia i n gerbils (Chen et al., 1998). Estradiol significantly reduces the number o f dentate gyrus hilar interneurons that die following the administration o f convulsive doses o f kainic acid in ovariectomized female rats (Azcoitia et a l , 1998, 1999). In addition, pretreatment with estradiol can prevent quinolinic acid-induced cell death i n the hippocampus o f adult male rats (Favata et al., 1998; K u r o k i et al., 2001). The most compelling evidence to suggest that estradiol could enhance the survival o f granule neurons produced in the adult rodent dentate gyrus is that estradiol has also already been shown to enhance the survival o f new neurons in the adult avian song circuit. More labelled cells were observed i n the song circuits o f female zebra finches surgically implanted with estradiol-filled versus empty silastic capsules 24 h after [ H]thymidine was injected 3  (assessed 18 days after implant; Burek et al., 1995), demonstrating that estradiol enhances the survival o f new neurons. Hidalgo and colleagues (1995) found more labelled cells i n the song circuit o f canaries implanted with an estradiol- versus cholesterol-filled silastic capsule 32 days after the onset o f 8 daily [ H]thymidine injections. Because Hidalgo et al. (1995) only found 3  estrogen receptors (ERs) on cells in the migratory pathway o f new neurons but not on the new neurons themselves, they concluded that estradiol indirectly influenced the survival o f new migrating neurons. Recent work by Loissant and his colleagues (2001) has demonstrated that estradiol enhances the survival o f cultured avian song circuit neurons by inducing B D N F expression i n endothelial cells. Concurrent changes in angiogenesis and neurogenesis have been reported i n the adult rodent dentate gyrus (Palmer et al., 2000) suggesting that estradiol could enhance the survival o f new granule neurons i n the dentate gyrus o f adult rodents and songbirds through a similar mechanism. Both estrogen receptor subtypes ERo. and ERp are expressed in the dentate gyrus (Shughrue et al., 1997; Milner et al., 2001). In fact, electron microscopy has revealed that not only are interneurons i n the dentate gyrus ERdimmunoreactive but the axons and dendrites o f granule neurons are also ER^-immunoreactive (Milner et al., 2001). Whether young neurons express either E R is unknown but the presence o f E R s in the dentate gyrus provides a means by which estradiol could enhance the survival o f young neurons, at least indirectly. Chapter 5 describes the effect o f estradiol on the survival o f young granule neurons.  1.7 O V E R V I E W A N D O B J E C T I V E S  The experiments described i n this thesis investigate the effect o f estradiol on components o f neurogenesis i n the adult rodent dentate gyrus and to determine whether changes i n young neuron number influence hippocampus-dependent behaviour. The reported  27 effects o f estradiol on progenitor cell proliferation i n the dentate gyri o f adult female rats and meadow voles are different. Elevated circulating estradiol levels are associated with increased proliferation in female rats (Tanapat et al., 1999) but decreased proliferation female meadow voles (Galea and M c E w e n , 1999). Several factors could account for the differences reported in the relationship between estradiol and cell proliferation i n the dentate gyri o f rats and voles. For example, Galea and M c E w e n (1999) used feral voles as subjects i n their study and many factors that alter adult neurogenesis cannot be controlled i n a feral sample. Moreover, rat and meadow vole reproductive physiologies differ such that each species would have experienced high circulating estradiol for different durations prior to the administration o f cell synthesis marker and estradiol could influence cell proliferation i n a time-dependent manner. Currently the effect o f estradiol on the survival o f young neurons produced i n the adult rodent dentate gyrus unknown. However, estradiol enhances the survival o f new neurons produced in the rodent C N S developmentally and in the avian song circuit during adulthood and, therefore, could enhance the survival o f young neurons produced i n the dentate gyrus. If estradiol enhances the survival o f young granule neurons, then hippocampus-dependent behaviour could be altered. Indeed, reports that hippocampus-dependent behaviour enhances the survival o f young neurons (Gould et al., 1999) and that some forms o f hippocampus-dependent behaviour are impaired when young granule neurons are depleted (Shors et al., 2001, 2002) have been published. Therefore, the objectives o f the present thesis are as follows:  1. To determine whether reproductive status influences the number of dividing and new cells in the dentate gyri of adult laboratory-reared female meadow voles (Chapter 2). A n attempt to replicate the finding that cell proliferation is suppressed i n the dentate gyri o f adult female meadow voles trapped during the breeding season when they are reproductively active compared to the non-breeding season when they are reproductively inactive w i l l be made using sample o f laboratory-reared voles to control  28 potentially confounding variables. Females w i l l be acclimated to a short- or longphotoperiod to simulate the winter non-breeding or summer breeding season, respectively, and a male or female cage partner w i l l be introduced to manipulate reproductive status, which w i l l be verified by ovary mass and serum estradiol level. B r d U or [ H]-thymidine w i l l be injected to assess the number o f dividing cells (2 h after B r d U ) and new cells (5 weeks after [ H]thymidine), respectively. Because cell 3  proliferation is elevated i n nonbreeding versus breeding feral adult female meadow voles, I expect to observe more labelled cells i n the dentate gyri o f reproductively inactive (with l o w circulating estradiol levels) versus active (with high circulating estradiol levels) laboratory-reared females, 2 h after a B r d U injection. Furthermore, because estradiol has been shown to promote the survival o f neurons i n other systems, I expect to observe more labelled cells i n the dentate gyri o f reproductively active versus inactive females, 5 weeks after a [ H]-thymidine injection. 3  2.  To determine whether the effect of reproductive status on cell proliferation  in the  dentate gyrus of adult female meadow voles can be mimicked by estradiol and whether estradiol influences cell proliferation  time-dependently (Chapter 2). Reproductively  inactive females w i l l be injected with estradiol four h (the time reported to increase cell proliferation i n rats) or 48 h (the time that female meadow voles are paired with a cage partner to manipulate reproductive status) before a B r d U injection. Voles w i l l be perfused 2 h later to assess the density o f proliferating cells i n their dentate gyri. These data w i l l address some o f the controversy i n the literature by determining whether the effect o f estradiol on cell proliferation i n the dentate gyrus o f adult female meadow voles is time-dependent. I hypothesize that female voles exposed to estradiol for 4 h w i l l have more labelled cells and females exposed to estradiol for 48 h w i l l have fewer labelled cells i n their dentate gyri than reproductively active females.  29  3.  To test the robustness of estradiol's time-dependent effects on cell proliferation acro species (Chapters 3 and 4). To test the robustness o f the time-dependent effect o f estradiol on cell proliferation, ovariectomized adult female rats and voles w i l l be given and injection o f estradiol or vehicle 4 h or 48 h prior to an injection o f B r d U . The animals w i l l be perfused 2 h, 24 h or 4 d later to determine the number o f dividing progenitors (2 h post-BrdU i n voles), new cells (24 h post-BrdU i n rats) and new neurons (4 days post-BrdU in rats). I expect that the number o f labelled cells w i l l increase i n the dentate gyri o f rats and voles treated with estradiol versus vehicle for 4 h but decrease i n the dentate gyri o f rats and voles treated with estradiol versus vehicle for 48 h prior to a B r d U injection. Because previous work has shown that estradiol does not alter the percentage o f BrdU-labelled cells that acquire a neuronal phenotype, I also expect to observe a similar percentage o f BrdU-labelled cells that express glial or neuronal protein i n estradiol- versus vehicle-treated rats (assessed 4 days post-BrdU).  4. To determine how estradiol suppresses cell proliferation in the adult rodent dentate gyrus (Chapters 3 and 4). Previous studies have shown that adrenal steroids and N M D A receptor activation suppress cell proliferation i n the dentate gyrus o f adult rats, and estradiol stimulates adrenal activity and increases both the number and sensitivity o f N M D A receptors. Therefore, estradiol could stimulate adrenal activity or N M D A receptor activity to suppress cell proliferation within 48 h. To test whether estradiol stimulates adrenal activity to suppress cell proliferation, ovariectomized/ adrenalectomized rats w i l l be exposed to estradiol or vehicle for 48 h and then injected with B r d U and perfused 24 h later to assess the density o f labelled cells. I predict that removing estradiol's influence over adrenal activity w i l l either eliminate (no difference between BrdU-labelled cell number w i l l be observed between groups) or reverse (more BrdU-labelled cells w i l l be observed i n the estradiol- versus vehicle-treated group) the  30 estradiol-induced suppression i n cell proliferation i n the dentate gyri o f adult female rats. To test whether estradiol influences N M D A receptor activity to suppress cell proliferation, ovariectomized female meadow voles w i l l be injected with estradiol or vehicle and then N M D A or saline 3 h later or M K - 8 0 1 or saline 47 h later. One h after the injection o f N M D A , M K - 8 0 1 or saline, B r d U w i l l be injected and the voles w i l l be perfused 1 h later. I predict that M K - 8 0 1 w i l l eliminate or reverse the estradiol-induced suppression i n cell proliferation. 5.  To determine whether estradiol influences the survival of young granule neurons produced in the dentate gyrus of adult rodents (Chapter 5). The effect o f estradiol the survival o f young granule neurons in the adult mammalian dentate gyrus has not been tested specifically. T o specifically test the effect o f estradiol on the survival o f new neurons independent o f its effects on cell proliferation, castrated male meadow voles w i l l injected twice with B r d U and then no treatment w i l l be administered for at least 24 h, as this is enough time for cells proliferating i n the adult rodent dentate gyrus to complete one mitotic division. This design enables the effect o f estradiol on the survival of young neurons independent o f its effects on cell proliferation, to be tested. Then males w i l l be injected once per day with estradiol or vehicle either over Days 1-5, Days 6-10 or Days 11-15 post B r d U and then perfused on D a y 16 to assess the total number of labelled cells (and their phenotypes). The time-periods were chosen to roughly correspond with differentiation and migration, axon extension and maturation, respectively. Because estradiol has been shown to promote the survival o f immature neurons during development and migrating neuroblasts i n the avian forebrain I hypothesize that more labelled neurons w i l l survive i n the dentate gyrus o f estradiolversus vehicle-treated males, possibly when estradiol is administered when cells are migrating (Days 1-5) similar to what has been observed i n the avian songcircuit.  31 6.  To determine whether increasing the number of young granule neurons influences hippocampus-dependent  behaviour (Chapter 5). Previous work has demonstrated that  hippocampus-dependent learning enhances the survival o f young neurons and that depleting the number o f young neurons with a cytostatic agent impairs some forms o f hippocampus-dependent behaviour. Therefore, i f estradiol increases the survival o f young neurons, then the increase may influence hippocampus-dependent behaviour. Castrated males w i l l be given two injections o f B r d U and then estradiol over Days 6-10 post B r d U (the time frame shown to enhance the survival o f new neurons i n Experiment 7; Chapter 5). Over Days 16-19, voles w i l l be trained i n the same spatial Morris water maze task shown previously to enhance the survival o f new neurons. O n Day 20, a probe trial w i l l be administered to test the voles' retention o f the platform location over training trials and then reversal trials w i l l be administered to test the voles' tendencies to perseverate. Because performance across training trials is unaffected when young neurons i n the dentate gyri o f adult rats are depleted, I predict that both groups may perform similarly across training trials but that estradiol-treated voles may outperform vehicle-treated voles on the probe trial. Chapters 2-5 w i l l describe experimental data collected to address the objectives described. A l l experimental data has been published or submitted for publication i n manuscript form. The details o f those findings w i l l not be reiterated i n the General Discussion, rather the findings w i l l be discussed generally and then i n terms o f how estradiol-induced changes i n neurogenesis may impact normal hippocampus function and may influence neuronal replacement strategies.  32  CHAPTER 2 REPRODUCTIVE STATUS INFLUENCES C E L L PROLIFERATION AND C E L L SURVIVAL IN THE DENTATE GYRUS OF ADULT F E M A L E M E A D O W VOLES: A POSSIBLE R E G U L A T O R Y R O L E FOR ESTRADIOL {published in Neuroscience 2001  102:369-379)  2.1 I N T R O D U C T I O N  Although most neurons are integrated into the central nervous system during discrete developmental periods, recent studies have shown that the hippocampus o f adult mammals retains the ability to produce and incorporate new granule neurons (Kaplan and Hinds, 1977; Kaplan and B e l l , 1984; Cameron et al., 1993b). N e w cells are produced when progenitor cells within the subgranular zone o f adult laboratory mammals (Altman and Das, 1967; Angevine, 1965; Gueneau et al., 1982; Wyss and Sripanidkulchai, 1985; Cameron et al., 1993b; Gould et al., 1997a; 1997b; Galea and M c E w e n , 1999; Gould et al., 1999a; 1999b; Kornack and Rakic, 1999) and humans (Eriksson et al., 1998) divide. Evidence suggests that the majority o f the progeny differentiate into neurons. N e w cells migrate into the granule cell layer, extend an axon into the C A 3 region by 10 days after birth and exhibit granule neuron morphology (Cameron et al., 1993b; Hastings et al., 1999). These new cells express immature neuronal markers, such as T O A D - 6 4 (turned on after division-64kD), within 24 hours after division (Minturn et al., 1995; Tanapat et al., 1999) and then mature neuron-specific proteins, such as neuron-specific enolase, 2 to 3 weeks later (Cameron et al., 1993b; G o u l d et al., 1997a; Eriksson et al., 1998). N e w granule neurons are likely functional as they receive synaptic input (Markakis and Gage, 1997), show paired-pulse facilitation comparable to mature granule neurons and enhanced electroplasticity (via lower threshold for the induction o f long-term potentiation in absence o f suppressed inhibition; Wang et al., 2000). In adult rodents, the rate o f cell proliferation is elevated by serotonin (Jacobs et al., 1998; Brezun and Daszuta, 1999; 2000)  33 and physical activity (van Praag et al., 1999a; 1999b) but diminished by dopamine agonists (Teucherdt-Noodt et al., 1997), TV-mefhyl-D-aspartate-receptor ( N M D A r ) activation (Cameron et al., 1995; Cameron et al., 1998) corticosterone ( C O R T ; Gould et al., 1992; Cameron and Gould, 1994; Cameron et al., 1995; Cameron and M c K a y , 1999; Cameron et al., 1998) and ageing (Seki and A r a i , 1995; K u h n et al., 1996; Montaron et al., 1998; Cameron and M c K a y , 1999). Although neurogenesis has been extensively studied i n laboratory animals, only a fewstudies have investigated the phenomenon i n natural populations (Barnea and Nottebohm, 1994; 1996; Galea and M c E w e n , 1999; Tramontin and Brenowitz, 1999; Lavenex et al., 2000). In the w i l d adult female meadow vole, Galea and M c E w e n (1999) found that the rate o f cell proliferation within the dentate gyrus fluctuates with season o f capture. Specifically, rates o f cell proliferation were significantly lower i n female meadow voles trapped during the breeding season than i n females trapped during the non-breeding season. Galea and M c E w e n (1999) also found that the rate o f cell proliferation was negatively correlated with serum estradiol level in these animals. Female meadow voles are reflex ovulators and thus, serum estradiol is rapidly elevated with the induction o f behavioural estrous and remains elevated throughout the breeding season as the majority o f female meadow voles are either i n behavioural estrous or are pregnant (Clulow and Mallory, 1970; Lee et al., 1970; Prentice and Shepherd, 1978; Boonstra and Boag, 1992). Therefore, the suppression i n cell proliferation observed by Galea and M c E w e n (1999) was likely mediated by estradiol. However, Tanapat et al. (1999) reported that estradiol transiently increased the level o f neurogenesis in the dentate gyrus o f adult female laboratory rats. Specifically, cell proliferation was increased during proestrous (associated with high serum estradiol levels) relative to either estrous or diestrous (associated with low or medium serum estradiol levels) in normally cycling rats (Buckingham et al., 1978; Butcher et al., 1974; Tanapat et al., 1999). In addition, Tanapat  34  et al. (1999) found that an ovariectomy-induced diminution i n granule neuron proliferation could be reversed i n the 2 hours following an injection o f estradiol benzoate. Therefore, while high levels o f endogenous estradiol i n w i l d female meadow voles are associated with suppressed rates o f granule cell proliferation (Galea and M c E w e n , 1999), high levels o f endogenous or exogenous estradiol in the laboratory-reared female rat are associated with increased rates o f granule cell proliferation (Tanapat et al., 1999). There are several possible explanations for the discrepant results found between the two studies, other than potential species' differences. The Galea and M c E w e n (1999) study utilised w i l d animals and therefore, many variables could have suppressed cell proliferation i n female meadow voles captured during the breeding season. For example, in Galea and M c E w e n ' s (1999) study, all o f the females captured during the breeding season were pregnant and pregnancy affects hippocampal morphology and could influence cell proliferation Galea et al., 2000). Ageing is associated with a decline i n the rate o f cell proliferation i n rodents (Kuhn et al., 1996; Seki and A r a i , 1995; Montaron et al., 1998; Cameron and M c K a y , 1999). Because reproductively successful adult female meadow voles establish and defend territories while inhibiting the reproductive status o f subadults within their territories (Madison, 1980; Madison et al., 1985), the pregnant females that Galea and M c E w e n (1999) trapped could have been older females. Alternatively, experience is associated with an increase i n cell proliferation and cell survival (Scott et al., 1998; Gould et al., 1999). Female meadow voles reduce their space use and territory size during the breeding season (Madison, 1985; Sheridan and Tamarin, 1988), which presumably changes experience within their environment relative to the nonbreeding season and relative to males in either season. Thus, while investigating cell proliferation i n natural populations is advantageous many uncontrolled variables in a w i l d sample could potentially influence the rate o f cell proliferation observed. Therefore, the present study was conducted to determine the effect o f reproductive status on cell proliferation and  35 survival i n a sample o f laboratory-reared meadow voles. B y using laboratory-reared meadow voles, the potentially confounding variables o f pregnancy, age, and experience could be controlled.  2.2 M E T H O D S  A l l animals were treated i n strict accordance with the guidelines set forth by the Canadian Council on A n i m a l Care and The University o f British Columbia regarding the ethical treatment o f animals used for the purposes o f research. Every effort was made to minimise the number o f animals used per group and their suffering.  Animals  Adult male (>35 g and 60 days old) and female (>25 g and 60 days old) voles from our breeding colony at The University o f British Columbia were used for all experiments. A l l voles were reared i n a 16 hr light/8 hr dark colony room (lights on at 0700h) and were given access to Lab Diet #5012 (Jamieson) and tap water, ad libitum. Twenty-one day old animals were weaned and housed i n a bedding-lined (Care Fresh; Absorption Corporation) polyurethane cage with a same-sex littermate until 60 days o f age. A t 60 days o f age, the voles were housed individually and kept i n either the long-photoperiod (16 hr light/8 hr dark) or moved to a shortphotoperiod (10 hr light/14 hr dark) to simulate breeding or non-breeding season day-length, respectively.  Procedure  After a 4-week acclimation period (to photoperiod), voles were paired with either a male or female cage partner for 48 hours to manipulate reproductive status. Previous studies  36 have shown that estradiol levels i n female meadow voles are elevated 12-18 hours after exposure to male pheromone only during the long-photoperiod (Lee et al., 1970) and remain elevated for at least 22 days (Seabloom, 1985). Similarly, exposure to a male vole induces a rapid increase in the serum estradiol level (about 200%; Cohen-Parsons and Carter, 1987) that persists for at least 3 weeks (Dluzen and Carter, 1979). Therefore, introducing a male cage partner for 48 hours was sufficient to shift female reproductive status and thereby elevate the associated hormones, particularly estradiol. Female-paired female meadow voles housed in either the long- or short-photoperiod were considered reproductively inactive (PJ) and malepaired female meadow voles housed i n the long-photoperiod were considered reproductively active ( R A ) . Serum estradiol levels further verified reproductive status. Experiment 1 was conducted to determine whether reproductive status influences the density o f proliferating cells within the dentate gyrus o f adult female meadow voles. In addition, separate groups o f R I females were given a single injection o f estradiol benzoate ( E B ) to determine whether estradiol mimicked the effect o f reproductive status because estradiol level is rapidly and dramatically increased with the onset o f behavioural estrous i n meadow voles (Prentice and Shepherd, 1978). Thus, female meadow voles were given a single injection o f estradiol benzoate ( E B ) 4 hours (EB4) or 48 hours (EB48) prior to bromodeoxyuridine (BrdU) labelling. These time points for E B administration were chosen to match the possible onset o f estradiol increase i n R A females (4 hours) and the maximum duration that R A females would be exposed to elevated estradiol prior to sacrifice (48 hours). R A females, R I females, E B 4 and EB48 (n=5 per group) were given a single intraperitoneal (i.p.) injection o f the thymidine analogue, B r d U (50 mg/kg; Sigma Aldrich Chemicals), between 1230 and 1300h). The voles were anaesthetised deeply with sodium pentobarbital (0.1 ml) and then perfused transcardially with 4.0% paraformaldehyde in 0 . 1 - M phosphate buffered saline (PBS), 2 h after B r d U was injected. A l l brains were extracted and post-fixed overnight i n perfusate. Unfixed adrenal  37 glands and ovaries were removed and weighed. The following day, the brains were sectioned and the sections were processed immunohistochemically for B r d U . Prior to perfusing the animals, blood samples were taken from the right ventricle and were stored at 4° Celsius. Twenty-four hours later, the blood samples were centrifuged at 4g for 10 minutes and then the serum was drawn and frozen at -70°Celsius until radioimmunoassays were performed. Experiment 2 was conducted to determine whether the initial difference i n cell proliferation observed i n R I versus R A females persisted for 5 weeks. Long-photoperiod and shortphotoperiod female meadow voles were housed with a cage partner for 48 hours and composed three experimental groups: 1) long-photoperiod male-paired females (n=7, reproductively active [RA]), 2) long-photoperiod female-paired females (n=5, reproductively inactive [RI]) and 3) short-photoperiod female-paired females (n=7, RI). Forty-eight hours after being paired, the voles were given a single i.p. injection o f [ H]thymidine (5 u C i / g ; between 1230 and 3  1300h). Five weeks after [ H]thymidine injection, the voles were deeply anaesthetised with 3  sodium pentobarbital (0.1 ml) and then perfused transcardially with 4.0% paraformaldehyde in 0.1-M phosphate buffered saline (PBS). Brains were extracted and post-fixed overnight in perfusate. The following day, the brains were sectioned and the sections processed autoradiographically for [ H]thymidine. B r d U was used i n Experiment 1, [ Hjthymidme was used i n Experiment 2 to label dividing cells. However, B r d U and [ Hjthymidine label proliferating cells at very similar relative rates in proliferation (the present study and see Galea and M c E w e n , 1999) and survival studies (Cameron and Gould, 1994 versus Cameron and M c K a y , 1999).  Drug  preparation  38 17B-Estradiol benzoate ( E B ; Sigma Aldrich Chemicals) was prepared by dissolving E B in sesame o i l (Sigma A l d r i c h Chemicals) to a concentration o f 10p,g o f EB/0.1 m l sesame oil. The solution was then stored i n a light insensitive container. A l l voles were given a 0.10 m l injection (subcutaneous) o f the solution (containing 10p.g o f E B ) . This dose was chosen because Carter and colleagues (1987) reported that lordosis behaviour could be induced in female prairie voles 48 hours after a single 10|ag injection o f E B . B r d U was prepared just prior to administration by dissolving B r d U in freshly prepared isotonic saline containing 0.7% 2 N N a O H to a concentration o f 10 mg B r d U / m l saline. B r d U was injected i.p. i n a volume o f 0.5ml/100g.  Histology  A l l brains were sliced into 40 um thick sections through the entire dentate gyrus with an oscillating tissue sheer ( O T S 3000, Electron Microscopy Sciences) using a bath o f 0.1-M phosphate buffer (PB). Slices prepared for peroxidase immunohistochemistry were pre-treated in a solution o f P B with 0.2% H2O2 for 20 m i n and then rinsed before being mounted on slides treated with 3% 3-aminopropyltriethoxy-silane in acetone. Slices prepared for autoradiography were mounted on 2 % gelatin-coated slides and left to dry overnight  Peroxidase immunohistochemistry  Tissue was processed for BrdU-immunoreactivity by applying solutions directly to the slide-mounted sections. Unless otherwise specified, phosphate buffered saline (0.1 M sodium phosphate heptahydrate i n 0.9% saline; p H 7.4) was used for all rinses and slides were rinsed repeatedly between each step. 1) Cells i n the sections were permeabilized with 0.05% Trypsin (Sigma Aldrich Chemicals) in T r i s - H C l buffer (pH 7.5) containing 0.1% C a C l for 10 min. 2) 2  39 D N A was denatured by applying 2 N HC1 for 30 m i n and then the sections were repeatedly rinsed (pH 6.0). 3) Sections were blocked i n 5.0% normal horse serum for 30 m i n and then incubated overnight i n mouse monoclonal antibody against B r d U (1:100 + 3% N H S + 0.5% Tween 20; Boehringer Mannheim) at room temperature. 4) Sections were incubated in mouse secondary antisera (1:29 + 3.0% normal horse serum; Vector Laboratories) for 4 hrs. 5) Sections were incubated i n avidin-biotin horseradish peroxidase ( A B ; 1:50; Vector Laboratories) for 60 min. 6) Sections were reacted for 10 m i n in 0.02% diaminobenzidine ( D A B ; Sigma A l d r i c h Chemicals) with 0.003% H2O2 and then counterstained with cresyl violet acetate (Baker), dehydrated and coverslipped with Permount (Fisher Scientific). For [ H]thymidine autoradiography, slides were dipped i n autoradiographic emulsion 3  ( N T B 2 , Kodak) and stored at 4 E C for 4 weeks. Then the slides were developed in Dektol (Kodak), fixed i n Ektaflo (Kodak), counterstained with cresyl violet acetate, dehydrated in ethanol, preserved with xylene and coverslipped with Permount.  Hormone assays Serum corticosterone ( C O R T ) levels were analysed in the Department o f Anatomy using a radioimmunoassay protocol described in detail by Weinberg and Nezio (1987). Briefly, antiserum was obtained from Immunocorp (Montreal, Canada) and tracer was obtained from Mandel Scientific (Guelph, Canada). Dextran-coated charcoal was used to adsorb and precipitate free steroids after incubation. Serum estradiol levels were analysed using a Coat-a-Count kit (Diagnostic Products Corporation, L o s Angeles, C A ) modified for low expected levels o f estradiol. The sensitivity o f the estradiol assay was 5 pg/ml. Values below the detection threshold o f the assay were arbitrarily given a value o f 0 pg/ml (n=3; R I females).  40 Data analyses  Slides were coded prior to the analysis to blind the experimenter to the treatment conditions. Six sections o f the middle portion o f the dentate gyrus (where the dentate gyrus is positioned horizontally beneath the corpus callosum and the suprapyramidal and infrapyramidal blades are joined at the crest; between A -3.3 and A -4.8 i n rats) were analysed, per subject (see Gould et al., 1992; Cameron et al., 1993b; Gould et al., 1997a; Cameron et al., 1998; Gould et al., 1999b), using a N i k o n Eclipse (C 600) Light microscope (100X objective). For the B r d U analyses, cells were considered immunoreactive i f they were intensely stained and exhibited appropriate morphology (medium-sized round or oval cell bodies; Cameron et al., 1993b; Figure 2 A ) . For the [ H]thymidine analysis, granule cells were considered labelled i f 3  the number o f silver grains was at least 20x the background level and the cell exhibited mature granule cell morphology (medium-sized round or oval cell bodies; Cameron et al., 1993b; see Figure 2B). Pyknotic cells were counted for all sections using the criterion set forth by Gould and colleagues (1991). Briefly, pyknotic cells lacked a nuclear membrane, had pale or absent cytoplasm and darkly stained spherical chromatin (Figure 2C). The total number o f [ H]thymidine-labelled or BrdU-immunoreactive and pyknotic cells found in the granule cell 3  layer or hilus was counted and divided by the total respective area. The area o f the granule cell layer and hilus was determined using the digitising software Analytical Imaging Station (Imaging Research Inc, Brock University, Ontario, Canada). The data are expressed as densities (the number o f labelled cells per m m ) . 2  Statistical analyses  The dependent variables from Experiment 1 (density o f BrdU-labelled cells and density of pyknotic cells) were each analysed using a one-way analysis o f variance ( A N O V A ) with  41 condition ( R A , R I , E B 4, and E B 48) as the independent variable. Pearson product-moment correlation tests were conducted on the density o f labelled and pyknotic cells and hormone levels. Data were derived from 19 females, as some sections from the brain o f one R I female were lost during processing. The data from Experiment 2 (density o f [ H]thymidine-labelled 3  cells and density o f pyknotic cells) were each analysed using an independent t-test with status (RI and R A ) as the independent variable. Data were derived from the brains o f 14 animals as some sections from two R A females and two R I females were damaged during processing. In addition, one R I female died prior to completing the experiment. Unless otherwise specified, Post hoc tests utilised the Newman-Keuls procedure. A l l statistical procedures set a = 0.05.  2.3 R E S U L T S  Experiment 1. Reproductive status, possibly via estradiol, influences cell proliferation in the dentate gyrus of adult female meadow voles.  Density of BrdU-labelled cells and pyknotic cells in the eranule cell layer and hilus Figure 2 A shows a BrdU-labelled cell found in the subgranular zone o f an adult female meadow vole. BrdU-labelled cells were observed to occur primarily i n clumps located in the subgranular zone. The density o f BrdU-labelled cells i n the granule cell layer was significantly greater i n R I and E B 4 females compared to R A females (p_ < 0.0007) and E B 4 8 females (p_ < 0.0013, F ,i5) = 21.016, p_ < 0.0001; see Table 1). Similarly, the density o f BrdU-labelled cells (3  in the hilus o f R I females was greater compared to all other groups; R A (p_ < 0.016), E B 4 (p_ < 0.027) and E B 4 8 females (p_ < 0.014; Fp.is) = 5.267, p < 0.011; see Table 1). N o significant difference was found between groups in the area o f the G C L (p_ < 0.09) or the hilus (p < 0.57).  42  Figure 2. Microphotographs of a bromodeoxv uridine (BrdU)-labeled cell, a [ H]thymidine-Iabeled cell and a pyknotic cell in Experiments 1 and 2. 3  A) Depicts a BrdU-labelled cell in the subgranular zone between the granule cell layer and hilus of the dentate gyrus of an adult female meadow vole. The majority of BrdU-labelled cells were observed to occur in clumps in the subgranular zone of the dentate gyrus. B) Depicts a [H]thymidine-labelled cell located within the granule cell layer of an adult female meadow vole. Most [ H]thymidine-labelled cells were located in the granule cell layer. C) Depicts a pyknotic cell located in the hilus of an adult female meadow vole. GCL = granule cell layer and H = hilus. Scale bar (lc) = 20 um. 3  3  43  Table 1. Mean (+SEM) density of BrdU-labelled and pyknotic cells in the granule cell layer and hilus of adult female meadow voles measured 2h after B r d U was injected in Experiment 1. Granule Cell Layer Hilus Group Labelled cells Pyknotic cells Labelled cells Pyknotic Cells RI (n=5) 88.61±18.81 19.31111.27 17.3116.29 0.9211.30 RA (n=4) 5.93±1.99** 13.0913.46 2.0910.50* 1.6110.76 EB4 (n=5) 126.22±16.99 23.6511.01 5.5911.01* 0.5310.93 EB48 (n=5) 3.6610.93** 21.3810.50 0.3510.16* 1.8411.60 * indicates a significant difference (p < 0.050) relative to the RI group **indicates a significant difference (p < 0.002) relative to the RI group * indicates a tendency toward a significant difference (p < 0.067) relative to the RI group f  A pyknotic cell is shown in Figure 2 C . There was no main effect o f condition on the density o f pyknotic cells i n the granule cell layer (p < 0.49) or the hilus (p < 0.51) o f female meadow voles (see Table 1). The density o f labelled cells was not correlated with the density o f pyknotic cells i n either group (Tables 1 and 2; granule cell layer [r i ) = 0.145; p < 0.62], hilus (  [r  (19)  9  =-0.287; p < 0.32]).  Table 2. Pearson product-moment correlations between dependent variables measured in adult female meadow voles in Experiment 1. BrdU-ir BrdU-ir Cells (GCL) Cells (hilus) Serum EB r =-0.657* R =-0.530 Serum CORT r = 0.362 R = -0.447 Adrenal Mass r = -0.692 R = -0.496 Ovary Mass r =-0.841** R =-0.672* Degrees of freedom for all r values = 9 * indicates a significant difference (p < 0.050) **indicates a significant difference (p < 0.005) P-values are one-tailed T  Pyknotic Cells (GCL) r =-0.177 r =-0.756 r = -0.400 r =-0.461  Pyknotic Cells (hilus) r = -0.074 r = -0.732 r =-0.175 r = 0.484  Adrenal Mass (mg) r = 0.733* r = 0.188  Ovary Mass (mg) r = 0.798** r = 0.314 r = 0.672*  r = 0.720*  Hormone levels and ovary and adrenal masses There was a significant effect o f condition on adrenal mass (F(3,i4) = 4.171, p < 0.026; Table 3). Post-hoc analyses revealed that R A females had significantly heavier adrenal masses than did R I females (p < 0.029) and E B 4 females (p < 0.013). Similarly, there was a significant effect o f condition on ovary mass (F(3,i5) = 28.87, p < 0.0001; Table 3). Post-hoc analyses  44 revealed that both R A females and E B 4 8 females had significantly heavier ovary masses than did R I females (p. < 0.0002 and p_ < 0.02, respectively) and E B 4 females (p_ < 0.0002 and p_ < 0.005, respectively and see Table 2). Due to heterogeneity o f variance between groups on serum estradiol levels (% (3) = 38.42,/? < 0.001) a non-parametric Kruskal-Wallis test was used 2  and revealed a significant difference between groups (x (3) = 12.842, p_ < 0.005). Post-hoc analyses revealed that all high estradiol groups ( R A , E B 4 and E B 4 8 ) had significantly greater serum E B levels than did the R I group (p_ < 0.05; Table 3). In addition, post-hoc analyses confirmed that R A females had significantly higher serum E B levels than did R I females (p_ < 0.05; Table 3) and that E B 4 females had significantly higher serum estradiol levels than did E B 4 8 females (p < 0.05; Table 3).  Table 3. Mean (±SEM) adrenal mass, gonad mass, serum C O R T level and serum estradiol level measured in adult female meadow voles in Experiment 1. Adrenal Mass (mg) RI (n=5) 17.6+1.8 RA (n=4) 28.5±5.8* EB4 (n=5) 12.3±4.1 EB48 (n=5) 21.0±5.2 indicates a significant difference (p < 0.050) **indicates a significant difference (p < 0.001) V J l  uuu  Ovary Mass Serum estradiol (pg/ml) (mg) 12.6+1.2 4.69+3.26 31.811.1** 22.0015.84* 9.812.9 250.52150.08** 19.015.7** 44.56+4.07* relative to the RI group relative to the R I group  Serum CORT (ng/ml) 634.32187.04 509.15192.63 719.70154.80 609.02139.04  Table 2 shows the correlations between hormone levels, adrenal and ovary masses, the density o f BrdU-immunoreactive (ir) cells and the density o f pyknotic cells i n the R I and R A female groups. The density o f BrdU-labelled cells i n the granule cell layer was negatively correlated with ovary mass and serum estradiol level (also see Tables 1 and 3). Serum estradiol was positively correlated with ovary and adrenal mass (also see Table 3). In addition, adrenal mass was positively correlated with ovary mass (also see Table 3).  45 Experiment 2. The elevated labelled cell density observed in RI adult female meadow voles persists for 5 weeks  Density of [3H]thymidine-labelled and pyknotic cells in the granule cell layer and hilus The majority o f [ H]thymidine-labelled cells were found within the granule cell layer, 3  consistent with the data reported by Cameron and colleagues (1993; see Figure 2B). There was no statistical difference between the R I female groups (female-paired females housed in the long-photoperiod versus the short-photoperiod) in the density o f [ H]thymidine-labelled cells (granule cell layer, p< 0.46; hilus, p < 0.67). Therefore, these groups were combined and referred to as R I females i n subsequent analyses. R I females had a significantly greater density of [ H]thymidine-labelled cells in the granule cell layer than did R A females (t(i i) = 2.944, p < 3  0.013, see Table 4).  Table 4. Mean (+SEM) density of [ H]thymidine-labelled and pyknotic cells measured in the granule cell layer and hilus 5 weeks after [ H]thymidine was injected in Experiment 2. Granule Cell Layer Hilus Group Labelled cells Pyknotic cells Labelled cells Pyknotic Ceils 3  3  RI RA  (n=9) (n=5)  5.9410.85 2.9311.99*  17.2413.00 22.2612.98  1.44+0.28 1.7210.60  1.4510.70 0.80+0.70  * indicates a significant difference (p < 0.050) relative to the RI group  However, densities o f [ H]thymidine-labelled cells i n the hilus (p < 0.44) and pyknotic cells in 3  the granule cell layer (p < 0.367) and the hilus (p < 0.109) did not statistically differ between groups (see Tables 3 and 4). The density o f pyknotic cells was not correlated with the number of labelled cells in either the granule cell layer (r i ) = 0.154,/> < 0.29]) or the hilus ( r ^ = (  4  0.458, p < 0.116; Table 4). The number o f pyknotic cells was = 285% and = 650% greater than the number o f [ H]thymidine-labelled cells in R I and R A voles, respectively (compare densities 3  in Table 4). There was 1 pyknotic cell labelled with [ H]thymidine i n the granule cell layer o f 3  46 an R A female. N o significant differences were found between the R I and R A groups in the area o f either the G C L (p < 0.99) or the hilus (p < 0.28).  2.4 DISCUSSION  The density o f proliferating cells in the dentate gyrus was elevated in reproductively inactive (RI) relative to reproductively active ( R A ) females and was negatively correlated with serum estradiol level (Tables 1 and 2). These findings indicate that reproductive status regulates cell proliferation i n female meadow voles. Exposing female meadow voles to E B for 48 hours, but not 4 hours could reproduce the degree o f suppression i n cell proliferation observed in R A versus R I females. In fact, exposure to E B for 4 hours tended to elevate the number o f proliferating cells i n the granule cell layer compared to R I females (Table 1). This finding demonstrates that the effect o f reproductive status can be mimicked b y estradiol i n a durationdependent manner. Similar patterns were observed with cell survival as the R I female meadow voles had greater densities o f labelled cells than did R A females, 5 weeks after the injection o f [ H]thymidine (Table 4). N o significant difference in pyknotic cell density was found i n either 3  the granule cell layer or the hilus between R I and R A female meadow voles, i n either experiment (Tables 1 and 4).  Reproductive status rapidly influences cell proliferation in the dentate gyrus of adult femal meadow voles  Reproductive status influences cell birth i n the dentate gyrus o f laboratory-reared female meadow voles. The density o f proliferating cells i n the granule cell layer and hilus o f R I females was approximately 9-times higher than i n R A females. These results replicate Galea and M c E w e n ' s (1999) finding that non-breeding female meadow voles had significantly greater  47 densities o f [ H]thymidine-labelled cells (approximately 10 times) i n the granule cell layer 3  compared to breeding female meadow voles. The density o f labelled cells i n the hilus o f the R I group was larger than i n the R A group (about 8x). This finding is also consistent with that o f Galea and M c E w e n (1999) that the density o f labelled cells in the hilus was larger in nonbreeding versus breeding voles, although the magnitude o f their difference was lower (about 3x). Typically, cells found i n the hilus express proteins consistent with a glial phenotype (Cameron et a l , 1993b) and, as observed i n the present study, fewer new cells are found in the hilus relative to the G C L . Interestingly, E B 4 (high estradiol) females i n this study tended to have more labelled cells in the G C L but fewer labelled cells i n the in the hilus relative to R I females. Based upon the findings o f the current and previous studies, it is tempting to speculate that different mechanisms may regulate the genesis o f cells found i n the G C L (mostly neuronal) versus the hilus (mostly non-neuronal). The density o f proliferating cells i n the dentate gyrus o f female laboratory-reared meadow voles was inversely related to serum estradiol level, which also complements Galea and M c E w e n ' s (1999) finding i n w i l d female meadow voles. In fact, the amount o f variance in proliferating cells accounted for by serum estradiol alone is 43%, and for ovary mass alone is 70%. The present study demonstrated that cell proliferation in the dentate gyrus o f laboratoryreared female meadow voles was initially increased but subsequently decreased by estradiol exposure. Specifically, a single high dose o f estradiol tended to increase cell proliferation 4 hours after exposure but significantly suppressed cell proliferation 48 hours after exposure relative to R I females. Females exposed to a high level o f estradiol for 4 hours exhibited rates o f cell proliferation similar to R I females, while females exposed to the same dose o f estradiol for 48 hours had rates o f cell proliferation similar to R A females. Therefore, the duration o f exposure to estradiol (endogenous or injected) appeared to produce the differences i n the rate o f cell proliferation observed at four versus 48 hours. The duration-dependent effect o f estradiol  48 on cell proliferation may explain the apparent discrepancies regarding the effects o f estradiol on neurogenesis i n the dentate gyrus o f rodents reported in the literature (Galea and M c E w e n , 1999; Tanapat et al., 1999). Tanapat and colleagues (1999) found that rats i n proestrous had significantly higher rates o f cell proliferation than rats i n estrous or diestrous. According to their protocol rats i n proestrous would have been exposed to high levels o f estradiol for approximately 2-7 hours before B r d U was injected while rats i n estrous would have experienced the proestrous estradiol surge approximately 30 hours before B r d U was injected (see Butcher et al., 1974). Thus, the meadow voles i n the present study were exposed to high a level o f E B for roughly the same durations that rats in the Tanapat et al (1999) study were exposed to proestrus level estradiol (E4 mimics proestrous and E48 mimics estrous). The duration-dependent effect o f estradiol on cell proliferation likely reflects an interaction between estradiol and other hormones or neurotransmitters. For example, breeding season-dependent increases i n estradiol enhance the secretion o f adrenocorticotropin hormone ( A C T H ) and corticoid binding thereby producing compensatory adrenal growth and secretion in female meadow voles after a lag time (Coyne and Kitay, 1969; Christian, 1975). This lag time must be between 4 and 48 hours, as changes i n adrenal mass were only observed, i n the present study, after 48 hours o f exposure to estradiol (either injected or induced). Estradiol may regulate C O R T level similarly i n rats as circadian fluctuations o f serum A C T H and C O R T are enhanced during proestrus (Buckingham et al., 1978) and estradiol upregulates glucocorticoid density in the hippocampus (Ferrini and DeNicola, 1991). H i g h levels o f circulating adrenal steroids suppress cell proliferation (Gould et al., 1992; Cameron and Gould, 1994; Cameron et al., 1995; Cameron et al., 1999; Cameron et a l , 1998). Therefore, estradiol could initially increase and subsequently decrease cell proliferation in the dentate gyrus o f adult female rodents by upregulating C O R T secretion from the adrenal glands (Christian, 1975) and C O R T receptor m R N A i n the brain (Ferrini and DeNicola, 1991). In the present study, adrenal masses  49 were significantly larger i n R A females compared to R I females which is i n agreement with the observations o f Christian (1975) and were positively correlated with ovary mass. Our method of blood removal (prior to perfusion) may have prevented accurate assessments o f pre-sacrifice differences i n serum C O R T level between groups by producing a ceiling effect. O f course, estradiol could influence cell proliferation by any number o f interactions within the dentate gyrus. Estradiol has been shown to increase the number of N M D A receptor binding sites on C A I pyramidal neurons (Weiland, 1992) and upregulate expression o f the obligatory N M D A receptor subunit N R 1 (Gazzaley et al., 1991). N M D A receptor activation also suppresses cell proliferation in the dentate gyrus o f adult rodents (Cameron et al., 1995; Cameron et al., 1998) and shrews (Gould et al., 1997a).  Estrogen receptor localization and effects within the hippocampus  Generally, estradiol both downregulates estrogen receptor expression in the a  hippocampus (Weiland et al., 1997) and can differentially regulate estrogen receptorp m R N A expression ( M c E w e n and Alves, 1999) which is more abundantly expressed i n the hippocampus (Shughrue et al., 1997). A n y estrogen receptor -mediated effect on cell a  proliferation is likely indirect as this receptor is expressed in G A B A e r g i c interneurons within the hippocampal formation o f rats (Weiland et al., 1997). Less is known about the phenotype o f cells that express estrogen receptorp but this receptor subtype is more abundant within the hippocampus o f rats and is expressed at low levels within the subgranular zone where the progenitors o f new granule neurons reside (Shughrue et al., 1997). O f course, definitive statements about the localization and regulation o f estrogen receptor subtypes cannot currently be made as studies utilising putatively specific antibodies, ligands and probes are quite recent (Shughrue et a l , 1997; M c E w e n and Alves, 1999). Thus, the effect o f E B on cell proliferation  50 in this study could have been mediated by either the a - or |3-receptor as both demonstrate high affinity for E B (Shughrue et al., 1999). Estrogen receptor subtypes activate different second messenger pathways ( M A P K and c A M P ) and activate gene transcription v i a different response elements ( C R E , S R E and A P - 1 ; for review see M c E w e n and Alves, 1999) and therefore, it would not be surprising to find that cell proliferation could be differentially influenced by estradiol along very different time lines. The female meadow vole is an advantageous model for investigating the mechanism(s) by which estradiol influences cell proliferation as estradiol i n this animal is maintained at either a high or low level (Seabloom, 1985) and estrogen receptor m R N A is distributed similarly across rodent species ( K o c h and Ehret, 1989; Simerly et al., 1989; Hnatchuk et al., 1994).  Neurogenesis in natural and laboratory  populations  While the labelling efficacy o f B r d U versus H-thymidine has not been directly 3  assessed, the densities o f labelled cells i n the granule cell layer o f both R A and R I laboratoryreared female meadow voles in the present study were very comparable to those found in w i l d voles by Galea and M c E w e n (1999). This finding also suggests that 10 to 12 generation w i l d th  th  female meadow voles that have been reared and housed in relatively impoverished laboratory conditions experience the same degree o f cell proliferation during the non-breeding and breeding season as meadow voles reared i n the w i l d (Galea and M c E w e n , 1999). Indeed, Kempermann and colleagues (1997) have shown that mice housed i n an enriched environment have more surviving new neurons but not more proliferating granule cells than mice housed i n impoverished conditions. Although they did not assess cell proliferation, Barnea and Nottebohm (1994) found that seasonal increases i n the density o f surviving neurons (labelled 6 or more weeks after [ Hjthymidine injection) were enhanced i n free-ranging relative to captive  51 black-capped chickadees, demonstrating that birds' natural environments enhanced cell survival beyond that observed in an enriched laboratory environment (a large outside aviary). Another study that directly assessed cell proliferation in w i l d grey squirrels reported seasonal fluctuations in brain volume but no change in cell proliferation in the dentate gyrus (Lavenex et al., 2000). However, the longevity o f w i l d grey squirrels (up to 6 years; Gurnell, 1987) increases the probability that senescent squirrels were included i n the sample analysed and cell proliferation is diminished i n senescent rats (Seki and A r a i , 1995; K u h n et al., 1996; Montaron et al., 1998; Cameron and M c K a y , 1999). In summary, while cell proliferation in adult w i l d animals may be seasonally regulated, the survival o f new neurons may be enhanced by experience.  Reproductive status influences granule cell survival in the dentate gyrus  In the present study, females were only paired with a cage partner for 48 hours, but cell survival was likely affected by reproductive status. Behavioural estrous, and therefore elevated estradiol levels, persist for at least 3 weeks following exposure to a male (Seabloom, 1985; Cohen-Parsons and Carter, 1987). In fact, R I females had more proliferating cells (Experiment 1) and more surviving new cells (Experiment 2) compared to R A females. However, the percentage o f cells labelled at 5 weeks (Experiment 2) versus 2 hours (Experiment 1) was lower in the R I female group (granule cell layer s 7%, hilus = 24%) compared to the R A female groups (see Tables 1 and 4; granule cell layer = 49%, hilus = 82%). This finding suggests that although R I females had more proliferating cells, R A females had a greater rate of cell survival (density o f labelled cells persisting 5 weeks). Studies have shown that survival can be regulated independent o f cell proliferation. For example, enriched housing (Kempermann et al., 1997) and hippocampus-dependent learning (Gould et al, 1999a) enhance the survival o f  52 new granule neurons but have no effect on cell proliferation. Interestingly, i n the avian higher vocal centre, the survival o f new neurons is modulated by older estrogen-ir neurons that lie within the migratory pathway (Goldman, 1999). There was no effect o f photoperiod on cell survival i n female meadow voles (the number o f new cells surviving 5 weeks) as R I females housed i n either the long- or shortphotoperiod had comparable densities o f both labelled and pyknotic cells. This finding coupled with previous work i n males suggests that there is a possible sex difference in the effect o f photoperiod on cell survival. For example, photoperiod has been shown to regulate the number o f new neurons surviving 7 weeks after B r d U injection in adult male golden hamsters (Huang et al., 1998). M a l e golden hamsters housed in a short-photoperiod had more seven week-old neurons than male golden hamsters housed i n a long-photoperiod. Observing the effect o f reproductive status on cell proliferation i n male meadow voles would be interesting because while reproductive status in females is dependent upon the environment (exposure to male pheromone or a male), reproductive status i n male meadow voles is photoperiod-dependent (Seabloom, 1985).  Reproductive status does not influence the density ofpyknotic  cells in the dentate gyrus of adult  female meadow voles  While reproductive status influenced cell proliferation i n the dentate gyrus o f female meadow voles, no effect on pyknotic cell density was found i n Experiment 1. Furthermore, the density o f labelled cells (proliferating or mature) within the dentate gyrus o f female meadow voles was not related to pyknotic cell density at either 2 hours or 5 weeks. Studies utilising experimentally induced pathologies, such as kindling (Bengzon et al., 1997; Parent et al., 1998; Scott et al., 1998), ischemia (Liu et al., 1995) and lesions (Gould and Tanapat, 1997) have found evidence that dying cells may induce the proliferation o f progenitor cells. Interestingly,  53 Galea and M c E w e n (1999) found that during the non-breeding season (when estradiol levels are low) the density o f pyknotic cells, along with the density o f proliferating cells was elevated. The R A females (present study) had the same density o f pyknotic cells as w i l d female meadow voles trapped during the non-breeding season by Galea and M c E w e n (1999). Interestingly, while pyknotic cell densities drop significantly during the breeding season i n w i l d voles (Galea and M c E w e n , 1999) they remain elevated i n R A females (present study). A s mentioned, serum C O R T level is inversely related to pyknotic cell density i n the dentate gyrus o f adult rodents (Sloviter et al., 1989; Gould et al., 1990; Gould et al., 1991) and serum C O R T levels measured in the present study were much lower than those reported by Galea and M c E w e n (1999). Therefore, pyknotic cell densities may have been artificially high i n the present study due to chronically low C O R T level.  Reproductive status-related changes in cell proliferation and/or survival in the female mead voles may be related to maternal behaviour in natural settings  The observation that reproductive status, possibly through estradiol, regulates the density o f proliferating cells within the dentate gyrus o f adult female meadow voles is intriguing, as these animals are very successful breeders. Behavioural estrous and subsequent ovulation can be induced i n female meadow voles on the day o f parturition (Lee et al., 1970). Because meadow voles are promiscuous, females are either in behavioural estrous or pregnant throughout much o f the breeding season (Anderson et al., 1976). The observation that the majority o f new granule neurons express mature neuronal markers (neuron-specific enolase, for example; Cameron et al., 1992) at approximately 21 days after their birth is compelling as gestation is 21 days i n meadow voles (and other rodents). Therefore, reproductive statusdependent shifts in cell proliferation might profoundly impact hippocampus-dependent behaviour at the time a mother meadow vole gives birth to her litter. I f the number o f new  54 granule neurons that form mature synapses affects hippocampal function, then hippocampusdependent behaviour should be affected about 21 days after any shift i n the rate o f proliferating cells within the dentate gyrus; i.e. during parturition i n the adult female meadow vole. Adult female meadow voles contract their home range size at parturition and expand their home range size during weaning (Madison, 1978). In addition, studies have shown that female meadow voles decrease their home range size during the breeding season (when cell proliferation rates are low) relative to the non-breeding season (Madison, 1985; Sheridan and Tamarin, 1988). Perhaps analogously, low estradiol female meadow voles learn a spatial version o f the water maze better than high-estradiol female meadow voles (Galea et al., 1995). Thus, the functional consequence o f reduced cell proliferation may be reduced spatial ability, which could be advantageous near parturition for female meadow voles. B y remaining close to her nest, a mother meadow vole likely reduces her probability o f predation and is better able to protect her pups. In fact, Sheridan and Tamarin (1988) found that smaller home range size is related to better reproductive success in female voles. In addition to influencing cell proliferation, estradiol produces morphological and electrophysiological changes within the hippocampus. For example, C A I pyramidal cell spine and synapse density is increased by estradiol (Woolley et al., 1990; Woolley and M c E w e n , 1992; Woolley and M c E w e n , 1993). Woolley (1998) has hypothesised that estradiol-induced changes i n hippocampal morphology may be related to maternal behaviour in female rodents. Early studies have demonstrated that aspects o f maternal behaviour are hippocampusdependent. Indeed, fimbria lesions impair pup retrieval, nest building and nursing (Kimble et al., 1967; Steele et al., 1978). Certainly, estradiol-induced morphological changes within the hippocampus could prime maternal behaviour and estradiol-induced decreases in the density o f proliferating cells could impair spatial ability.  55  2.5 I M P L I C A T I O N S  In conclusion, reproductive status, possibly through estradiol, regulates cell proliferation and survival i n the dentate gyrus o f adult female meadow voles. Initially, estradiol tends to increase cell proliferation i n the dentate gyrus o f rodents but subsequently induces a mechanism (perhaps C O R T mediated) that suppresses cell proliferation. Although the density of labelled cells remains elevated in R I females relative to R A females, 5 weeks later, the rate o f survival o f newly formed granule cells i n the granule cell layer is enhanced in R A females, indicating that estradiol may enhance cell survival. Because this change is reproductively linked, the long-term functional consequence o f a changed rate o f cell proliferation could be that maternal behaviour is enhanced and spatial ability is decreased, both o f which could potentially enhance the reproductive success o f female meadow voles.  56  CHAPTER 3 E S T R A D I O L I N I T I A L L Y E N H A N C E S B U T S U B S E Q U E N T L Y SUPPRESSES (VIA A D R E N A L STEROIDS) P R O G E N I T O R C E L L P R O L I F E R A T I O N W I T H I N T H E DENTATE GYRUS OF ADULT F E M A L E RATS (published in the Journal of Neurobiology  2003 55:247-260)  3.1 I N T R O D U C T I O N The dentate gyrus o f the hippocampus is a structure that exhibits neurogenesis throughout adulthood (for review see Kempermann and Gage, 1999; Fuchs and Gould, 2000; Ormerod and Galea, 2000). Estrogen is one factor that can regulate neurogenesis within the adult rodent dentate gyrus (Galea and M c E w e n , 1999; Tanapat et al., 1999; Ormerod and Galea, 2001). H i g h levels o f estradiol increase progenitor cell proliferation in intact female rats relative to vehicle controls (Tanapat et al., 1999). In contrast, the density o f new cells is correlated negatively with plasma estradiol levels in w i l d adult female meadow voles (Galea and M c E w e n , 1999). This differential nature o f estradiol's regulation o f cell production within the rat versus vole dentate gyrus could be time-dependent. W e have found that estradiol exposure for 4 h enhances whereas estradiol exposure for 48 h suppresses cell proliferation in the dentate gyrus o f adult laboratory-reared female meadow voles (Ormerod and Galea, 2001). Understanding how estradiol dynamically regulates granule cell production in the rodent hippocampus w i l l facilitate our ability to understand and potentially control neurogenesis. The differential time-dependent nature by which estradiol regulates the production o f new cells may be mediated by its stimulatory effects on the hypothalamic-pituitary-adrenal ( H P A ) axis. One possibility may be that estradiol-induced high levels o f circulating adrenal steroids suppress the number o f new cells produced in the female rodent dentate gyrus. Indeed, high levels o f adrenal steroids suppress cell proliferation i n the dentate gyrus o f adult male rats (Gould et al., 1992; Cameron and Gould, 1994; Cameron et al., 1995; Cameron and M c K a y ,  57 1999). In addition to stimulating corticosterone ( C O R T ) secretion i n adult male and female rodents, estradiol elevates adrenocorticotropin hormone ( A C T H ) levels, which gradually produce adrenal enlargement (Coyne and Kitay, 1969; Christian, 1975; Burgess and Handa, 1992; Handa et al., 1994). Consistent with these findings, we (2001) found larger adrenal masses in adult female meadow voles exposed to estradiol for 48 versus 4 h and adrenal mass size is indicative o f H P A activity (Akana et al, 1983; Lemaire et al, 1997; Lemaire et al, 2000). Because C O R T suppresses cell proliferation estradiol could, in turn, suppress cell proliferation indirectly by stimulating adrenal activity. The present study investigated whether estradiol influences the production o f new cells time-dependently i n the dentate gyrus o f adult female rats and whether the estradiol-induced suppression in cell proliferation depends on estradiol-stimulated adrenal activity. W e hypothesized that the number o f new cells observed i n the dentate gyrus o f adult female rats would increase following a 4 h but decrease following a 48 h exposure to estradiol. Because estradiol stimulates adrenal function, we hypothesized that the suppression o f new cells could be eliminated or reversed by adrenalectomizing animals to prevent estradiol-induced stimulation o f adrenal activity. Understanding the interaction between estradiol and adrenal steroids on neuron production may provide insight into how different estrogen replacement therapies can either benefit or exacerbate the symptoms o f Alzheimer Disease (Hogervorst et al., 2000). Indeed the cognitive deficits and hippocampus atrophy associated with Alzheimer disease may be associated with abnormal levels o f Cortisol (Lupien et al., 1998,1999,2002).  3.2 M E T H O D S  A l l animals were treated i n strict accordance with the guidelines set forth by the Canadian Council on A n i m a l Care and The University o f British Columbia regarding the ethical  58 treatment o f animals used for the purposes o f research. Every effort was made to minimise the number o f animals used per group and their suffering.  Animals  Sixty-six female Sprague-Dawley rats (225-250 g) were obtained from Charles River Canada. U p o n arrival, the rats were housed i n groups o f four i n hanging metal cages i n a temperature controlled colony room (21±1°C) with a 12:12 h lightdark cycle (lights on at 0700h). Beginning the day after arrival, rats were handled for 5 m i n daily. One week after arrival, rats were ovariectomized (Experiment 3) or ovariectomized and adrenalectomized (Experiment 4) and then given another week to recover from surgery before testing began. During recovery, the rats were housed singly i n bedding-lined (Care Fresh; Absorption Corporation) polyurethane cages. Food (Lab Diet #5012; Jamieson) and tap water were available, ad libitum throughout the experiments. To maintain salt balance, adrenalectomized rats were given 0.9% NaCI i n their drinking water.  Procedure  Experiment 3 was conducted to determine whether exposure to estradiol for 4 h increases and exposure to estradiol for 48 h decreases the number o f new (post-mitotic) cells in the dentate gyrus o f adult female rats. Previously, Tanapat et al (1999) reported that rats i n the proestrus (a high estradiol phase) had more dividing (assessed 2 h post-BrdU) and more new (assessed 2-14 d post B r d U ) cells i n their dentate gyri than rats i n the estrus (low estradiol) phase. In addition, they found that estradiol reversed an ovariectomy-induced reduction in the number o f new neurons. These results suggest that estradiol increases neurogenesis. Ormerod and Galea (2001) then demonstrated that female meadow voles exposed to estradiol for 4 h before B r d U  59 was injected had more but females exposed to estradiol for 48 h had significantlyfewer dividing (assessed 2 h post-BrdU) cells than females that were reproductively inactive (with undetectable circulating estradiol levels). These results suggest that estradiol enhances then suppresses neurogenesis by increasing and then decreasing the number o f progenitor cells that leave the cell cycle to complete mitosis. Interestingly, the observation made b y Tanapat and colleagues (1999) that estrous versus proestrous rats had fewer dividing and new cells i n their dentate gyri supports the hypothesis that estradiol influences cell proliferation time-dependently because rats i n estrus would have experienced their proestrus surge 24-48 h before receiving B r d U . Therefore, we hypothesized that females exposed to estradiol for 4 h prior to an injection of B r d U would have more new or labelled cells and that females exposed to estradiol for 48 h before an injection o f B r d U would have fewer new cells than females exposed to vehicle. To test whether estradiol time-dependently influences the number o f new cells produced, O V X d female rats were given a subcutaneous injection o f either estradiol benzoate ( E B ; 10 \xg in 0.10 m l o f sesame oil) or vehicle ( V ; 0.10 m l sesame oil) either 4 h ( E B 4 group, n=7 and V 4 group, n=6) or 48 h (EB48 group and V 4 8 group, n=6 per group) before a single injection o f the cell synthesis marker, bromodeoxyuridine (BrdU; 200 mg/kg, i.p.). W e were interested in determining whether estradiol influenced the number o f new rather than dividing cells because Nowakowski and Hayes (2001) argue that treatment-induced changes i n cell proliferation could actually reflect changes s-phase length that would artificially suggest the number o f dividing cells has changed. Because progenitor cell division is complete within « 24 h o f an injection o f B r d U (Cameron and M c K a y , 2001) we perfused rats 24 h after B r d U was injected to determine the number o f newly divided cells after one mitotic division. To investigate whether the expression o f immature neuronal protein ( T U C - 4 and doublecortin; D C X ) increases in B r d U labelled cells with a longer survival time, a subset o f E B 4 and V 4 rats were sacrificed 4 days  60 (or 96 h) after B r d U was injected (n=4 per group). In addition, we sacrificed separate groups o f ovariectomized animals either 4 or 48 h after an injection o f E B or vehicle (n=4 per group) so that we could assess the relationship between serum estradiol and C O R T at the time that animals i n the other groups were injected with B r d U . Experiment 4 was conducted to determine whether adrenal steroids mediate the suppressive effect o f estradiol on the production o f new cells. Previous work has shown that estradiol increases cell proliferation through a serotonin-mediated mechanism (Banasr et al., 2001). Therefore, we investigated whether we could eliminate (no difference i n BrdU-labelled cell number would be observed between estradiol- and vehicle-treated groups) or reverse (the estradiol-induced increase in BrdU-labelled cell number would persist) the estradiol-induced suppression in the number o f new cells by removing estradiol's influence on circulating adrenal steroid levels (via adrenalectomy). Exposure to high-level estradiol for 48 h induces adrenal enlargement i n meadow voles (Coyne and Kitay, 1969; Ormerod and Galea, 2001) and increases C O R T secretion i n rats (Burgess and Handa, 1992) and i n dispersed adrenocortical cells (Nowak et al., 1995). In addition, circadian fluctuations i n serum A C T H and C O R T are potentiated during proestrus i n the rat (Buckingham, 1978). H i g h levels o f the adrenal steroid C O R T have been found to suppress cell proliferation within the dentate gyrus o f adult male rats (Gould et al., 1992; Cameron and Gould, 1994; Cameron and Gould, 1996; Cameron et al., 1999). Thus, a plausible hypothesis is that estradiol suppresses cell proliferation 48 h after exposure by increasing circulating adrenal steroids. O V X d + A D X d female rats received an injection o f either estradiol ( A D X + E 4 8 group; 10 ug i n 0.10 m l o f sesame oil, s.c; n=9) or vehicle ( A D X + V 4 8 group; 0.10 m l sesame oil, s.c; n=8) 48 h prior to receiving a single injection o f B r d U (200 mg/kg, i.p.). A low oral dose o f C O R T (25ug per m l 0.9% saline) was added to the rats' drinking water over 4 days beginning the day prior to the estradiol injection.  61 This dose has been shown to produce low circulating physiological levels o f C O R T (Gould et al., 1990; Cameron and Gould, 1994). Low-level C O R T was administered orally to maintain basal levels because we wanted to prevent a possible ceiling effect with an enhancement o f cell proliferation by both A D X and high-level estradiol that would mask a potential reversal o f the suppression. In addition, low level C O R T replacement eliminates the enhanced granule cell death response to A D X in the dentate gyrus o f adult rats (Gould et al., 1990; Cameron and Gould, 1998) and cell death stimulates the division o f neuronal progenitors (Gould and Tanapat, 1997). Rats were perfused 24 h after receiving B r d U to analyse the number o f new cells produced by a 48 h exposure to estradiol.  Histology  Twenty-four h or 4 d after B r d U was injected, rats were anaesthetized with sodium pentobarbital and then perfused with 4% paraformaldehyde. A 24 h survival time after B r d U injection was chosen to allow for one mitotic division. The phenotype o f B r d U - i r cells was verified in both the 24 h and the four-day survival group by assessing anti-TUC-4 (Turned on After D i v i s i o n - 6 4 k D / U L I P / C R M P ; T U C - 4 is a protein expressed by postmitotic granule neurons; Quinn et a l , 1999; Tanapat et al., 1999; Shors et al., 2001), anti-doublecortin (a protein expressed by migrating and differentiating granule neurons; J i n et al, 2001; Gleeson et al, 1999; Francis et al, 1999; used only i n 4 d survival group) and anti-glial fibrillary acidic protein ( G F A P ; an astroglia marker; Cameron et al., 1993b) immunoreactivity. Before perfusing the rats, blood samples were taken transcardially to verify serum estradiol and C O R T levels by radioimmunoassay. B l o o d samples were taken at the time o f perfusion to minimize any effect o f stress that may occur by taking blood at the time o f B r d U injection. Intact adrenal glands were removed and immediately weighed. Adrenal mass is expressed as mg/lOOg body  62 mass. Following perfusion, brains were extracted and stored overnight in perfusate at 4°C. The following day, the brains were sliced into 40 um thick sections through the entire dentate gyrus (8-9 sections per rat) an oscillating tissue sheer (Leica VT1000S) in a bath o f 0 . 1 - M phosphate buffer (PB). Slices prepared for peroxidase immunohistochemistry were pre-treated in a solution o f 0.2% H2O2 i n P B for 20 m i n and then rinsed i n P B before being mounted on slides treated with 3% 3-aminopropyltriethoxy-silane in acetone (Sigma Chemicals). BrdU-labelled cells were counted on peroxidase treated tissue and the phenotype o f new cells was determined on fluorescent probe-treated tissue.  Peroxidase immunohistochemistry  Tissue was processed for BrdU-immunoreactivity by applying solutions directly to the slide-mounted sections. Unless otherwise specified, phosphate-buffered saline (0.1 M sodium phosphate heptahydrate i n 0.9% saline; p H 7.4) was used for all rinses and slides were rinsed repeatedly between each step. 1) Cells in the sections were permeabilized with 0.05% Trypsin (Sigma A l d r i c h Chemicals) i n T r i s - H C l buffer (pH 7.5) containing 0.1% C a C l for 10 min. 2) 2  D N A was denatured by applying 2 N HC1 for 30 m i n and then the sections were repeatedly rinsed (pH 6.0). 3) Sections were blocked in 5.0% normal horse serum for 30 m i n and then incubated overnight i n mouse monoclonal antibody against B r d U (1:100 + 3% N H S + 0.5% Tween 20; Boehringer Mannheim) at room temperature. 4) Sections were incubated in mouse secondary antisera (1:29 + 3.0%> normal horse serum; Vector Laboratories) for 4 hrs. 5) Sections were incubated in avidin-biotin horseradish peroxidase ( A B C Elite K i t ; 1:50; Vector Laboratories) for 60 m i n . 6) Sections were reacted for about 10 m i n i n 0.02% diaminobenzidine ( D A B ; Sigma A l d r i c h Chemicals) with 0.003% H2O2 and then counterstained with cresyl violet acetate (Baker), dehydrated and coverslipped with Permount (Fisher Scientific).  63  Fluorescence immunohistochemistry  Separate sets o f slide-mounted sections were triple-stained with fluorescent probes to assess B r d U - , T U C - 4 - and GFAP-immunoreactivity (ir) or double-stained with fluorescent probes to assess B r d U - and doublecortin-ir. Unless stated otherwise all sections were rinsed several times with tris-buffered saline ( T B S ; p H 7.5) between steps. 1) Endogenous peroxidase was quelched for 10 m i n with 2 % H2O2 i n T B S . 2) Sections were incubated for 2 h in a solution o f deionized formamide i n 2 X S S C at 65°C. 3) D N A was denatured by applying 2 N HC1 for 30 min at 37°C. 3) Sections were incubated in 0.1 M borate buffer for 10 min. 4) Sections were blocked i n 5.0% normal donkey serum (Jackson Immunoresearch) for 30 m i n and then incubated overnight i n a cocktail containing rat anti-BrdU (ascites 1:100; Oxford Biochemicals Incorporated), rabbit monoclonal anti-TUC-4 (1:500; Chemicon) and mouse monoclonal antiG F A P (1:2000; Novacastra) or were incubated i n a cocktail o f rat anti-BrdU and goat antidoublecortin (1:2000; Santa Cruz) at 4°C. 5) 5% normal donkey serum (Jackson Immunoresearch) was applied to slides. 6) Sections were incubated in a cocktail o f donkey anti-rat flourescein (FITC; to visualize anti-BrdU) and donkey anti-rabbit Cy5 (to visualize T U C - 4 ) and donkey anti-mouse Cy3 (to visualize G F A P ; all diluted 6ul/ml; Jackson Immunoresearch) or in a solution o f donkey anti-rat F I T C (to visualize B r d U ) and donkey antigoat Cy3 (to visualize doublecortin) for 4 hrs. 5) Sections were then rinsed and coverslipped with the anti-fading agent diazobicyclooctane ( D A B C O ; 2.5% D A B C O , 10% polyvinyl alcohol and 20% glycerol i n T B S ; Sigma).  Hormone assays  Serum estradiol and C O R T assays were performed as described i n Ormerod and Galea (2001). Briefly, blood samples were stored overnight at 4 ° C and then were centrifuged at lOg  for 10 min. Serum estradiol was assayed using a Coat-A-Count kit (Diagnostic Products Corporation, L o s Angeles, C A ) modified for low expected levels o f estradiol. The intra-assay coefficient o f variation was 1.35 %. Serum corticosterone levels were analysed using a radioimmunoassay protocol described by Weinberg and Nezio (1987). Briefly, antiserum was obtained from Immunocorp (Montreal, Canada) and tracer was obtained from Mandel Scientific (Guelph, Canada). Dextran-coated charcoal was used to adsorb and precipitate free steroids after incubation. The intra-assay coefficient o f variation was 1.55 %.  Data analyses  Prior to analysis, slides were coded so that the experimenter was blind to the treatment conditions. Total B r d U - i r and pyknotic cells through the granule cell layer and subgranular zone (defined as approximately the 50 urn band between the granule cell layer and the hilus; Palmer et al., 2000) were stereologically estimated using peroxidase-treated tissue. O n peroxidase-treated  tissue, B r d U - i r and pyknotic cells were counted on every 10 section th  through the dentate gyrus per rat. Cells were considered BrdU-labelled i f they were intensely stained and exhibited medium round or oval cell body morphology (see Figure 3 A and Cameron et al., 1993b; Ormerod and Galea, 2001). Cells were considered pyknotic i f they lacked a nuclear membrane, had pale or absent cytoplasm and darkly stained spherical chromatin (see Figure 3B and Gould et al., 1991; Ormerod and Galea, 2001). Pyknotic and BrdU-ir cells were counted with a 100X objective on a N i k o n Eclipse (e600) light microscope and the total number o f cells was estimated using a modified version o f the optical fractionator method (West et al., 1991) for an estimate o f total cell counts:  65  HILUS  D  «-  C  f  ^  '  ^  j  j  j  \  i  .  -r\^*^  s  ^ Hilus  ^  Figure 3. Photomicrographs of B r d l -labeled and pyknotic cells and confocal images of neurons or glia in the dentate gyri of adult female rats in Experiments 3 and 4. A) Photomicrograph of a BrdU-labelled cell clump in the subgranular zone (SGZ) of an OVXd female exposed to estradiol for 4h (lOOx objective). B) Photomicrograph of a pyknotic (dying) cell in the SGZ (lOOx objective). C) Confocal image (63X objective) of neurons in the granule cell layer of a female rat exposed to estradiol for 48h. The white arrows point to cells expressing the neuronal protein doublecortin (indicated by the red label). The yellow arrow points to BrdU-ir cell (green label; a new cell) expressing the neuronal protein doublecortin. The cell is therefore a new neuron. D) Confocal image of cells expressing the glial protein GFAP (red label). The white arrows point to glia and the yellow arrow shows a new cell (that is not a new glia). Scale bar represents 10 um.  66  where, ECellsBrdu is the total number o f BrdU-labelled cells counted on all sections, t = section thickness (0.04mm), h = height o f the counting frame (plane o f focus, 0.005mm), asf = area sampling fraction (in our case the section area o f dentate gyrus), and ssf = sections sampled fraction (1/10). Areas were measured using the digitizing software Analytical Software Imaging Station (Imaging Research, Brock University, Ontario, Canada) and dentate gyrus volume estimates were made using Cavalieri's principle (Gunderson et al., 1988). Because we have previously reported B r d U - i r cell densities (Ormerod and Galea, 2001; Galea and M c E w e n , 1999) we also calculated B r d U - i r cell densities (# o f cells/area) on 6 anatomically matched sections per rat (where the dentate gyrus lies just beneath the corpus callosum and the infrapyramidal and suprapyramidal blades are joined at the crest; between A -3.3 and A -4.8 in rats) in order to compare the density o f B r d U - i r cells with stereo logical estimates o f total B r d U ir cells i n the dentate gyrus. BrdU-ir cell phenotypes were analyzed on fluorescent probe-treated tissue. Twenty-five B r d U labelled cells on four sections per brain (400 um apart; n = 3 per group) taken where the infrapyramidal and suprapyramidal blades j o i n at the crest were identified on a Zeiss fluorescence microscope and their phenotype verified using a confocal laser scanhead (BioRad 2000) and a 6 3 X objective. The percentage o f B r d U - i r cells that expressed a neuronal ( T U C - 4 ir or D C X - i r ; Figure 3C) or glial phenotype ( G F A P - i r ; Figure 3D) was determined. Z-sections at 0.4 um intervals were taken and optical stacks o f 10 images were created with NTH Image for P C (http://www.scioncorp.com/pages/menu.htm) and imported into Adobe Photoshop for channel merging. Digital manipulations were restricted to contrast enhancements and colour level adjustments.  Statistical analyses  67 In Experiment 3, the dependent variables (total B r d U - i r cells, B r d U - i r cell density, total pyknotic cells, percentage B r d U / G F A P - i r cells, percentage B r d U / T U C - 4 - i r cells or B r d U / D C X - i r cells; serum hormone levels) were analysed using an analysis o f variance ( A N O V A ) with group ( V 4 , E B 4 , V 4 8 and E B 4 8 , respectively) as the independent variable. Newman-Keuls procedure were used i n post-hoc analyses unless the groups violated the assumption o f homogeneity o f variance and in this case, the W e l c h ' s t' test was used. In Experiment 4, dependent variables were analyzed with Student's independent t-tests with group ( A D X + V 4 8 versus A D X + E 4 8 ) as the independent variables. In Experiment 3, Spearman Rank correlations were conducted between the dependent variables ( V 4 vs. E 4 and V 4 8 vs. E48) because comparisons included hormone assay values that fell below the threshold o f detection and were arbitrarily given a value o f zero. In Experiment 4, Pearson product-moment correlations were run between dependent variables with the exception o f analyses that included serum hormone levels. A l l statistical procedures set a = 0.05.  3.3 R E S U L T S  Experiment 3. Relative to vehicle, the number of BrdU-labelled gyrus of adult female rats increases following  cells observed in the dentate  exposure to EB for 4 h but decreases following  exposure to EB for 48 h.  The number o f BrdU-labelled cells significantly differed between groups (£(3,21) = 4.49, P < 0.014). E B 4 females had significantly more labelled cells (p_< 0.04) and E B 4 8 females had significantly fewer BrdU-labelled cells (p < 0.006) relative to the V 4 and V 4 8 groups, respectively (see Figure 4). W e found similar results when comparing the density o f B r d U labelled cells between groups and found a significant positive correlation between total labelled cell estimates and labelled cell densities (ls(38) 0.95; p < 0.001). Neither total number o f =  68  pyknotic cells (p_ > 0.23; see Figure 4) nor pyknotic cell density (p_ > 0.97) significantly differed between groups. N o significant linear relationship between BrdU-labelled cell number  7000 i  V4  EB4  V48  V48  EB48  EB48  Figure 4. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female rats given B r d U either 4 h or 48 h after estradiol or vehicle and sacrificed 24 h later in Experiment 3. White bars represent the data of females given sesame oil vehicle either 4 or 48 h before BrdU (n=6 per group) and grey and black bars represent data of females given estradiol either 4 h (n=7) or 48 h (n=6) before BrdU, respectively. A ) Mean number new (BrdU-ir) cells observed in the dentate gyrus of adult female rats when BrdU was injected 4 h or 48 h after an injection of estradiol or vehicle. Estradiol significantly increased the number of BrdU-ir cells (approximately 316%) found within the dentate gyrus of adult female rats within 4 h (p_= 0.01) but significantly decreased the number of cells (approximately 60%) within 48 h (p = 0.006). B) Mean number of pyknotic cells in the dentate gyrus of adult female rats given estradiol or vehicle. Estradiol exposure for either 4 or 48 h did not influence the number of pyknotic cells relative to vehicle. **denotes p < 0.01 *denotes p < 0.05 "denotes 0.10 > p < 0.05  and pyknotic cell number was observed between groups (p < 0.57). Total volume o f the granule cell layer and subgranular zone did not statistically differ between groups (F(3,2l) 0-63, p_ < =  0.63; V4=1.360 ± 0.05 m m ; E B 4 = 1.47 ± 0.05 m m ; V48=1.40 ± 0.08 m m ; EB48=1.42 ± 3  3  3  0.08 mm ). N o significant difference was observed between groups i n the percentage o f B r d U 3  69 ir cells expressing T U C - 4 or G F A P i n animals perfused 24 h ( T U C - 4 ~22%, = 0.21 and G F A P ~ 17%, p = 0.48; see Table 5) after B r d U was injected.  Table 5. Mean (+SEM) % B r d U - i r cells expressing a neuronal (TUC-4-ir) or glial ( G F A P ir) phenotype measured 24 h after B r d U was injected did not significantly differ in adult female rats exposed to estradiol or vehicle for either 4 or 48 h in Experiment 3. Group  TUC-4 ir  GFAP-ir  V4 (n=6) V48 (n=6) EB4 (n=7) EB48 (h=6)  14.7±4.9 18.7±3.5 19.3±4.7 25.3±3.5  17.0±2.9 18.7±2.7 14.7±3.5 18.7+2.4  ~  _=  A s expected, E B 4 (p < 0.0003) and E B 4 8 (p < 0.04) females had higher serum estradiol levels than V 4 and V 4 8 females, respectively (F 2i) = 29.09, p < 0.001; see Table 6). Planned (3)  comparison revealed that adrenal mass was significantly heavier i n the E48 versus V 4 8 females (p < 0.02) but was similar i n E 4 and V 4 females (p < 0.54; F , i ) = 2.26, p < 0.11; see Table 6). (3  2  Serum C O R T levels tended to differ between groups (F( ,2i) = 2.66, p < 0.07) and interestingly, 3  planned comparisons showed that levels did not differ between E48 and V 4 8 females (p = 0.38) but were higher i n the E 4 versus V 4 females (p < 0.02; see Table 6).  Table 6. Mean (±SEM) serum hormone levels and adrenal masses in adult female rats injected with B r d U either 4 or 48 h after estradiol administration in Experiments 3 and 4.  Experiment 1  Experiment 2  _  Serum Estradiol (pg/ml)  Serum C O R T (ng/ml)  Adrenal Mass (mg/lOOkg bw)  V4 (n=6) EB4 (n=7) V48 (n=6) EB48 (n=6) A D X V 4 8 (n=9) ADXE48(n=8)  N/A 28.97±3.91** N/A* 7.13±1.62* N/A 8.84±0.94*  377.9±7.99 595.9+2.93* 558.713.33 482.318.16 96.412.74 155.613.72  27.2110.96 25.46+2.19 23.9312.64 31.0111.68*  v  v  * indicates a significant difference (p < 0.050) relative to the control group **indicates a significant difference (p < 0.001) relative to the control group ^ N/A indicates that serum hormone levels were below the detection threshold of the radioimmunoassay (5 pg/ml)  Because blood samples were taken 28 and 72 h after E B was injected and serum C O R T assay values are likely not reflective o f the values that would be observed at the time that B r d U  70 was injected (4 or 48 h after E B was administered). Thus, w e assessed the relationship between serum estradiol and serum C O R T level in animals that were sacrificed, rather than injected with B r d U , either 4 or 48 h after E B was injected. In animals that were sacrificed 4 h after E B was injected, serum C O R T assay values were 100% o f control values whereas in animals sacrificed 48 h after E B was injected, serum C O R T assay values were 175% o f control values (Table 7).  Table 7. Mean (+SEM) serum hormone levels and adrenal masses in samples taken from animals either 4 or 48 h after an estradiol injection in Experiment 3 (no B r d U injected). _ Condition Vehicle-4 h Estradiol-4h Vehicle-48 h Estradiol - 48 h  Serum Estradiol . , ...  Serum CORT . ,  (pg/ml)  (ng/ml)  7.14±2.41 339.08166.29** N/A 20.5114.52*  247.1±68.28 248.9191.94 68.4116.62 120.3143.56*  =  =  =  =  =  =  =  =  =  =  =  * indicates a significant difference (p < 0.050) relative to the control group """indicates a significant difference (p < 0.001) relative to the control group * N/A indicates that serum hormone levels were below the detection threshold of the radioimmunoassay (5 pg/ml)  In E B 4 and V 4 females, serum estradiol was significantly correlated positively with total B r d U - i r cell count (R(i3)=0.61; p_ < 0.02) with higher levels o f estradiol being associated with more B r d U - i r cells (Figure 4 and Table 6). In E B 4 8 and V 4 8 females, serum estradiol was significantly correlated negatively with total B r d U - i r cell count ( R ( i 2 ) = -0.63; g < 0.03), with more B r d U - i r cells associated with lower levels o f serum estradiol (Figure 4 and Table 6). There were no other significant correlations observed between any o f the dependent variables. In animals perfused 4 days after B r d U was injected, E B 4 females had significantly more labelled cells than V 4 females (p < 0.02; vehicle = 888.2±333.4 and E B 4 = 4052±1322.23). N o difference was observed between vehicle- and EB-treated rats in the percentage o f B r d U labelled cells expressing the neuronal protein T U C - 4 (-27%, p_ = 0.56) or in the percentage o f BrdU-labelled cells expressing G F A P (-23%, p = 0.86; see Table 8). A greater percentage o f BrdU-labelled cells expressed the neuronal protein D C X (-65%) relative to T U C - 4 or G F A P  71 but no difference i n the percentage o f B r d U / D C X - l a b e l l e d was observed between vehicle- and EB-treated animals (p < 0.77).  Table 8. Mean (±SEM) % B r d U - i r cells expressing a neuronal (TUC-4-ir or doublecortinir) or glial ( G F A P - i r ) phenotype measured 4 days after B r d U was injected did not significantly differ in adult female rats exposed to estradiol for 4 h in Experiment 3. Group TUC-4 ir Doublecortin-ir GFAP-ir =  V4(n=4) EB4 (n=4)  28.0±2.3 26.711.3  Experiment 4. The Estradiol-induced  64.0±4.6 66.0±2.0  22.7+5.3 24.0±4.6  Suppression in Cell Proliferation  Within the Dentate  Gyrus of Adult Female Rats is Reversed by Adrenalectomy.  Total B r d U - i r and pyknotic cells did not significantly differ between groups (p = 0.43 and p = 0.54, respectively; Figure 5) and the density o f BrdU-labelled cells was similar between groups (p < 0.82; data not shown). Non-significant differences were observed between groups in the percentage o f cells expressing T U C - 4 (~ 19%, p = 0.56) or G F A P (~ 18%, p = 0.72; Table 9).  Table 9. Mean (+SEM) % B r d U - i r cells expressing a neuronal (TUC-4-ir) or glial ( G F A P ir) phenotype in adrenalectomized females following a 24 h survival time after B r d U injection in Experiment 4 did not significantly differ between groups. Group TUC-4 ir GFAP-ir V48(n=9) E B (n=8)  17.3+3.5 20.012.3  18.7±2.7 20.0±2.4  Serum estradiol (measured 24 h after B r d U ) was elevated i n estradiol-treated rats versus controls (p < 0.015; Table 6). However, no significant differences between groups in serum C O R T (measured 24 hr after B r d U ) were observed. Table 6 shows that the dose o f C O R T replaced i n the drinking water o f rats did produce circulating levels that were lower than the endogenous levels observed i n Experiment 3. In addition, there were no significant correlations  72 found between the dependent variables. Total volume o f the granule cell layer and subgranular zone did not differ between adrenalectomized rats exposed to vehicle 48 h before B r d U ( A D X V 4 8 : 1.580 ± 0.103 m m ) and adrenalectomized rats exposed to E B 48 h before B r d U 3  ( A D X E 4 8 : 1.586 ± 0.104 m m ) groups (p < 0.97). 3  10000 -i  8000  ~  6000  4000  ADXV48  ADXEB48  ADXV48  ADXEB48  Figure 5. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adrenalectomized adult female rats when B r d U was administered 48 h after estradiol or vehicle in Experiment 4. White bars represent the data of females given sesame oil vehicle (n=8) and black bars represent data of females given estradiol (n=9). A) Mean number new (BrdU-ir) cells in the dentate gyrus of adult female rats 4 h after an injection of estradiol. No difference was observed between groups given estradiol versus vehicle in the number of BrdU-ir cells found within the dentate gyrus of adult female rats following adrenalectomy (p= 0.43). B) Mean number of pyknotic cells in the dentate gyrus of adult female rats given estradiol or vehicle. Estradiol did not influence the number of pyknotic cells (p=0.54). Note, however that more pyknotic cells were observed in adrenalectomized animals (compare with Figure 4).  3.4 D I S C U S S I O N In adult female rats, we found that estradiol dramatically alters the number o f new (BrdU-labeled cells) in a biphasic manner. Relative to vehicle, exposure to a high dose o f estradiol for 4 h increases whereas exposure for 48 h decreases the both the number and density of BrdU-labeled cells. The dual effect o f estradiol on the number o f new cells could be either duration or dose-dependent, however several lines o f evidence suggest the effect is dosedependent. First, in the present study, estradiol levels remained elevated i n the group that received estradiol 48 h before B r d U (Experiment 3; Table 6), relative to the group that received  73 vehicle, but the number o f labeled cells was significantly lower. This result is similar to what we have observed previously in adult female meadow vole. W e found that although females exposed to estradiol for 48 h had serum levels intermediate to females exposed to estradiol for 4 h and females with low circulating estradiol levels, they had significantly fewer labeled cells in their dentate gyri (Ormerod and Galea, 2001). In fact, the number o f labeled cells was suppressed to the level observed i n the dentate gyri o f females with chronically high circulating serum estradiol levels (Ormerod and Galea, 2001). Second, Tanapat and colleagues (1999) found that the rate o f cell proliferation was similar during the diestrus and estrus phase although estradiol levels differ during these phases. Finally, we have preliminary data to suggest that prolonged exposure to estradiol suppresses the production o f new cells within the dentate gyrus o f adult female rats ( E . M . Falconer and L . A . M . Galea, personal communication). Estradiol suppresses the number o f new cells, at least i n part, by stimulating adrenal activity because adrenalectomy eliminated the estradiol-induced decrease i n the number o f labeled cells observed (Experiment 4). Previous work has shown that estradiol stimulates adrenal activity (Coyne and Kitay, 1969; Buckingham, 1978; Burgess and Handa, 1992; Nowak et a l , 1995; Ormerod and Galea, 2001) and that adrenal steroids suppress cell proliferation i n the dentate gyrus o f adult male rats (Gould et al., 1992; Cameron and Gould, 1994; Cameron and Gould, 1996; Cameron et al., 1999). Here we show adrenalectomized females exposed to estradiol have similar numbers o f BrdU-labeled cells in their dentate gyri as females exposed to vehicle. To fully understand how adult neurogenesis is regulated, it is important to determine how a factor, such as estradiol, dynamically regulates components o f the process. This study shows that estradiol first enhances and then suppresses the production of new cells by stimulating adrenal activity. This finding is important because the manipulation o f endogenous progenitor cells could lead to advances i n therapy geared toward alleviating neurodegenerative disease.  74 Stress, working through an elevation in adrenal steroids, has been shown to suppress cell proliferation in the dentate gyrus o f adult male rats (Tanapat et al., 2001). Thus, the possibility that subcutaneous injection is stressful enough to elevate C O R T and diminish the production o f new cells exists. In fact, a previous study has shown that the number o f new cells produced i n the dentate gyrus o f adult rats may be reduced by a subcutaneous injection (Cameron and Gould, 1994). That finding is consistent with our study, i n which we noted that serum C O R T levels were higher 4 h ( V 4 and E4) versus 48 h (V48 and E48) after a subcutaneous injection o f estradiol (Table 6). In addition, the number o f BrdU-labeled cells tended to be suppressed i n the V 4 group relative to the V 4 8 group (p_ < 0.06; see Figure 4). However, relative to their controls, E48 rats had higher levels o f C O R T than E 4 rats, suggesting again that estradiol has a stimulatory effect on adrenal function 48 h after exposure. In addition, the enhanced level o f C O R T i n the E B 4 group did not block the estradiol-induced increase in cell proliferation. Interestingly, we did not find a significant relationship between cell birth and cell death in our experiments, however more pyknotic cells were observed following adrenalectomy, which is consistent with previous reports (Gould et al., 1992; Cameron and Gould, 1994; Cameron et al., 1995; Cameron and M c K a y , 1999). Across experiments estradiol did not alter the phenotype o f new cells.  Estradiol interacts with adrenal steroids to suppress cell  proliferation  In the present study adrenal steroids mediated the estradiol-induced suppression in the number o f new cells observed. H i g h levels o f adrenal steroids suppress cell proliferation in the dentate gyrus o f adult male rats (Gould et al., 1992; Cameron and Gould, 1994). Previous work has shown that estradiol increases serum C O R T levels i n female rodents (Christian, 1975;  75 Coyne and Kitay, 1969; Buckingham, 1978; Handa and Burgess, 1994) and increases adrenal mass in adult female meadow voles 48 h but not 4 h after estradiol is administered (Ormerod and Galea, 2001). Consistent with this literature, we found heavier adrenals in E48 but not E 4 females, relative to controls. Relative to vehicle, rats sacrificed 4 h after estradiol was injected (when V 4 and E 4 rats would have been injected with B r d U ) had similar serum C O R T values whereas rats sacrificed 48 h after estradiol was injected (when V 4 8 and E48 rats would have been injected with B r d U ) had serum C O R T values that were 175% o f their controls' values. The estradiol-induced suppression i n cell proliferation i n the dentate gyrus o f adult female rats was eliminated but not reversed by adrenalectomy (Experiment 4). This observation suggests that the estradiol-induced suppression i n cell proliferation may be mediated by another factor. Estradiol upregulates Type I glucocorticoid receptor expression in the hippocampus (Ferrini and De N i c o l a , 1991). Thus the low level replacement o f C O R T used i n this study could have masked a possible adrenalectomy-induced reversal o f the estradiol-mediated suppression in cell proliferation. In addition, Cameron et al (1995) found that N-mefhyl-Daspartate receptor ( N M D A r ) activation suppresses cell proliferation i n the dentate gyrus o f adult male rats downstream o f C O R T . Estradiol increases N M D A r binding sites i n the hippocampus (Weiland, 1992) and upregulates the N M D A r N R 1 subunit (Gazzaley et al., 1996). Therefore i n addition to increasing serum C O R T levels, estradiol could mediate the synergistic effect o f C O R T and N M D A r activation by increasing N M D A r and/or Type I glucocorticoid receptor expression to suppress cell birth at 48 h after its administration.  Estradiol could act time-dependently through numerous pathways  The current study found that exposure to estradiol for 4 h increases and for 48 h decreases the number o f new cells (assessed 24 h post-BrdU) complementing previous work (Ormerod and Galea, 2001; Tanapat et al., 1999). Thus, estradiol appears to influence  76 neurogenesis by altering cell proliferation or the number o f progenitor cells that exit the cell cycle to produce new cells as an estradiol-induced change i n the length o f s-phase in progenitor cells already dividing would not presumably change the number o f daughter cells produced (see Nowakowski and Hayes, 2001). A previous study has shown that estradiol initially enhances cell proliferation by influencing serotonin activity (Daszuta et al., 2001) and in the present study we have evidence that estradiol subsequently reduces cell proliferation by influencing adrenal steroids.  Because adrenalectomy eliminated but did not completely reverse the estradiol-induced suppression we observed i n the number o f new cells produced, estradiol may also work through other mechanisms to suppress the production o f new cells. There are two estrogen receptor isoforms ( E R a and E R p ) expressed within the hippocampus (Shughrue et al., 1997; Milner et al., 2001). Although progenitor cell E R expression has not been investigated, weak E R p receptor m R N A expression and extranuclear E R a expression is present i n the dentate gyrus including the subgranular zone (Shughrue et al., 1997; Weiland et al., 1997; M i l n e r et al., 2001). F U R T H E R M O R E , E R p has been localized on astrocytes i n the subgranular zone (Garcia-Segura et al., 1999), and astrocytes have been putatively identified as neural stem cell precursors (Seri et al., 2001). Based upon E R localization, the estradiol-induced increase in cell proliferation could be directly mediated by activation o f the E R p isoform or indirectly by the E R a isoform. Estradiol could regulate cell proliferation time-dependently by changing E R expression (Shughrue et al., 1992) or by activating second messenger pathways and gene transcription as estradiol activates c A M P (Gu and Moss, 1996), calcium (Improta-Brears et al., 1999), inositol-3 phosphate kinase, diaglycerol, src (Arnold et al., 1995), C R E B , E R K - 1 and E R K - 2 (Sing et al., 1999) cascades. Moreover, both ligand bound E R isoforms can stimulate gene transcription o f an estrogen-response element (ERE)-driven reporter gene and can  77 modulate the activity o f other transcription factors ( N F K B , A P - 1 and S F R E ; Shyamala and Guiot, 1992; Webb et al., 1999; M c D o n n e l l and Norris, 2002). A plausible hypothesis is that estradiol enhances cell proliferation v i a second messengers and eventually suppresses cell proliferation by interacting with transcription factors or that ER<xs and E R p differentially s  influences adult neurogenesis. Clearly, the time-dependent effect o f estradiol on cell proliferation could be mediated by several factors.  Estradiol time-dependently influences many forms ofplasticity  within the hippocampus  In addition to neurogenesis, estradiol mediates several forms o f plasticity within the adult rodent hippocampus. High-level estradiol enhances both long-term potentiation ( L T P ) and long-term depression i n hippocampal C A I pyramidal neurons (Cordoba Montoya and Carter, 1997; Desmond, Zhang, & Levy, 2000; Good, Day, & M u i r , 1999; Warren et al., 1995). However, high levels o f estradiol reduce L T P i n the dentate gyrus (Gupta et al., 2001). H i g h estradiol levels increase dendritic spine and synapse density on hippocampal C A I pyramidal neurons but not i n the dentate gyri o f young female rats (Woolley et al., 1990). Interestingly 410 d old granule neurons extend axons to C A 3 pyramidal neurons (Hastings and Gould, 1999), which i n turn synapse with C A I pyramidal neurons (Amaral and Witter, 1995). Increases in C A I pyramidal dendritic spine density are maximal from 48 h to 96 h after estradiol administration before gradually declining (Woolley and M c E w e n , 1993). Thus, increases in C A I dendritic synapse density coincide with the time that estradiol-induced new granule neurons begin to extend axons into the C A 3 region. Similarly, C A I dendritic spine density recedes at the time when estradiol-induced decreased numbers o f new neurons would begin to extend their axons into the C A 3 region. Thus estradiol may regulate plasticity o f hippocampal circuitry  time-dependently.  3.5 I M P L I C A T I O N S  Estradiol increases but then decreases cell proliferation i n the dentate gyrus o f adult female rats and voles (Ormerod and Galea, 2001; current study) suggesting that this timedependent effect is robust across rodent species. Neurogenesis occurs i n the dentate gyrus during adulthood i n many species (Cameron et al., 1993b; Kempermann et al., 1997; Gould et al., 1997, 1998, 1999a, 1999b; Kornack and Rakic, 1999; Ormerod and Galea, 2001) including humans (Eriksson et al., 1998). Putative younger neurons are more electrophysiologically plastic than older granule neurons (Wang et al., 2000). In fact recent electrophysiological work has shown that new neurons born i n adulthood mature into functional granule neurons i n the mouse dentate gyrus (van Praag et al, 2002). Recent work has shown that hippocampusdependent learning requires and enhances the survival o f 1-2 week old neurons (Gould et al., 1999; Shors et al., 2001). Therefore, estradiol-induced changes i n the number o f new cells produced could profoundly influence hippocampus-dependent behavior. Indeed, hippocampusdependent learning is impaired by a high dose but enhanced by a low dose o f estradiol in female rats (Galea et al., 2001; Holmes et al., i n press).  These findings suggest that there may  be an optimal level o f neuron production and survival for optimal performance on hippocampus-dependent tasks. O f course, the effect o f estradiol on cell proliferation would have to be dissociated from its effect on learning before definitive statements about the functional role o f estradiol-induced changes in cell proliferation can be made. Intriguingly, high levels o f estradiol for 48 h decreases cell proliferation i n the dentate gyrus o f adult female meadow voles (Ormerod and Galea, 2001) and increases cell proliferation in the subventricular zone (but not the dentate gyrus) o f adult female prairie voles (Fowler et al., 2001; Smith et al., 2001), indicating that the functional role o f estradiol-induced changes in neurogenesis may differ among strains o f voles with very different social patterns (meadow  voles are promiscuous and prairie voles are monogamous). Both the subventricular zone and dentate gyrus are likely to mediate different subsets o f behaviours and while E R p is expressed in both the rat dentate gyrus and the olfactory area, E R a is expressed at very low levels i f at all in the olfactory area (Shughrue et al., 1997). Future work could take advantage o f this dissociation in the effect o f estradiol on cell proliferation to investigate the functional role o f these new neurons.  80  CHAPTER 4 NMDA RECEPTOR ACTIVITY AND ESTRADIOL: INDEPENDENT REGULATION OF C E L L P R O L I F E R A T I O N IN T H E DENTATE GYRUS O F A D U L T F E M A L E MEADOW VOLES (In press, Journal of Endocrinology) 4.1 I N T R O D U C T I O N N e w granule neurons are added to the dentate gyri o f all mammalian species that have been studied, including humans throughout adulthood (Altman and Das, 1965; Cameron et al., 1993; Gould et a l , 1997, 1998, 1999; Eriksson et al, 1998; Kornack and Rakic, 1999). The number o f granule neurons added to the mammalian dentate gyrus appears substantial. In rats, approximately 9,000 new cells are produced daily and many o f these new cells differentiate into granule neurons (Cameron and M c K a y , 2001). Altering progenitor cell proliferation, the fate o f daughter cells or the survival o f new granule neurons could increase or decrease neurogenesis i n the dentate gyrus. Understanding how different components o f adulthood neurogenesis are influenced by a single factor is important because a factor that both increases cell proliferation and decreases the survival o f young neurons could produce no net change in new neuron number. Estradiol dynamically influences neurogenesis within the adult rodent dentate gyrus by first increasing and then decreasing cell proliferation (Ormerod and Galea, 2001; Ormerod et al., 2003) as well as by enhancing the survival o f young neurons (Ormerod et al., 2002). This study was designed to better understand how estradiol dynamically influences cell proliferation i n the adult rodent dentate gyrus. Several studies have shown that estradiol influences cell proliferation i n the dentate gyri o f adult rodents. For example, short-term exposure to estradiol (2-4 h) stimulates cell proliferation i n the dentate gyri o f adult ovariectomized ( O V X d ) female rats (Tanapat et al., 1999; Banasr et al., 2001; Ormerod et al, 2003). Interestingly, we have previously shown that estradiol initially enhances cell proliferation (within 4 h) but subsequently suppresses cell  81 proliferation (within 48 h) i n the dentate gyrus o f O V X d adult female rats, suggesting that estradiol time-dependently influences cell proliferation (Ormerod et al., 2003). In fact, estradiol appears to dynamically regulate cell proliferation across rodent species. Ormerod and Galea (2001) found that a 4 h exposure to estradiol tended to increase whereas a 48 h exposure significantly decreased the density o f proliferating cells i n the dentate gyrus o f reproductively inactive female meadow voles (with low circulating estradiol levels). However, i n that study all females had intact ovaries and the possibility that other ovarian steroids could also have influenced cell proliferation exists. Therefore, one objective o f this study was to investigate whether the dynamic changes i n cell proliferation observed i n the dentate gyri o f O V X d adult female rats and intact female meadow voles following estradiol administration also occur in dentate gyri o f adult O V X d female meadow voles. This finding would verify that the dynamic effects o f estradiol are robust across rodent species with diverse reproductive strategies and physiologies. The mechanisms by which estradiol differentially influences cell proliferation in the adult rodent dentate gyrus are beginning to be explored. Banasr and colleagues (2001) have shown that the estradiol-induced increase i n cell proliferation is mediated by serotonin. Specifically, the estradiol-induced increase i n cell proliferation i n the dentate gyrus o f adult O V X d female rats is abolished by the administration o f the serotonin synthesis inhibitor P C P A (Banasr et al., 2001). W e found that the estradiol-induced suppression i n cell proliferation is partially mediated by adrenal steroids because the suppression is eliminated (but not reversed) in adult female rats that are adrenalectomized (Ormerod et al., 2003). Therefore, estradiol must influence another factor, i n addition to stimulating adrenal activity, to suppress cell proliferation with longer exposure. Estradiol exposure for 48 h increases the number and sensitivity o f A^-methyl-Daspartate ( N M D A ) receptors i n the hippocampus o f adult rats (Weiland, 1992; Gazzaley et al.,  82 1996). Furthermore, N M D A receptor activation (via N M D A ) decreases and N M D A receptor inactivation (via M K - 8 0 1 or CGP43487) increases cell proliferation i n the dentate gyri o f adult male rats, tree shrews and aged female rats (Cameron et al, 1994, 1995; Gould et al., 1997; Bernabau and Sharp, 2000; Nacher et al., 2001; Nacher et al., 2003 but see Bernabau and Sharp, 2000 and Arvidsson et al., 2001). Interestingly, Cameron and colleagues (1998) demonstrated that N M D A receptor activation works downstream o f adrenal steroids to suppress cell proliferation as the effects o f low-level or high-level corticosterone can be blocked by N M D A receptor activation or inactivation, respectively. Therefore, another objective o f the current study was to investigate whether longer exposure to estradiol influences N M D A receptor activity to suppress cell proliferation. We investigated whether estradiol influences neurogenesis i n the dentate gyrus o f adult O V X d female meadow voles by first increasing (within 4 h) and then decreasing (within 48 h) cell proliferation and whether estradiol stimulates N M D A receptor activity to suppress cell proliferation. W e also examined whether N M D A receptor activation and inactivation influenced cell proliferation as it does i n adult male and aged female laboratory rats and tree shrews. Based upon our previous work using adult O V X d rats and intact adult female meadow voles, we hypothesized that estradiol would increase cell proliferation within 4 h but decrease cell proliferation within 48 h i n the dentate gyri o f OVXd adult female meadow voles. W e also hypothesized that because estradiol influences N M D A receptor number and sensitivity and N M D A receptor activation influences cell proliferation, administration o f the N M D A receptor antagonist M K - 8 0 1 could reverse the estradiol-induced suppression i n cell proliferation. Discovering how estradiol mediates its diverse effects on neurogenesis in the adult dentate gyrus could promote the development o f strategies to control the process i n order to replace neurons lost in disease or trauma. Moreover, fully characterizing the time-dependent effects o f estradiol on neurogenesis may be important for patients on long-term estrogen replacement  83 therapies, given that neurogenesis has been linked to hippocampus-dependent behavior (Gould et al, 1999; Shors et al., 2001, 2002). For example, estrogen replacement therapy has been purported to reduce the risk and severity o f Alzheimers disease as w e l l as the associated cognitive impairment (Henderson et al., 1994; Sherwin, 1997; K a w a s et al., 1997).  4.2 M E T H O D S  A l l animals were treated in strict accordance with the guidelines set forth by the Canadian Council on A n i m a l Care and The University o f British Columbia regarding the ethical treatment o f animals used for the purposes o f research. Every effort was made to minimise the number o f animals used per group and their suffering.  Animals  Forty-one adult female meadow voles (at least >25 g and 60 d old) reared i n our breeding colony at The University o f British Columbia were used as subjects. The voles were bred and reared i n a colony room that was temperature controlled (21±1°C) with a 16:8 h (lights on at 0700h) light:dark cycle. A l l animals were housed i n polyurethane paper beddinglined (Carefresh; Absorption Corporation) cages that contained enrichment supplies (plastic and/or cardboard containers). A t 21 days o f age, the voles were weaned and housed either with same sex siblings or individually (if the vole was the only female sibling o f her litter) until 60 days o f age when all voles were housed individually. The voles had free access to tap water and Jamieson Lab Diet #5012 for the duration o f the experiment and were given weekly sunflower seed, alfalfa pellet, carrot, and apple food supplements. A l l voles were ovariectomized ( O V X d ) using sterile surgical techniques under Halothane anaesthesia delivered at 3% (reduced as  84 required to maintain stable respiration) and given one week to recover from surgery before participating i n an experiment.  Procedure  Experiment 5 was conducted to determine whether 1) short-term (4 h) exposure to estradiol increases cell proliferation, 2) N M D A receptor activation decreases cell proliferation, and 3) estradiol influences N M D A receptor activity within 4 h to alter cell proliferation i n the dentate gyri o f adult O V X d female meadow voles. O V X d female meadow voles were injected either with estradiol benzoate (EB4; 10 ug) or sesame o i l (OIL; 0.05 ml) and then either N M D A (30 mg/kg) or vehicle ( V E H ; 0.05 m l saline) 3 h later. Four hours after the injection o f estradiol or oil, the voles were given a single injection o f the cell synthesis marker, bromodeoxyuridine ( B r d U ; 50 mg/kg) and were perfused 1 h later to assess cell proliferation (see Figure 6 for experiment timeline). 1 WEEK  OVX  3h  1h  1h  OIL or VEHICLE BrdU PERFUSE ESTRADIOL OR NMDA  Figure 6. Time line of Experiment 5. Voles were injected with oil (0.05 ml) or estradiol (10 ug) and then either with vehicle or N M D A (30mg/kg) 3 h later and BrdU (50 mg/kg) 4 h later.  Therefore, effects on cell proliferation were tested i n four groups in Experiment 1: O I L 4 + V E H (n=5), E B 4 + V E H (n=6), O I L 4 + N M D A (n=5) or E B 4 + N M D A (n=5). If estradiol influenced N M D A receptor activity within 4 h to alter cell proliferation, then we would expect to observe no difference or perhaps a suppressed number o f labelled cells i n the EB4+NMDA-treated versus the OIL4+NMDA-treated voles.  85 Experiment 6 was conducted to investigate whether 1) estradiol exposure for 48 h suppresses cell proliferation in the dentate gyrus o f adult O V X d female meadow voles, 2) M K - 8 0 1 stimulates cell proliferation, and 3) estradiol stimulates N M D A activity to suppress cell proliferation in the dentate gyri o f adult O V X d female meadow voles. To test whether estradiol exposure for 48 h decreases cell proliferation, O V X d voles were injected (s.c.) with either estradiol benzoate (EB48; 10 ug) or sesame o i l (OIL; 0.05 ml) and then either M K - 8 0 1 (1 mg/kg) or vehicle ( V E H ; 0.05 m l saline) 47 h later. Forty-eight hours after the injection o f estradiol or sesame o i l vehicle, the voles were given a single injection o f the cell synthesis marker, bromodeoxyuridine ( B r d U ; 50 mg/kg, i.p.) and were perfused 1 h later to assess cell proliferation (see Figure 7). 1 WEEK  OVX  47h  OIL or ESTRADIOL  1h  VEHICLE BrdU OR MK-801  1h  PERFUSE  Figure 7. Time line of Experiment 6. Voles were injected with oil (0.05 ml) or estradiol (10 ug) and then either with vehicle or MK-801 (1 mg/kg) 47 h later and BrdU 48 h later. In both experiments, voles were perfused 1 h after BrdU was injected.  Therefore, effects on cell proliferation were tested i n four groups i n Experiment 2: O I L 4 8 + V E H , E B 4 8 + V E H , OIL48+MK-801 or E B 4 8 + M K - 8 0 1 (n=5 per group). If estradiol stimulates N M D A receptors to suppress cell proliferation i n the dentate gyrus o f adult O V X d female meadow voles, then we would expect to find that antagonizing N M D A receptor activity with M K - 8 0 1 would eliminate or reverse the estradiol-induced suppression i n cell proliferation.  Drug preparation  Estradiol benzoate ( E B ; Sigma A l d r i c h Chemicals) solution was prepared by dissolving E B in sesame o i l (Sigma A l d r i c h Chemicals) to a concentration o f 10 ug o f EB/0.05 m l sesame oil.  86 The solution was then stored i n a light insensitive container and used for all experiments. A l l voles were subcutaneously injected with 0.05 m l o f the solution (10 p.g E B per vole). Although estradiol-induced changes i n blood-brain permeability could, i n theory, account for differences in BrdU-labeling between groups, estradiol only alters rat blood brain barrier permeability after at least 3 weeks o f exposure (Ziylan et al., 1990). In addition, previous work has shown that the number BrdU-labeled cells is elevated i n the rostral migratory stream (but not dentate gyrus) o f adult female prairie voles with high- versus- low estradiol levels (Smith et al., 2001; Fowler et al., 2001). /V-methyl-D-aspartate ( N M D A ; Tocris) was prepared just prior to its use i n Experiment 1. N M D A was dissolved i n isotonic saline to a concentration o f 30 mg/ml and was injected intraperitoneally (i.p.) in a volume o f O.lml/lOOg body weight making the dose o f N M D A 30 mg/kg. The noncompetitive N M D A receptor antagonist M K - 8 0 1 (Tocris) was prepared just prior to its use Experiment 2. M K - 8 0 1 was dissolved in isotonic saline to a concentration o f 1 mg/ml and was injected i.p. i n a volume o f O.lml/lOOg body weight making the dose o f M K - 8 0 1 1 mg/kg. The doses and durations o f exposure to N M D A or M K - 8 0 1 were chosen because they have been shown previously to influence cell proliferation in the dentate gyri o f adult rats (Cameron et al., 1995, 1998). Bromodeoxyuridine ( B r d U ; Sigma Aldrich Chemicals) was prepared just prior to use by dissolving B r d U i n freshly prepared saline buffered with 0.7% 2 N N a O H to a concentration o f 10 mg B r d U / m l saline. Voles were injected intraperitoneally with 0.5ml/100g o f the solution (50 mg/kg). This dose has been used to label cells dividing i n the dentate gyrus o f mice (Kempermann et al., 1997) and voles (Ormerod and Galea, 2001; Smith et al., 2001; Fowler et al., 2002).  Figure 8. Photomicrographs of a BrdU-labelled cells and a pyknotic cell. A) Microphotograph of BrdU-labelled cells located in the subgranular zone of an O V X d female exposed to estradiol for 4 h before BrdU was injected. These cells are representative of those counted in the dentate gyrus of all groups. B) Microphotograph of a representative pyknotic or dying cell in the subgranular zone (SGZ) between the G C L and hilus. Scale bar represents 10 um.  88  Histology  A t the end o f each experiment, voles were anaesthetized with sodium pentobarbital and then perfused with 4% paraformaldehyde, 1 h after B r d U was injected. Following perfusion, brains were extracted and refrigerated overnight i n perfusate at 4 ° C . The following day, the brains were sectioned 40 u m thick sections through the entire dentate gyrus using an oscillating tissue sheer (Leica V T 1 0 0 0 S ) in a bath o f 0 . 1 - M phosphate buffer (PB). Sections were pretreated i n a solution o f 0.2% H2O2 i n P B for 20 m i n and then rinsed i n P B before being mounted on slides treated with 3% 3-aminopropyltriethoxy-silane i n acetone (Sigma Chemicals) to enhance slide adherence.  Peroxidase immunohistochemistry  Tissue was processed to reveal B r d U labelling by applying solutions directly to the slide-mounted sections as described previously (Cameron et al., 1993b; Gould et al., 1999; Tanapat et al., 1999; Ormerod and Galea, 2001; Ormerod et al., 2003). The sections were rinsed repeatedly between steps i n phosphate-buffered saline (0.1 M sodium phosphate heptahydrate i n 0.9% saline; p H 7.4) unless stated otherwise. Sections were incubated in 0.05% Trypsin (Sigma A l d r i c h Chemicals) in 0.1% C a C l T r i s - H C l buffer ( p H 7.5) for 10 m i n to 2  permeabilize cells. D N A was then denatured by applying 2 N HC1 for 30 m i n and then the sections were repeatedly rinsed i n P B S ( p H 6.0). Sections were blocked with 5.0% normal horse serum for 30 m i n and then incubated overnight i n mouse monoclonal antibody against B r d U (1:100 + 3% N H S + 0.5% Tween 20; Boehringer Mannheim) at room temperature. The following day, sections were incubated i n mouse secondary antisera (1:29 + 3.0% normal horse serum; Vector Laboratories) for 4 hrs and then i n avidin-biotin horseradish peroxidase complex  89 ( A B C Elite K i t ; 1:50; Vector Laboratories) for 60 min. Sections were reacted for about 10 m i n in 0.02% diaminobenzidine ( D A B ; Sigma Aldrich Chemicals) and 0.003% H 0 i n Tris2  2  buffered saline and then counterstained with cresyl violet acetate (Baker), dehydrated and coverslipped with Permount (Fisher Scientific) so that pyknotic cells could also be counted.  Data Analyses  Prior to analysis, slides were coded to blind the experimenter to the treatment conditions. Total B r d U - i r (intensely stained medium round or oval cells; F i g 8 A and Cameron et al., 1993b; Ormerod and Galea, 2001; Ormerod et al., 2003) and pyknotic cells (with pale or absent cytoplasm, dark spherical chromatin and no nuclear membrane; F i g 8B and Gould et al., 1991; Ormerod and Galea, 2001) through the granule cell layer and subgranular zone (the « 50 um border between the hilus and granule cell layer; Palmer et al., 2000) o f the dentate gyrus were stereologically estimated. To stereologically estimate cell numbers, total B r d U - i r and pyknotic cells were counted on every 10 section (8 sections per vole; p=1.00) through the th  rostral-caudal extent o f the dentate gyrus per rat using a 100X objective under a N i k o n Eclipse (€600) light microscope. The counts were then applied to a modified version o f the optical fractionator formula (West et al., 1991; described in Ormerod, Lee and Galea, i n press) to project what was counted on every 10 section to what would be counted on the entire dentate th  gyrus. Areas were measured using the digitizing software Analytical Software Imaging Station (Imaging Research, Brock University, Ontario, Canada) and dentate gyrus volume was estimated using Cavalieri's principle (Gunderson et al., 1988). Because we have previously reported B r d U - i r cell densities (Ormerod and Galea, 2001; Galea and M c E w e n , 1999) we also calculated B r d U - i r cell densities (# o f cells/area) on 5 anatomically matched sections per rat (where the dentate gyrus lies just beneath the corpus callosum and the infrapyramidal and  90 suprapyramidal blades are joined at the crest; between A -3.3 and A -4.8 i n rats) in order to compare the density o f B r d U - i r cells with stereological estimates o f total B r d U - i r cells in the dentate gyrus. Our relative densities and stereologically estimated total BrdU-labelled cell numbers were similar to those reported by Cameron and M c K a y (2001).  Statistical analyses  In Experiment 5, the dependent variables (total B r d U - i r cells, B r d U - i r cell density, and total pyknotic cells) were analysed using an analysis o f variance ( A N O V A ) with hormone (EB4, OIL4) and drug ( N M D A , V E H ) as the independent variables. In Experiment 6, the dependent variables (total B r d U - i r cells, B r d U - i r cell density, and total pyknotic cells) were analyzed using an A N O V A with hormone (EB48, OIL48) and drug ( M K 8 0 1 , V E H ) as the independent variables. For both experiments, Pearson product-moment correlations were run between dependent variables and the Newman-Keuls procedure was used as the post-hoc analysis. A l l statistical procedures set a = 0.05.  4.3 R E S U L T S  Experiment 5. Relative to vehicle, estradiol increased and NMDA decreased cell proliferation and a 4h estradiol exposure did not appear to stimulate NMDA receptor activity to influence cell proliferation.  Estradiol significantly increased (main effect o f hormone: N M D A significantly decreased (main effect o f drug: F (  U 7  £(1,17)  = 14.27, p < 0.001) and  ) = 9.62, p < 0.006) the total number  of BrdU-labelled cells i n the dentate gyri o f adult female meadow voles (Figure 9 A ) . Estradiol did not stimulate N M D A receptors within 4 h to alter cell proliferation because hormone treatment did not interact with drug treatment to influence labelled cell number (p < 0.77;  91  **  1 6 0 0 -|  1400  -  1200  -  1000  -  o3 o  T3 CD CD JQ _ro  Z>  m  800 -  o  L_  CD  -O  600 -  E C  o H  400 -  200 -  0 OIL4+VEH  E4+VEH  OIL4+NMDA  E4+NMDA  Condition  4000  g  3000  >* CL o  2000  i—  CD  -Q E  =J  c ~  1000  OIL4+VEH  E4+VEH  OIL4+NMDA  E4+NMDA  Condition  Figure 9. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female voles injected with B r d U 4 h after estradiol or oil and 1 h after N M D A or vehicle in Experiment 5. The white bar represents the data of OIL4+VEH females (n=5), the light gray bar represents the data of EB4+VEH females (n=6), the dark gray bar represents the data of OIL4+NMDA (n=5) females and the black bar represents the data of E B 4 + N M D A females (n=5). A ) Mean number new (BrdU-ir) cells observed in the dentate gyrus of adult female voles. Relative to oil, estradiol increased (p_< 0.001) and N M D A decreased (p_< 0.006) BrdU-labelled cell number in the adult female vole dentate gyrus. Estradiol increased the number of BrdU-labelled cells both in the absence (OIL4+VEH versus E4+VEH; p_< 0.01) and in the presence (OIL4+NMDA versus E4+NMDA; p_< 0.03) of N M D A . B) Mean number of pyknotic cells in the dentate gyrus of adult female voles. The number of pyknotic cells did not differ between groups. * Newman-Keuls result p<0.001 * Newman-Keuls result p<0.05  Figure 9 A ) . In fact, Figure 9 A shows that estradiol enhanced cell proliferation regardless o f drug condition. W e found similar results when comparing BrdU-labelled cell density (OIL4+VEH=1.20 ± 0 . 1 8 cells; E B 4 + V E H = 2.28 ± 0.34 cells; O I L 4 + N M D A = 0 . 3 2 ± 0.40 cells; E B 4 + N M D A = 1 . 4 7 ± 0.20 cells) between groups; estradiol increased (F(i,n) = 21.17, p < 0.0003) and N M D A decreased ( F i (  = 11.96, p < 0.003) the density o f labelled cells but no  > 1 7 )  interaction effect was observed (p < 0.95). In fact, BrdU-labelled cell density was strongly correlated positively with total BrdU-labelled cell number (r i) = 0.93; p < 0.001). Neither the (2  total number o f pyknotic cells (p > 0.68; see Figure 9B) nor pyknotic cell density (p > 0.79) significantly differed between groups. The total area on which BrdU-labelled cells were counted (granule cell layer + subgranular zone) was similar between groups ( F ( i , i ) = 0.70, p < 7  0.54; OIL4+VEH=2.51 ± 0 . 1 7 m m ; E B 4 + V E H = 2.63 ± 0 . 1 0 m m ; O I L 4 + N M D A - 2 . 3 8 ± 0.09 3  3  m m ; E B 4 + N M D A = 2 . 4 1 ± 0.18 m m ) , verifying that differences observed between groups in 3  3  BrdU-labelled cell number were not volumetric.  Experiment 6. Relative to vehicle, estradiol significantly decreased and MK-801 increased cell proliferation proliferation  but estradiol did not interact with NMDA  significantly  receptors to alter cell  within 48 h.  Estradiol significantly decreased (main effect o f hormone: JL(\,\6) 10.13, p < 0.006) and =  M K - 8 0 1 significantly increased (main effect o f drug: F(ij6) = 36.37, p < 0.0001) the number o f BrdU-labelled cells. Similar to Experiment 5, estradiol did not appear to stimulate N M D A receptors within 48 h to influence cell proliferation because the hormone x drug interaction effect was non-significant (p_ < 0.80; Figure 10A). In fact, Figure 10A shows that estradiol suppressed cell proliferation, both i n the absence and presence o f M K - 8 0 1 . BrdU-labelled cell  93  OIL48+VEH  E48+VEH  OIL48+MK801  E48+MK801  Condition  3000 -i  2500 4  OIL48+VEH  E48+VEH  OIL48+MK801  E48+MK801  Condition  Figure 10. Mean (±SEM) number of new cells or pyknotic cells in the dentate gyrus of adult female voles injected with B r d U was administered 48 h after estradiol or oil and 1 h after MK-801 or vehicle in Experiment 6. The white bar represents the data of OIL48+VEH females, the light gray bar represents the data of E48+VEH females, the dark gray bar represents the data of OIL48+MK-801 females and the black bar represents the data of E48+MK-801 females (n=5 per group). A) Mean number new (BrdU-ir) cells in the dentate gyrus of adult female voles. Relative to oil, estradiol significantly decreased (p_ < 0.006) and MK-801 significantly increased (p_ < 0.0001) the number of BrdU-labelled cells in the dentate gyrus of adult female voles. Relative to oil, coadministration of estradiol and MK-801 tended to increase (p_ <0.06) the number of labelled cells. B) Mean number of pyknotic cells in the dentate gyrus of adult female voles. The treatments did not influence pyknotic cell number. Newman-Keuls result p<0.001 Newman-Keuls result p<0.05 Newman-Keuls result p<0.06 a  94 density differed similarly to BrdU-labelled cell number between groups (OIL48+VEH=1.56 ± 0.30 cells; E B 4 8 + V E H = 0.57 ± 0.09 cells; OIL48+MK801=3.89 ± 0.58 cells; EB48+MK801=2.86 ± 0.37 cells); estradiol decreased ( F increased  (£(1,16)  ( U 6 )  = 7.19, p < 0.016) and M K - 8 0 1  = 37.64, p < 0.0001) the density o f labelled cells but no significant interaction  effect was observed (p < 0.95).In fact, BrdU-labelled cell density was strongly correlated positively with total BrdU-labelled cell number  (r o) (2  = 0.94; p < 0.001). Neither the total  number o f pyknotic cells (p > 0.91; see Figure 10B) nor pyknotic cell density (p > 0.89) significantly differed between groups. The total area that BrdU-labelled cells were counted on did not differ between groups ( F (  = 1.17, p < 0.30; OIL48+VEH=2.76 ± 0.16 m m ; 3  1>16)  E B 4 8 + V E H = 2.45 ± 0.15 m m ; OIL48+MK801=2.54 ± 0.17 m m ; EB48+MK801=2.54 ± 0.06 3  3  mm ), verifying that differences observed between groups i n BrdU-labelled cell number were 3  not related to volumetric differences.  4.4 DISCUSSION  These data demonstrate that estradiol dynamically influences cell proliferation i n the dentate gyrus o f adult female meadow voles but does not interact with N M D A receptors to mediate its effects on cell proliferation. Estradiol increased cell proliferation within 4 h but decreased cell proliferation within 48 h i n the dentate gyri o f O V X d adult female meadow voles, consistent with what we have reported previously i n the dentate gyrus o f adult female O V X d rats and intact meadow voles (Ormerod and Galea, 2001; Ormerod et al., 2003). In addition, N M D A receptor activation (via N M D A ) suppressed and N M D A receptor blockade (via M K 8 0 1 ) enhanced cell proliferation which extends previous findings showing that N M D A receptor activity regulates cell proliferation in the dentate gyri o f adult rats and tree shrews (Cameron et al, 1994; Cameron et al., 1995; Gould et al., 1997; Bernabau and Sharp, 2000; Nacher et  95 al.,2001; Nacher et al., 2003). Estradiol did not appear to influence N M D A receptors to alter cell proliferation at either the 4 h or 48 h time point because estradiol increased proliferation i n the presence or absence o f N M D A (within 4 h) and decreased proliferation in the absence or presence o f M K - 8 0 1 (within 48 h; see Figures 8 A and 9 A ) . Pyknotic cell number did not differ between groups and total pyknotic cell number was similar to what we have reported previously (Ormerod and Galea, 2001; Ormerod et al., 2003).  W e believe that the differential effects o f estradiol on cell proliferation observed within 4 versus 48 h o f its administration are time- rather than dose-dependent. Serum estradiol levels are high 4 h after an estradiol injection, intermediate 48 h after an estradiol injection and low or undetectable following a vehicle injection but the number o f labeled cells is elevated 4 h after an estradiol injection and suppressed 48 h after an estradiol injection relative to a vehicle injection (Ormerod and Galea, 2001; Ormerod et al., 2003). In addition, cell proliferation is suppressed 4 h after an estradiol injection (same dose used as the present study) in the dentate gyri o f adult female rats exposed to low dose-estradiol (via silastic implant) for 1 week (Falconer and Galea, personal communication). Future work could verify whether the differential effect o f estradiol on cell proliferation is time-dependent by keeping dose constant over 48 h.  Estradiol suppresses cell proliferation by stimulating adrenal activity but not NMDA recepto activity  Previous work has shown that estradiol increases cell proliferation i n the dentate gyrus of adult rats by stimulating serotonin synthesis (Banasr et al., 2001) and suppresses cell proliferation partially b y stimulating adrenal steroids i n the adult rodent dentate gyrus (Ormerod et al., 2003). However, removing estradiol's stimulatory effect on the H P A axis via  96 adrenalectomy eliminates but does not reverse the suppression i n cell proliferation observed to occur i n the dentate gyrus o f adult female rats 48 h after an estradiol injection (Ormerod et al., 2003), suggesting that estradiol stimulates a factor, perhaps i n addition to adrenal steroids, to suppress cell proliferation. Similar to effects shown previously i n the dentate gyri o f adult male rats (Cameron et al, 1994; Cameron et al., 1995; Gould et al., 1997; Bernabau and Sharp, 2000; Nacher et al., 2001), we found that N M D A receptor activation decreased and N M D A receptor blockade increased cell proliferation i n adult female meadow voles. One other study, demonstrated that N M D A receptor blockade enhances cell proliferation in the dentate gyri o f gonadally intact adult female rats (Nacher et al., 2003). Our results demonstrated similar effects in adult O V X d female meadow voles. However, N M D A receptor-mediated effects on cell proliferation observed in the current study occurred independently o f estradiol's effects on cell proliferation. This result was somewhat surprising given that estradiol administration for 48 h has also been shown to increase the sensitivity and number o f N M D A receptors i n the hippocampus o f adult female rats (Weiland 1992; Gazzaley 1996) and N M D A receptors work downstream o f corticosterone to suppress cell proliferation (Cameron et al., 1998). In fact, Cameron and colleagues (1998) postulated that another factor likely operates the suppressive corticosterone/NMDA receptor pathway because dividing progenitor cells do not appear to express glucocorticoid receptors (Cameron et al., 1993a). Because estradiol stimulates both the H P A axis and N M D A receptor activity, in theory, estradiol could have modulated cell proliferation through its effects o f N M D A receptor activity. O f course, we cannot completely dismiss that estradiol interacts with N M D A receptors. Perhaps the use o f CGP43487, a longer-lasting N M D A receptor antagonist than M K - 8 0 1 (Schmutz et al., 1990; Cameron et al., 1995) would have yielded different results. However, our finding that similar relative estradiol-induced increases (within 4 h) i n cell proliferation in the presence or absence o f N M D A and similar relative estradiol-induced decreases (within 48 h) in  97 presence or absence o f M K - 8 0 1 exist suggests that the effects we observed are straightforward. Within our experimental parameters estradiol did not work through N M D A receptors to influence cell proliferation. Although we found that estradiol and N M D A receptors influenced proliferation independently, the effects o f co-administering estradiol+NMDA or estradiol+MK801 on cell proliferation were additive. Remembering that the effects o f stress (Gould et al., 1997, 1998; Tanapat et al., 2001; Holmes and Galea, 2002), some forms o f learning (Gould et al., 1999), exercise (van Praag et al., 1999a,b) and hormones (Cameron and Gould, 1994; Cameron et al., 1998; Cameron and M c K a y , 1999; Tanapat et al., 1999; Banasr et al., 2001; Ormerod et al., 2003) can all influence cell proliferation, perhaps independently, is important as most non-laboratory-reared mammals likely experience all these phenomena daily. The effects could be additive or cancel one another out.  Estrogen could time-dependently influence cell proliferation in the dentate gyrus of adult rodents through numerous pathways.  Both known estrogen receptors (ER) subtypes ERa and ERp are expressed in the hippocampus, including the subgranular zone (Shughrue et al., 1997; Weiland et al., 1997; Milner et al., 2001) and can activate numerous second messenger pathways as well as stimulate gene expression (Kawata, 1995; Beyer, 1999 for review). A recent study has shown that progenitor cells derived from the adult rat ventricular lining express ERp and to a lesser extent ERd (Brannvall et al., 2002). In these progenitors, estradiol reversed an epidermal growth factor-stimulated increase i n proliferation (Brannvall et al., 2002) suggesting that estradiol mediates differential effects upon adult-derived progenitor cells in vitro depending upon the presence o f other factors. O f course, cells derived from the ventricular subependyma appear to  98 exhibit stem cell properties but the subgranular zone derived cells appear to exhibit the properties o f more restricted progenitors (Seaberg and van der K o o y , 2002). Interestingly, E R Q is expressed at very low levels i n the olfactory area o f rats (Shughrue et al., 1997) and cell proliferation is elevated i n subventricular zone and the rostral migratory stream (but not dentate gyrus) o f adult female prairie voles 60 h after the onset o f daily estradiol versus vehicle treatments (Smith et al., 2001). In addition, estradiol does not influence the proliferation o f progenitors i n the avian song circuit (Burek et al., 1995; Hidalgo et al., 1995; Loissant et al., 2002). Relative differences i n the distribution o f E R a and E R p may produce differences i n the effect o f estradiol on cell proliferation i n these systems. In addition, the interesting dichotomy between the effects o f estradiol on cell proliferation in the dentate gyri o f adult prairie versus meadow voles may reflect the w e l l known differences i n mating and affliliative systems between these two species (Dewsbury, 1987). Changes i n cell proliferation within the dentate gyri o f intact females support that estradiol alters cell proliferation dynamically Natural fluctuations i n cell proliferation within the dentate gyri o f intact adult female rodents also suggest that estradiol regulates the process time-dependently. For example, cell proliferation increases i n the rat dentate gyrus during proestrus relative to diestrus or estrus but increases i n the dentate gyrus o f adult female meadow voles that are reproductively inactive versus active (Galea and M c E w e n , 1999; Tanapat et al., 1999; Ormerod and Galea, 2001). Whereas female rats experience elevated estradiol levels only on the afternoon o f proestrus (Buckingham et al., 1978), female meadow voles experience elevated circulating estradiol levels that persist for up to 45 d only after ovulation is induced by male contact (Lee et al., 1970; Seabloom et al., 1985; Nubbemeyer, 1999). Tanapat and colleagues (1999) injected rats with B r d U at 2 pm, and therefore proestrus females would have likely been exposed to high  99 circulating estradiol for a few hours (see Buckingham et al., 1978). Ormerod and Galea (2001) injected females with B r d U 48 h after introducing a male or female cage partner and, therefore, reproductively active females would likely have been exposed to high circulating estradiol levels for approximately 30-36 h as estradiol levels rise between 12-16 h upon male contact (Lee et al., 1970; Seabloom et al., 1985; Nubbermeyer, 1999). Therefore, the duration o f exposure that intact rats and meadow voles would be exposed to elevated circulating estradiol levels to increase (Tanapat et al., 1999) and then decrease (Ormerod and Galea, 2001) cell proliferation are similar to the durations found i n this study to increase and then decrease cell proliferation i n the dentate gyrus o f adult O V X d female meadow voles. In addition to influencing cell proliferation in the adult rodent dentate gyrus (Ormerod and Galea, 2001; Ormerod et al., 2003; current study), estradiol also has been shown to enhance the survival o f young neurons. Estradiol enhances the survival o f neurons migrating i n the adult avian songbird forebrain (Burek et al., 1995; Hidalgo et al., 1995) by upregulating the production o f brain-derived growth factor i n endothelial cells (Loissant et a l , 2002). W e have found that when estradiol is administered 6-10 days, but not 1-5 or 11-15 days post B r d U , twice as many new neurons survive i n the dentate gyrus o f adult male meadow voles (Ormerod et al., 2002 and submitted for publication). Clearly, the observations that estradiol time-dependently influences cell proliferation as w e l l as enhances the survival o f young neurons could complicate the interpretation o f results from studies utilizing intact female animals to investigate the effect o f estradiol, or other factors, on neurogenesis.  Changes in cell proliferation alters cell proliferation  within the dentate gyri of intact females support that estradiol  dynamically  Approximately 270,000 new cells are produced monthly within the dentate gyri o f adult rats and many o f these cells differentiate into neurons (Cameron and M c K a y , 2001). These new  100 granule neurons extend axons to the C A 3 region within 4-10 days after birth (Hastings and Gould, 1999) and resemble mature granule neurons electrophysiologically by 4 weeks after birth (van Praag et al., 2002). Therefore, young granule neurons could contribute rapidly to the influence that the dentate gyrus has over hippocampus activity and therefore, hippocampusdependent behaviour. Because a large number o f new granule neurons are added to the dentate gyrus monthly, the possibility that new granule neurons influence hippocampus-dependent behaviour seems plausible. In fact, Gould and colleagues (1999) found that more new neurons survive in the dentate gyrus o f rats that engage in hippocampus-dependent (trace eyeblink conditioning and spatial Morris water maze acquisition trials) versus non-hippocampusdependent (delay eyeblink conditioning and cued water maze trials) behaviour. Then, Shors and colleagues (2001, 2002) found that animals with depleted young neuron number following the administration o f a cytostatic agent exhibited impaired performance on training trials in some hippocampus-dependent tasks (trace eyeblink conditioning and context conditioning) but not others (spatial Morris water maze training trials). W e recently discovered that young granule neuron number i n the dentate gyrus o f adult male meadow voles is not related to performance on spatial Morris water maze training trials but increased young neuron number is related to improved performance on a probe trial (Ormerod et al., 2002 and submitted for publication). Because estradiol does not appear to alter the differentiation o f new daughter cells produced in the dentate gyrus o f adult rodents (60-70% neuronal differentiation; Tanapat et al., 1999; Ormerod et al., 2003; Ormerod et al., 2002), the estradiol-induced increase and then decrease in cell proliferation likely results i n an increased and then decreased number o f new granule neurons that are integrated into existing hippocampal circuitry and could influence hippocampus-dependent behaviour.  New neurons appear functional  and influence hippocampus-dependent  behaviour  101 Approximately 270,000 new cells are produced monthly within the dentate gyri o f adult rats and many o f these cells differentiate into neurons (Cameron and M c K a y , 2001). These new granule neurons extend axons to the C A 3 region within 4-10 days after birth (Hastings and Gould, 1999) and resemble mature granule neurons electrophysiologically by 4 weeks after birth (van Praag et al., 2002). Therefore, young granule neurons could contribute rapidly to the influence that the dentate gyrus has over hippocampus activity and therefore, hippocampusdependent behaviour. Because a large number o f new granule neurons are added to the dentate gyrus monthly, the possibility that new granule neurons influence hippocampus-dependent behaviour seems plausible. In fact, Gould and colleagues (1999) found that more new neurons survive in the dentate gyrus o f rats that engage i n hippocampus-dependent (trace eyeblink conditioning and spatial Morris water maze acquisition trials) versus non-hippocampusdependent (delay eyeblink conditioning and cued water maze trials) behaviour. Then, Shors and colleagues (2001, 2002) found that animals with depleted young neuron number following the administration o f a cytostatic agent exhibited impaired performance on training trials i n some hippocampus-dependent tasks (trace eyeblink conditioning and context conditioning) but not others (spatial Morris water maze training trials). W e recently discovered that young granule neuron number in the dentate gyrus o f adult male meadow voles is not related to performance on spatial Morris water maze training trials but increased young neuron number is related to improved performance on a probe trial (Ormerod et al., 2002 and submitted for publication). Because estradiol does not appear to alter the differentiation o f new daughter cells produced i n the dentate gyrus o f adult rodents (60-70% neuronal differentiation; Tanapat et al., 1999; Ormerod et al., 2002, Ormerod et al., 2003), the estradiol-induced increase and then decrease in cell proliferation likely results i n an increased and then decreased number o f new granule neurons that are integrated into existing hippocampal circuitry and could influence hippocampus-dependent behaviour.  102 4.5 I M P L I C A T I O N S  Estradiol diversely influences neurogenesis i n the adult rodent dentate gyrus by first increasing and then decreasing progenitor cell proliferation as well as by enhancing the survival of young granule neurons. Estradiol can increase cell proliferation by stimulating serotonin activity and suppress cell proliferation by stimulating adrenal steroids (but not v i a N M D A receptors). Discovering the mechanisms by which estradiol mediates its diverse effects over the production and survival o f new neurons in the dentate gyrus may facilitate our understanding o f how to control the process to perhaps restore the neuronal loss associated with disease or trauma in the hippocampus and other areas o f the adult central nervous system. Hippocampusdependent behavior enhances the survival o f young granule neurons and young granule neurons appear necessary for the successful performance o f some hippocampus-dependent tasks. Estradiol-induced increases i n neuron number are associated with improved retention in the Morris water maze i n meadow voles. Clearly understanding estradiol's role over adult neurogenesis could improve neuronal replacement strategies and assist the development o f strategies that manipulate new neurons in situ. This study provides insight into the mechanism by which estradiol suppresses cell proliferation i n the adult rodent dentate gyrus and demonstrates that estradiol robustly influences cell proliferation i n the dentate gyri o f rodents with diverse cycles (reflex ovulating voles and cycling rats) i n a similar fashion.  103  CHAPTER 5 ESTRADIOL ENHANCES NEUROGENESIS BY INCREASING T H E SURVIVAL OF Y O U N G N E U R O N S A N D T H E I N C R E A S E IS R E L A T E D T O B E T T E R S P A T I A L M E M O R Y IN A D U L T M A L E RODENTS (submitted to  Hippocampus)  5.1 I N T R O D U C T I O N  The discoveries that cells derived from the adult mammalian central nervous system ( C N S ) exhibit stem cell properties i n culture (Reynolds and Weiss, 1992) and that many cells born within the neurogenic adult rat dentate gyrus express neuronal protein (Cameron et al., 1993) verified Altman's (Altman and Das; 1965) early observation that new neurons are produced in the adult mammalian C N S . Eriksson and colleagues' (1998) discovery that new granule neurons are produced i n the adult human dentate gyrus fuelled extensive research into discovering how the production and survival o f neurons is regulated in the adult mammalian C N S (see K u h n et al., 2001 for review). Clearly, the ability to regulate and manipulate neurogenesis i n neurogenic regions or to induce neurogenesis in non-neurogenic regions o f the adult rodent central nervous system could lead to strategies for replacing neurons lost in diseases such as Alzheimers (West et al., 1994; Guela, 1998) or Parkinson's ( U h l et a l , 1985) to potentially promote restoration o f function (for review see Kempermann and Gage, 1999). A m o n g the many factors that influence neurogenesis i n the adult rodent hippocampus (Kuhn et al., 2001), estradiol has been shown to regulate neurogenesis dynamically by affecting both the production and possibly the survival o f new granule neurons. For example, estradiol dramatically alters the rate o f cell proliferation in the dentate gyrus o f adult female rats. In the dentate gyrus o f adult female rats and voles, the number o f dividing progenitor cells increases following short-term (4 h) but decreases following longer-term (48 h) estradiol exposure (Ormerod and Galea, 2001; Ormerod et al., 2003). In the song circuit o f adult canaries and  104 zebra finches, estradiol promotes the survival o f new neurons as they differentiate and migrate to their final destinations (Hidalgo et al., 1995; Burek et al., 1995; 1997). Previous evidence from our laboratory suggests that estradiol also promotes the survival o f new neurons born in the dentate gyrus o f adult rodents. W e found a higher proportion o f new cells surviving 5 weeks versus 2 h i n the dentate gyrus o f adult female meadow voles with high versus low circulating estradiol (Ormerod and Galea, 2001). However, in that study, we did not directly manipulate cell survival as estradiol levels were different between the groups both before and after the injection o f cell synthesis markers, and therefore cell proliferation as well as cell survival was affected. Treatments can influence cell proliferation (the division o f progenitor cells) and/or the survival o f young neurons and increasing either cell proliferation or survival could produce a net increase net neurogenesis (the number o f neurons produced). In the present study, we directly tested whether estradiol could directly influence the survival o f new neurons independent o f its effects on cell proliferation. Therefore, the first objective o f this study was to determine whether estradiol specifically enhances the survival o f new granule neurons i n the dentate gyrus o f adult male meadow voles. To this end, we injected adult male meadow voles with bromodeoxyuridine (BrdU) and waited 24 h before administering estradiol or vehicle as most progenitor cells complete mitosis within 24 h (Cameron and M c K a y , 2001). Because previous work with avian species (Hidalgo et a l , 1995; Burek et al., 1997) has shown that estradiol enhances the survival of new migrating neurons, we tested whether new neurons born i n the dentate gyrus o f adult meadow voles were more vulnerable to the trophic effect o f estradiol at various time points roughly associated with different stages o f their maturation. Therefore, we injected voles with estradiol over three different 5 day periods after B r d U was administrated; either Days 1-5 (which coincide with the time that proteins associated with differentiation and migration begin to be expressed; Tanapat et al., 1999; Quinn et al., 1999; Cameron and M c K a y , 2001; Jin et al.,  105 2001), Days 6-10 (which coincide with axon extension; Hastings and Gould, 1999) and Days 11-15 (which coincide with the onset o f the expression o f mature neuronal protein; Cameron et al., 1993b; Kempermann et al., 1997; Tanapat et al., 1999). Based upon the work done by Hidalgo and colleagues (1995) and Burek and colleagues (1995; 1997) showing that more new migrating neurons survived i n the avian forebrain o f estradiol-treated songbirds, we expected that estradiol may increase the number o f young neurons i n the dentate gyrus o f adult meadow voles treated with estradiol just after the administration o f B r d U . The functional consequences o f changes in neuron number are just beginning to be explored. Recent work suggests that hippocampus-dependent behaviour influences the number and relies upon the presence o f relatively young dentate gyrus granule neurons. For example, animals that engage in hippocampus-dependent Morris water maze place training or traceeyeblink conditioning have more new granule neurons surviving i n their dentate gyri than animals that engage i n non-hippocampus-dependent visible platform training or delay-eyeblink conditioning (Gould et al., 1999; Ambrogini et al., 2000). Moreover, Shors and colleagues (2001) found that when progenitor cell proliferation is reduced 80%, by the administration o f a cytostatic agent over 14 days but not over 6 days just prior to training, trace- but not delayeyeblink conditioning is impaired. However, recent work by Shors and colleagues (2002) showed that performance on training trials i n the Morris water maze is not affected when progenitor cell proliferation is reduced by the same treatment, suggesting that young granule neurons influence learning i n some but not all hippocampus-dependent tasks. These studies suggest that hippocampus-dependent behaviour increases the number o f young granule neurons and that hippocampus-dependent behaviour is impaired by a significant reduction i n the number o f 1-2 week old neurons. In rats, estradiol has been shown to influence both neurogenesis i n the dentate gyrus (Galea and M c E w e n , 1999; Tanapat et al., 1999; Ormerod and Galea, 2001; Ormerod et al., 2003) and learning and memory (Galea et al., 2001; Holmes  106 et al., 2002). Therefore, estradiol may produce some o f its effects on hippocampus-dependent behaviour by influencing neurogenesis. Our second objective, therefore, was to determine whether any estradiol-induced change i n the number o f young neurons influences hippocampus-dependent behaviour. To test whether estradiol-induced changes in neuron number influence hippocampus-dependent behaviour, we observed the performance o f animals that had been treated with either estradiol or vehicle over Days 6-10 post B r d U (with hormone treatment ending 6 days prior to training) i n the standard Morris water maze place task. W e found that estradiol increased the number o f young neurons surviving i n the dentate gyrus o f adult male meadow voles, but only when administered over Days 6-10 after B r d U when the young neurons are 6-10 days old (presumably in the process o f extending axons; Hastings and Gould, 1999). Furthermore during a probe trial (on D a y 20 after B r d U administration), voles treated with estradiol over Days 6-10, spent significantly more time in the quadrant o f the Morris water maze that had previously housed the platform than vehicletreated males. The results suggest that spatial memory is improved by an increased number o f young neurons.  5.2 M E T H O D S  A l l animals were treated i n strict accordance with the guidelines set forth by the Canadian Council on A n i m a l Care and The University o f British Columbia regarding the ethical treatment o f animals used for the purposes o f research. Every effort was made to minimise the number o f animals used per group and their suffering.  Animals  107 W e used adult male meadow voles (n=45; >35 g and 60 d old) reared i n our breeding colony at The University o f British Columbia as subjects. The voles were bred and reared i n polyurethane bedding-lined (Carefresh; Absorption Corporation) cages that contained plastic and cardboard containers. A t 21 days o f age, the voles were weaned and housed either with same sex siblings or individually (if no sibling o f the same sex existed) until 60 days o f age when they were all housed individually. The colony room was temperature controlled (21±1°C) colony room and the lightdark cycle was set at 16:8 h (lights on at 0700h). The voles had free access to tap water and Lab Diet #5012 (Jamieson) for the duration o f the experiment and were given sunflower seed/alfalfa pellet supplements once per week.  Surgery  A t 70-80 d old, male meadow voles (5-6 per group) were castrated, using sterile surgical techniques. First animals were put into a chamber to which halothane was delivered at a flow rate o f 5% (flow rate o f 0 was 2%) until anaesthesia was visibly induced. Then animals 2  were transferred to a nose cup to which halothane was delivered initially at a flow rate o f 3% but was reduced to as low as 1% to maintain a stable respiratory rate. A small incision was made through each scrotal sac to extrude the testis. Each vas deferens was then tied off using surgical silk and the testis was surgically removed before the scrotal sac was sutured. Animals recovered for one week prior to the onset o f each experiment.  Drug preparation  The cell synthesis marker bromodeoxyuridine ( B r d U ; Sigma A l d r i c h Chemicals), a marker o f dividing cells (Nowakowski et al., 1989), was prepared just prior to injection. B r d U was dissolved to a concentration o f 10 mg/ml freshly prepared 0.9% saline (buffered with 7ul  108 2 N N a O H / m l saline) and injected intraperitoneally i n a volume o f 0.5ml/100 g body weight (50 mg/kg). This dose is typically used to investigate neurogenesis i n mice (Kempermann et al., 1997; Kempermann et al., 1998; Kempermann and Gage, 2002) and meadow voles (Ormerod and Galea, 2001; Smith et al., 2001; Fowler et al., 2002). Estradiol (17-f3 estradiol benzoate; Sigma A l d r i c h Chemicals) was dissolved over low heat i n sesame o i l (Sigma Aldrich Chemicals) to a concentration o f 10p,g estradiol/0.5ul sesame oil. Once dissolved, the estradiol solution was stored i n a light insensitive container. Subcutaneous injections o f the estradiol solution or sesame o i l vehicle were given in a volume o f 0.5ul.  Procedure  Experiment 7 was conducted to investigate whether estradiol influences the survival o f new neurons. Because previous work i n canaries has shown that estradiol improves the survival o f new neurons within the song circuit as they migrate (Hidalgo et al., 1995; Burek et al., 1997), we were interested i n determining whether young neurons i n adult male meadow vole dentate gyrus were more vulnerable to the trophic effect o f estradiol at different stages o f their maturation. To test the effect o f estradiol on the survival o f new neurons, voles were injected twice with B r d U on D a y 0, once at 0800 h and then again at 1000 h. W e then waited 24 h after the second injection as that amount o f time allows progenitor cells that have incorporated B r d U to complete one mitotic division (Cameron and M c K a y , 2001). To test whether new (BrdU containing) cells were more vulnerable to the trophic effect o f estradiol at different stages o f their maturation, voles were injected with either vehicle or estradiol for 5 consecutive days either over Days 1-5 ( V i . and E ] . ; n=5 and n=6, respectively), Days 6-10 5  5  (V -io 6  and  E -io; 6  n=6  and n=5, respectively), or Days 11-15 (Y\\.\ and E n . i ; n=5 and n=6, respectively). These time 5  5  periods were chosen to roughly correspond with the onset o f migration and differentiation  109 (Days 1-5; Tanapat et al.', 1999; Quinn et al., 1999; Cameron and M c K a y , 2001; J i n et al., 2001), to roughly correspond with the time that new cells extend axons (Days 6-10; Hastings and Gould, 1999) or to roughly correspond with maturation (Days 11-15; Cameron et al., 1993b; Kempermann et a l , 1997; Tanapat et al., 1999) o f new granule neurons. A l l animals were perfused on D a y 16 post-BrdU administration. A l l animals were perfused on Day 16 so that differences i n new neuron number between groups could be compared independent o f differences that would likely occur due to time-dependent naturally occurring cell death observed to occur over time (Cameron et al., 1993b; Cameron and M c K a y , 2001). The time line o f the experiment is depicted i n Figure 11.  Groups  V,.  5  E,.  5  Day 0 (BrdU)  Days 6-10 (estradiol or vehicle injections)  Days 11-15 (estradiol or vehicle injections)  sssss  SS  SS  VMO ^6-10  v„.,  Days 1-5 (estradiol or vehicle injections)  S  SSSSS  S  SS  5  Day 16 (Perfuse)  s  SSSSS  E11-15  Figure 11. Timeline of Experiment 7. Voles were injected with either vehicle or estradiol either over Days 1-5 (Vi_ and E i . ; n=5 and n=6, respectively), Days 6-10 ( V . and E . ; n=6 and n=5, respectively), or Days 11-15 (Vu_i and E . i ; n=5 and n=6, respectively) to test whether new (BrdU containing) cells were more vulnerable to the trophic effect of estradiol at different stages of their maturation. A l l voles were perfused on Day 16. 5  6  10  6  1 0  5  5  u  5  Experiment 8 was conducted to investigate whether an estradiol-induced increase in the number o f new granule neurons influences hippocampus-dependent behaviour. Previous work has shown that adult male rats that engage in water maze place training (standard Morris water maze training) have more new neurons than males that engage i n a cued version o f the task (Gould et al., 1999) when training occurs 7-10 days after B r d U is administered (i.e. when the new neurons are 7-10 days old). Because the place version o f the task relies upon the presence  110 of an intact hippocampus whereas the cued version does not (Morris et al., 1982), the results o f that study suggest that the survival o f young neurons is influenced by hippocampus-dependent behaviour. Furthermore, Shors and colleagues (2001) found that rats treated with the cytostatic agent methylazoxymethanol ( M A M ) for 14 days but not 6 days prior to training exhibited impaired trace-eyeblink and trace fear conditioning but not impaired Morris water maze acquisition or contextual fear conditioning. Interestingly, in Experiment 7, we found that estradiol increases the number o f young neurons in the dentate gyrus o f adult male meadow voles when administered Days 6-10 after their birth (after B r d U was injected). Thus, we used the same injection paradigm to investigate whether the estradiol-induced increased number o f new neurons influences learning and/or retention i n the standard Morris water maze task. W e injected voles with B r d U twice on D a y 0, once at 0800 h and again at lOOOh and then with either estradiol (E6-10; n=6) or vehicle (V6-10; n=6) over Days 6-10 post B r d U . Voles then began Morris water maze training hormone free on Day 16-post B r d U (6 days after the last estradiol or vehicle injection). A l l animals were perfused on Day 24 after the completion o f Morris water maze training. The time line o f this experiment is depicted i n Figure 12.  Groups  Ve-io  Day 0 (BrdU)  Days 1-5 (Estradiol or Veh)  SS  Days 6-10 (Estradiol or Veh) SSSSS  Days 11-15 (Estradiol or Veh)  Day 16 (MWM ) S  Ee-10  Figure 12. Timeline of Experiment 8. Voles were injected twice with BrdU on Day 0, once at 0800 h and again at lOOOh and then with either estradiol (E _i ; n=6) or vehicle (V 6 _i 0 ; n=6) over Days 6-10 post BrdU. This was done to test the effect of changes in young neuron number on hippocampus-dependent behaviour. Voles then began Morris water maze training drugfreeon Day 16-post BrdU (6 days after the last E B or vehicle injection). 6  0  Morris water maze training  A l l animals i n Experiment 8 were trained in the standard Morris water maze task (Morris et al., 1982; Morris, 1984). The maze (90cm diameter x 45cm height) was filled with  Ill water (20±1°C) that was rendered opaque with Tempera nontoxic white paint (Demco) to a height o f (30cm). The maze was divided into quadrants and four equally spaced release points were designated N , E , S and W . A platform (7 cm diameter) was hidden 1 cm beneath the water in the centre o f the northwest quadrant. Cues (camera, coloured cardboard symbols, television, door, etc) were visible throughout the room. The first day o f training consisted o f a probe trial, in which swim speed was assessed i n absence o f the platform. Twenty-four hours after the first probe trial, the voles were given four training trials per day over 4 days, i n which they were given 60 s to swim to the platform hidden in the northwest quadrant. If the voles did not find the platform i n the allotted time, they were placed on the platform for 15 s (ITI 60s). Over training trials, latency and path-length to reach the platform and average swim speed were assessed. O n the fifth day o f behavioral testing, a probe session was administered i n which the platform was removed from the maze and percent time spent i n each quadrant was assessed. Then, the following day the platform was moved to the southwest (opposite) quadrant and the voles were given four reversal-learning trials. Behaviour i n the water maze was videotaped and then analyzed with an H V S tracking system ( H V S Image, Hampton, U K ) . Blocks were the average o f four trials.  Histological  procedures  O n D a y 16 (Experiment 7), voles were anaesthetized with sodium pentobarbital and then perfused with 4% paraformaldehyde. Their brains were extracted and stored overnight in perfusate at 4°C. The following day, 40um sections were sliced through the entire dentate gyrus with an oscillating tissue sheer (Leica OTS1000) i n 0.1 M phosphate buffer (PB). Sections were incubated i n a 0.2%  H2O2  P B solution for 20 m i n and then rinsed before being  112 mounted on slides treated with a solution o f 3% 3-aminopropyl-triethoxysilane (Sigma Aldrich Chemicals) in acetone. Sections were dried overnight then processed immunohistochemically. Peroxidase  immunohistochemistry  Peroxidase immunohistochemistry was performed as described previously to stereologically estimate total BrdU-labelled cell number on slide-mounted tissue (Cameron et al., 1993b; Gould et al., 1999; Tanapat et al., 1999; Ormerod and Galea, 2001; Ormerod et al., 2003). Sections were rinsed repeatedly i n phosphate-buffered saline ( P B S ; p H 7.4) between steps. Cells were permeabilized using a solution o f 0.05% trypsin (Sigma A l d r i c h Chemicals) in TRIS-buffered saline for 10 m i n and D N A was denatured by incubating tissue in 2 N HC1 for 30 m i n at 37°C. Nonspecific antigens were then blocked by incubating the tissue in a solution of 5% normal horse serum ( N H S ; Vector Laboratories) in P B S ( P B S ) for 20 m i n and then the +  tissue was incubated overnight i n mouse monoclonal anti-BrdU (Boehringer Mannheim; 1:400). The next day, the tissue was rinsed repeatedly and then incubated i n mouse secondary antisera (Vector Laboratories; 1:129) i n P B S  +  for 4 h and then i n avidin-biotin horseradish  peroxidase complex ( A B C Elite; Vector Laboratories) for 1 h. Finally, tissue was reacted for 10 min in 0.2% 3,3'-diaminobenzidine (Sigma Aldrich Chemicals) TRIS-buffered saline and counterstained with cresyl violet (Baker) to assess the number o f pyknotic cells before being coverslipped with permount.  Immunofluorescent  labelling  Immunofluorescent labelling was done on slide-mounted tissue to verify the phenotype of BrdU-labelled cells as described previously (Gould et al., 1999; van Praag et al., 1999a; van Praag et al., 2002; Ormerod et al., 2003). Sections were rinsed i n TRIS-buffered saline (TBS) between steps. Sections were incubated i n deionized formamide for 2 h at 65°C and D N A was  113  Figure 13. Microphotographs of cells analyzed in Experiment 7. A) A clump of BrdU-labelled cells in the dentate gyrus of a male meadow vole injected with estradiol over Days 6-10. B) A pyknotic cell in the dentate gyrus of an animal treated with vehicle. C) Neurons (immunoreactive for Cy3 conjugated anti-NeuN) are shown in red and a new cell (immunoreactive for FITC conjugated anti-BrdU) is shown in green. The arrow points to a new neuron (immunoreactive for both anti-NeuN and anti-BrdU). No significant difference in the number of new cells expressing NeuN was observed between groups (p>0.80). D) Glia (immunoreactive for Cy3 conjugated anti-GFAP) are shown in red and a new cell (immunoreactive for anti-BrdU conjugated with FITC) is shown in green. The arrow points to a new glia (immunoreactive for both anti-GFAP and anti-BrdU). No significant difference was observed between groups in the number of new cells that expressed G F A P (p>0.38).  114 denatured i n 2 N HC1 at 37° for 30 min. Sections were blocked i n 5% normal donkey serum ( N D S ; Jackson immunoresearch) i n T B S ( T B S ) for 30 m i n and then overnight i n a cocktail o f +  rat anti-BrdU (ascites 1:100; Oxford Biochemical Incorporated), rabbit monoclonal neuron specific enolase ( N S E ; 1:2000; Santa Cruz) and mouse monoclonal anti-glial fibrillary acidic protein ( G F A P ; 1:2000; Novacastra) in T B S or rat anti-BrdU and mouse a n t i - G F A P or rat +  anti-BrdU and mouse anti-neuronal nuclei (NeuN; Chemicon). N S E and N e u N label mature neurons (Cameron et al., 1993b; van Praag et al., 1999; Palmer et al., 2000) and G F A P labels glial cells (Debus et al., 1983; Gould et al., 1999; Smith et al., 2001). The following day, the sections were rinsed and blocked i n 5% N D S and then for 4 h i n a cocktail o f donkey anti-rat fluorescein (FITC; to visualize B r d U ) , donkey anti-rabbit Cy5 (to visualize N S E ) and donkey anti-mouse Cy3 (to visualize G F A P or N e u N ; all 6 ul/ml) i n T B S . Slides were rinsed and then +  coverslipped with the anti-fading agent diazobicyclooctane.  Data analyses  A l l slides were coded to blind the experimenter from the treatment conditions. On th  peroxidase-treated  tissue, B r d U - i r and pyknotic cells were counted on every 10  section  through the subgranular zone ( « t h e 50 um band between the granule cell layer and the hilus; Palmer et al., 2000) and granule cell layer using a lOOx objective on a N i k o n Eclipse G600 light microscope. Cells were considered BrdU-labelled i f they were intensely stained and exhibited medium round or oval cell body morphology and pyknotic i f they lacked a nuclear membrane and had condensed chromatin (Cameron et al., 1993b; Ormerod and Galea, 2001; Figure 13A and 13B). Areas counted were measured using the digitizing software Analytical Software Imaging Station (Imaging Research, Brock University) so that the total number o f BrdU-ir and pyknotic cells that would be present throughout the complete dentate gyrus could be estimated  115 using a modified version o f the optical fractionator method (West et al., 1991). W e estimated the total number o f BrdU-labelled cells using a modified version o f the optical fractionator method as outlined i n Ormerod, Lee and Galea (2003). Areas were obtained using the digitizing software Analytical Software Imaging Station (Imaging Research, Brock University, Ontario, Canada) and dentate gyrus volume estimates were made using Cavalieri's principle (Gunderson et al., 1988). Because we have previously reported B r d U - i r cell densities (Galea and M c E w e n , 1999; Ormerod and Galea, 2001; Ormerod et al., 2003) we also calculated B r d U - i r cell densities (# o f cells/area) on 6 anatomically matched sections per rat (where the dentate gyrus lies just beneath the corpus callosum and the infrapyramidal and suprapyramidal blades are joined at the crest; between A -3.3 and A -4.8 in rats) in order to compare the density o f B r d U ir cells with stereological estimates o f total B r d U - i r cells in the dentate gyrus. B r d U - i r cell phenotypes were analyzed on fluorescent probe-treated  tissue. Twenty-five BrdU-labelled cells  on 4-6 sections per animal (n=3 per group) were identified using a Zeiss fluorescent microscope and their phenotype analyzed using a confocal laser scanning head (BioRad 2000) with U V (red diode), green H e N e and argon lasers. Z-sections at 0.4 um were taken and optical stacks o f 10 images were created with NTH image for P C so that cells could be rotated in orthogonal planes to verify double labelling. Neurons were double-labelled with B r d U + N S E or B r d U + N e u N (Figure 13C) and glia were double-labelled with B r d U + G F A P (Figure 13D). NTH images were imported into Adobe Photoshop for channel merging and digital manipulations were restricted to contrast enhancements and color level adjustments.  Statistical analyses  In Experiment 7, the dependent variables (number o f BrdU-labelled cells, pyknotic cells, density o f both BrdU-labelled and pyknotic cells, and percentage o f B r d U -  116 labelled/GFAP-ir, BrdU-labelled/NSE-ir or BrdU-labelied/NeuN-ir labelled cells) were each analyzed using an analysis o f variance ( A N O V A ) with condition (vehicle, E1.5, E 6 - 1 0 and E i 1-15) as the between-subjects factor. Pearson product-moment correlations were conducted to assess the relationship between BrdU-labelled cell number and density. In Experiment 8, the dependent variables (latency, path-length and average swim-speed to reach the platform) were each analyzed using repeated-measures A N O V A s with condition (vehicle, E .io) as the 6  between-subjects factor and session (1, 2, 3, 4) or quadrant (training, adjacent 1, adjacent 2, opposite) as the within-subjects factor. Post-hoc analyses utilized the Newman-Keul's procedure.  5.3 R E S U L T S  Experiment 7. Estradiol promotes the survival of new neurons The number o f labelled 16-day old BrdU-labelled cells did not differ between the V 1 . 5 , V6-10  and  Vii-15  groups (p > 0.86) and therefore the data for these groups were collapsed into  one group (Vehicle) for subsequent analyses. Relative to vehicle, estradiol increased the total number o f 16 d-old BrdU-labelled cells (F(3,29) = 4.55; p < 0.01) when administered over Days 6-10 (p < 0.009) but not over either Days 1-5 (p > 0.41) or Days 11-15 (p > 0.70; see Figure 14A). Similarly, estradiol increased the density o f BrdU-labelled cells but only when administered over Days 6-10 (4.82 + 1.58; p > 0.01) and not Days 1-5 (2.46 + 0.34; p > 0.72) or Days 11-15 (1.45 + 0.32; p > 0.41) relative to vehicle (2.16 + 0.23; F  ( 3 > 2 9 )  = 4.96; p < 0.007).  Our measures o f total number and density o f BrdU-labelled cells were highly positively correlated  (r 3) (3  = 0.91; p < 0.001).  117  B  ®  5000  5000  4000  4000  E  -z. O  =  3000  3000  "D  Q) <D  J5 CD  =>  2000  2000  CQ  "ro o 1000 \  1000  Vehicle  E,.  5  Eg.  1 0  E ^ j  Vehicle  Condition  E..  5  Eg.,,,  E„.,  5  Condition  Figure 14. Stereological estimates of BrdU-labelled and pyknotic cells in the dentate gyrus of adult male meadow voles following estradiol or vehicle treatment in Experiment 7. The white bars depict the data of voles treated with vehicle over Days 1-5, 6-10 or 11-15 after BrdU (data were collapsed as the number of BrdU-labelled cells did not statistically differ between groups). The grey bars depict the data of voles treated with estradiol over Days 1-5, 6-10 or 11-15 after BrdU was injected. A). Stereological estimate of total BrdU-labelled cell number in the granule cell layer of male voles. Relative to vehicle, estradiol nearly doubled the number of BrdU-labelled cells in the dentate gyrus of adult male meadow voles but only when administered over Days 6-10 after BrdU was injected (p<0.009). B) Stereological estimate of total pyknotic cell number in the granule cell layer of adult male meadow voles. More pyknotic cells were found in the granule cell layer of voles treated with estradiol over Days 1-5 (p<0.06) or Days 6-10 (p<0.03) than vehicle-treated voles, ••denotes p O . O l *denotes p<0.05 "denotes 0.10>p<0.05  There were no group differences in dentate gyrus volume indicating that the difference in labelled cell number between estradiol- and vehicle-treated voles was not related to a volumetric difference (£0,29) = 0.56; p's > 0.60; see Table 10).  Table 10. Mean (±SEM) dentate gyrus volume in vehicle- and estradiol-treated adult male meadow voles in Experiment 7. Group Vehicle (n=16) E,- (n=6) E . (n=5)  Volume (mm ) 2.34±0.95 2.4410.14 2.37±0.10 E i 1-15(11=6) 2.5310.75 Dentate gyrus volume did not significantly differ between groups 5  6  10  118 The majority o f 16 day-old BrdU-labelled cells expressed the mature neuronal protein, N S E ( « 60%) or N e u N ( « 66%) and this percentage was Consistent across groups (F( ) = 0.034; p > 3j8  0.99 and  = 0.033; p > 0.80, respectively; see Table 11). Fewer BrdU-labelled cells  F 3, ) (  8  expressed the glial marker G F A P ( « 16%) and this percentage was similar between groups ©3,8)=  1.17; p > 0.38; see Table 11).  Table 11. Mean (±SEM) % B r d U - i r cells expressing a neuronal (NSE- or NeuN-ir) or glial (GFAP-ir) phenotype measured 16 d after B r d U was injected did not significantly differ in adult male meadow voles treated with estradiol or vehicle in Experiment 7. Group NSE-ir NeuN-ir GFAP-ir =  Vehicle (n=16) E!. (n=6) E .io(n=5) En. (n=6) 5  6  l5  59.2±3.2 60.0+2.3 60.3±3.2 58.711.3  66.2±2.5 63.3±4.1 67.311.8 59.2±1.3  16.0±2.3 18.7+2.7 12.1±1.3 17.3±1.3  The total number o f pyknotic cells tended to differ between vehicle- and estradioltreated groups  (F(3,29)  = 2.45; p > 0.08; see Figure 14B). Because previous work has shown that  high estradiol levels are associated with lower number o f pyknotic cells (Tanapat et al., 1999) and because we observed differences i n the number o f new (16 d old) neurons i n the dentate gyri o f estradiol-treated animals (see above), we planned a priori to compare the number o f pyknotic cells between the vehicle-treated group and each o f the estradiol-treated groups. Relative to vehicle, estradiol reduced the number o f pyknotic cells when administered either over Days 1-5 (p < 0.06) or over Days 6-10 (p < 0.03) but not when administered over Days 11-15 (p > 0.17; see Figure 14B).  Experiment 8. Estradiol-induced performance  changes in young neuron number did not influence  on water maze acquisition  trials  Both groups exhibited similar swim speeds (t(io) = 1.68;p > 0.12) and pathlengths (t(io> = 0.98; P > 0.35) on a baseline trial (Day 16) in which the platform was absent from the pool (data not  119 shown). Over training trials (Days 16-19), we observed that average latency for each session was significantly positively correlated with the respective pathlength for each session (r's (n) > 0.80; p's < 0.002). W e found that latency to reach the platform significantly decreased across sessions (main effect o f session: FQ^7) - 8.86; p < 0.00003) i n both groups (main effect o f condition: p > 0.55) and that performance did not differ between groups across session (session by group interaction p > 0.90; see Figure 15 A ) . Newman Keuls post hoc tests revealed that both groups exhibited significantly shorter average latencies during training sessions 2, 3 and 4 relative to session 1 (p's < 0.002). Similarly, the distance traveled to reach the platform (pathlength) decreased across training sessions (main effect o f session: F(3,27) = 15.04; p > 0.00002) i n both groups (main effect o f condition: p > 0.82) and that pathlength did not differ between groups across sessions (condition by session interaction: p > 0.72; see Fig. 15B). N o difference i n swim speed between groups was observed across training sessions (p > 0.96).  Estradiol-induced  changes in neuron number did influence performance  on a water maze  retention trial  O n probe trial performance (Day 20), a A N O V A on percent time spent each quadrant revealed a significant main effect o f quadrant (£(1,10) 112.75; p < 0.001) and a significant =  interaction effect o f condition and quadrant (interaction F(i ) = 8.16; p < 0.03). Newman>10  Keuls comparisons revealed estradiol-treated voles (E6-10) spent significantly more time there than did vehicle-treated voles (p < 0.015) i n the quadrant that had previously held the platform (Figure 16). Neither total pathlength (p > 0.96) nor average swim speed (p > 0.92) varied between groups on probe trial performance. To ensure that the observation that estradiol-treated males more time spent i n the training quadrant than vehicle-treated males was better explained by improved memory versus perseveration, we administered reversal trials i n which the  120  Figure 15. Performance of vehicle- versus estradiol-treated meadow voles in training trials of the Morris water maze in Experiment 8. In both graphs, white circles depict the drug-free performance of voles treated with vehicle over Days 6-10 and black circles depict the data of voles treated with estradiol over the same period. A ) Latency did not differ between groups over training trials. Note that both groups acquired the task at the same rate and to the same degree. B) Pathlength was highly correlated with latency (p<0.002) and demonstrates that both groups acquired the task at the same rate and to the same degree.  121 platform was hidden i n the opposite quadrant to that o f the training trials one day after the probe trial. Neither a significant main effect o f condition (p > 0.45; data not shown) nor a condition by trial interaction (p > 0.29) was observed, suggesting that both groups learned the new location o f the hidden platform at the same rate.  60 Vehicleg.,, Estradiol.  nl 50 "O CO  I  cr  sz 40 o CD CD  c  CD CL CO CD  30  X  H  E 20 A CD  0-  10  Training  Opposite  JL  Adjacent 1  Adjacent 2  Quadrant  Figure 16. Performance of vehicle- versus estradiol-treated meadow voles on a probe trial in the Morris water maze in Experiment 8. White bars depict the drug-free performance of voles treated with vehicle over Days 6-10 and black bars depict the data of voles treated with estradiol over the same period. Estradiol-treated voles spent significantly more time in the training quadrant than did vehicle-treated voles (p<0.015).  5.4 D I S C U S S I O N  We designed our study to specifically test whether estradiol influences the survival o f new neurons, independent o f any effect on the proliferation o f progenitor cells by administering our  122 treatment at a time when progenitor cells would have completed mitosis. W e found that relative to vehicle, estradiol approximately doubled the number o f 16 d old neurons i n the dentate gyrus of adult male meadow voles but only when administered over Days 6-10 after the new neurons are born. Estradiol did not influence the differentiation o f new daughter cells (approximately 60% differentiated into neurons across groups) but enhanced neurogenesis by increasing the number o f young neurons that survived 16 d i n the dentate gyrus o f adult meadow voles. Furthermore, estradiol-treated voles outperformed vehicle-treated voles on a probe trial i n the Morris water maze despite both groups acquiring the task and the post-probe trial reversal training trials at the same rate. Because the voles were trained and tested hormone free in the Morris water the improved retention trial performance observed could be related to the estradiol-induced increase i n young neuron number. Our results show that estradiol does indeed promote the survival o f young neurons born in the dentate gyrus o f adult meadow voles and that this change i n neuron number did not influence learning, but was related to improved hippocampus-dependent memory.  Estradiol influences the survival of neurons in various systems  This study shows that estradiol influences the survival o f new granule neurons. Relative to vehicle, estradiol approximately doubles the number o f new granule neurons surviving 16 days i n the dentate gyrus o f adult male meadow voles. This is consistent with our previous work showing that the ratio o f new cells surviving 5 weeks relative to 2 h was higher in the dentate gyrus o f adult female meadow voles with chronically high versus l o w endogenous estradiol levels (Ormerod and Galea, 2001). However in the previous study, estradiol level was related to rates o f cell proliferation as more dividing progenitors were observed i n the dentate gyri o f the low estradiol females compared to high estradiol females 2 h after B r d U was injected. Because females with chronically high estradiol had more cells surviving 5 weeks,  123 despite having fewer new cells produced initially, estradiol likely enhanced the survival o f young cells i n that studyl. In the current study all animals presumably had the same number o f proliferating progenitor cells at the time o f B r d U injection (they were castrated and housed in standard conditions) and no treatment was administered until at least 24 h after B r d U was injected. Because progenitor cells complete division i n approximately 24 h (Cameron and M c K a y , 2001), we can conclude that i n this study, estradiol enhanced neurogenesis by increasing the survival o f new granule neurons independent o f an increase in the number o f progenitor cells undergoing mitosis. Estradiol enhances the survival but not production o f new neurons i n the higher vocal centre ( H V C ) o f the adult songbird forebrain (Hidalgo et al., 1995; Burek et al., 1995; Burek et al., 1997). Recent work has shown elegantly that estradiol induces angiogenesis in the adult songbird H V C and by upregulating the expression o f vascular endothelial growth factor ( V E G F ) , its receptor V E G F - R 2 / Q u e k l and induces brain-derived neurotrophic factor ( B D N F ) expression i n endothelial cells which promotes the survival o f migrating neurons (Loissant et al., 2002). Estradiol could influence the survival o f new granule neurons produced in the dentate gyrus o f adult voles v i a a similar mechanism. In the hippocampus o f adult rats and voles, B D N F and its receptor T r k B is widely expressed (Barbacid, 1994; Lindsay et al., 1994; McAllister et al., 1999; L i u et al., 2001) and estradiol significantly increases B D N F m R N A expression most prominently in the dentate gyrus (Gibbs, 1998; L i u et al., 2001). Palmer and colleagues (2000) have shown that angiogenesis and neurogenesis occur concurrently in the dentate gyrus o f adult female rats substantiating that estradiol could enhance the survival o f new neurons produced there i n the same manner observed i n the avian H V C . Future work could investigate the link between angiogenesis/neurogenesis to determine whether estradiol upregulates B D N F i n endothelial cells to influence the survival o f new cells in neurogenic regions o f the adult mammalian central nervous system.  124 Interestingly, the survival-promoting effect o f estradiol i n male meadow voles is temporally discrete because the number o f new granule neurons is increased when estradiol is administered 6-10 days but not 1-5 days or 11-15 days after the neurons are produced. Hastings and Gould (1999) have shown that young granule neurons extend their axons between 4 and 10 days after birth. Thus, the time when new granule neurons are extending their axons is coincident with the time that these new neurons appear receptive to estradiol's survivalpromoting effect. Developmentally, the estrogen receptor subtype a is expressed transiently in various brain areas including the hippocampus thereby promoting neuronal survival and growth as well as neurite extension (Kawata, 1995; Beyer, 1999). Moreover, during development estradiol enhances the activity o f survival promoting neurotrophins and most prominently, B D N F and its receptor T r k B m R N A in the dentate gyrus (McAllister et al., 1999; Gibbs, 1998; Davies, 1994). Both B D N F and T r k B are expressed developmentally at times associated with maximal neuronal growth, differentiation and synaptogenesis (Davies et al., 1994). Thus, estradiol could enhance the survival o f new granule neurons by providing trophic support, possibly by either increasing growth factor production in the microenvironment o f new granule neurons or by inducing the expression o f growth factor receptors i n new neurons. In addition to increasing neuronal survival v i a growth factors, estradiol has also been shown to prevent the death o f hippocampal neurons both in vitro and in vivo. Estradiol rescues cultured hippocampal neurons from oxidative stress-, excitotoxicity- and P-amyloid toxicityinduced death (Goodman et al., 1996; Green et al., 1996) and decreases cell death in the hippocampus o f rats that undergo experimentally induced ischemia and excitotoxicity (Hall et al., 1991; Simpkins et al., 1997; Dubai et al., 1998; Azcoitia et al., 1999). Thus, estradiol could have increased the number o f 16-day old neurons in the present study by decreasing the expression o f proteins associated with cell death. For example, estradiol has been shown to  125 increase the expression o f negative cell death regulators such as B c l - 2 i n adult neurons in vivo (Garcia-Segura et al., 1998) and B c l - X L in cultured hippocampal neurons (Pike, 1999). In fact, the bcl-2 promoter contains several putative estrogen response elements with which estradiol could directly interact to influence transcription (Teixeira et al., 1995). Our results do suggest that estradiol influenced the death o f young neurons. First, we found that the number o f pyknotic cells was significantly lower in voles treated with estradiol on either Days 1-5 or Days 6-10 days after B r d U injection (but not Days 11-15) suggesting that prolonged (5 d) exposure to estradiol reduces the number o f dying cells and that the effects may be somewhat delayed and temporally discrete. Second, although we did not note any BrdU-labelled pyknotic cells, we did note that many o f the pyknotic cells were located near or i n the subgranular zone that harbours presumably young neurons (Wang et al, 1999; van Praag et al., 2002).  Estradiol regulates different components of neurogenesis that occurs in adulthood  In addition to influencing the survival o f new granule neurons, estradiol dynamically regulates the production o f new neurons i n the adult rodent dentate gyrus (Tanapat et al., 1999; Galea and M c E w e n , 1999; Ormerod and Galea, 2001; Ormerod et al., 2003). Specifically, more new neurons are produced i n the dentate gyrus o f adult female rats on the day o f proestrus (when estradiol levels are highest) than on the days o f estrus or diestrus and an ovariectomyinduced decrease i n the production o f new neurons can be reversed by estradiol administration (Tanapat et al., 1999). W e found previously that while short-term (4 hour) exposure to estradiol increases the number o f new neurons produced longer exposure (48 hour) decreases the number o f new neurons produced i n the dentate gyrus o f both adult female meadow voles and rats and that the decrease is due, at least i n part, to estradiol-stimulated adrenal activity (Ormerod and Galea, 2001; Ormerod et al., i n press). More new cells are observed i n the dentate gyrus o f reproductively inactive (low estradiol) versus reproductively active (high estradiol) adult  126 female meadow voles (Galea and M c E w e n , 1999; Ormerod and Galea, 2001). Interestingly, the number o f BrdU-labelled cells observed is elevated in the rostral migratory stream (but not dentate gyrus) o f adult female prairie voles with high versus low estrogen levels (Smith et al., 2001; Fowler et al., 2002). Prairie voles and meadow voles distinctly differ i n their affiliative behaviour, spatial behaviour including space use in their native environments and hippocampal volume, indicating that species-specific changes i n neurogenesis may support species-specific behaviors. Therefore, estradiol dynamically influences neurogenesis i n the dentate gyrus o f adult rodents by first increasing and then decreasing (via adrenal steroids) the production o f new granule neurons and by increasing their survival. Intriguingly, estradiol influences the survival o f new neurons in the avian song circuit and the rodent hippocampus and influences the production o f new neurons i n the rodent hippocampus but not the avian song circuit (Hidalgo et al., 1995, Burek et al, 1995; 1997; Tanapat et al., 1999; Ormerod et al., 2003; Loissant et al., 2002). Comparative studies investigating differences i n the localization o f estradiol-induced mitogenic factors between the species may provide insight into the mechanisms that control the proliferation o f adult neural stem cells i n general.  New neurons have functional  characteristics  N e w neurons are produced i n the dentate gyrus throughout adulthood in all mammalian species studied (see Ormerod and Galea, 2001 for review) including humans (Eriksson et al., 1998). In fact, several thousand new cells are produced daily i n the dentate gyrus o f adult rats (Cameron and M c K a y , 2001). M a n y o f the new cells produced within the dentate gyrus extend axons through the mossy fibre pathway within 4-10 days to the C A 3 region (Stanford and Trice, 1988; Markakis and Gage, 1999; Hastings and Gould, 1999), express immature and then mature neuronal markers (Cameron et al., 1993b; Palmer et al., 2002; Tanapat et al., 1999; for example) and acquire synapses on their dendrites and soma (Kaplan and Hinds, 1977; Kaplan  127 and B e l l , 1984). Electrophysiological studies i n rats have shown that presumably young granule neurons with cell bodies located near the neurogenic subgranular zone exhibit greater plasticity than presumably older neurons located deeper in the granule cell layer (Wang et al., 2000). Recent work using a GFP-tagged retrovirus to birth-date granule neurons produced in the dentate gyrus o f mice found electrophysiological responses typical o f mature granule neurons 4 weeks after infection and a mature granule neuron morphology (dendritic complexity and similar spine counts) 4 months after infection (van Praag et al., 2002). Taken together, this evidence suggests that new granule neurons could rapidly influence and be influenced by activity within the hippocampus. Most attention has been focused upon the role o f the hippocampus i n spatial processing, although evidence suggests a role for the hippocampus in declarative memory (Cohen and Eichenbaum, 1993; Bunsey and Eichenbaum, 1996), working memory (Olton, 1983), stimulusstimulus learning (McDonald and White, 1993), and data-based memory (Kesner, 1998). Destruction o f the hippocampal formation impairs rats' performances i n the spatial (platform hidden) but not the nonspatial version (platform visible) o f the Morris water maze (Kelsey and Landry, 1988; Morris et al., 1982; Morris et al., 1990; Schenk and Morris, 1985; Taube et al., 1992) and in spatial versions o f the radial arm maze (Olton and Samuelson, 1976; Jarrard, 1983; Jarrard et al., 1984). Indeed, the performance o f rats with fairly specific granule neuron depletion is impaired on hippocampus-dependent tasks (Sutherland et al., 1983; Whishaw et al., 1987; M c L a m b et al., 1988; McNaughton et al., 1989; Nanry et al., 1989; Armstrong et al., 1993; Conrad and Roy, 1993; Schuster et al., 1997). Recently, Xavier and colleagues (1999) reported that fairly complete and specific colchicine-induced granule neuron destruction severely impaired performance across acquisition trials and on a probe trial in the standard Morris water maze task.  128 Neurogenesis and hippocampus-dependent  behaviour in adulthood  A few studies have shown hippocampus-dependent behaviour can increase young neuron number and that a change i n young neuron number can alter the ability o f rats to learn some hippocampus-dependent tasks. Gould and colleagues (1999) found that rats trained in trace eye-blink conditioning trials or standard Morris water maze training trials had more new (7-14 day old) dentate gyrus granule neurons (but not subventricular zone-produced neurons) relative to rats trained in delay eye-blink conditioning or cued water maze training trials. Both standard Morris water maze training and trace eyeblink-conditioning requires an intact hippocampus whereas cued water maze training and delay eyeblink-conditioning do not (Morris et al., 1982; Solomon et al., 1986; Moyer et al., 1990; Clark and Squire, 1998). Another study found some evidence to suggest that rats exhibiting good performance in the Morris water maze have more young neurons surviving than rats exhibiting poorer performance, although some rats trained on the cued platform version o f the task had as many new cells as the good learners (Ambrogini et al., 2000). Further evidence that new neurons participate in successful hippocampus-dependent performance has been derived from studies utilizing the cytostatic agent methylazoxymethanol ( M A M ) to halt the proliferation o f progenitor (and other) cells. M A M treatment for 14 days (but not 6 days) prior to training impairs trace eyeblink conditioning but delay eye-blink conditioning (Shors et al., 2001). Recent work has shown that M A M administration impairs performance on another associative task, trace fear conditioning, but does not influence performance on Morris water maze acquisition trials, contextual fear conditioning trials or on elevated plus maze exploration (Shors et al., 2002). Taken together, this evidence suggests that more young neurons are found i n the dentate gyri o f animals that engage i n hippocampus-dependent learning and that young neurons participate in  129 hippocampus-dependent associative tasks but not necessarily acquisition o f spatial information used for navigation. Interspecies comparisons have also shown that baseline neurogenesis and performance in hippocampus-dependent tasks are related. Strains o f mice with high levels o f baseline neurogenesis (C57BL/6) outperform strains with low levels (129/SvJ and D B A / 2 , for example) on standard Morris water maze training trials (Kempermann et al., 1997; Kempermann and Gage, 2002). Interestingly, seasonal changes i n baseline neurogenesis may relate to seasonal changes i n spatial ability. Adult female meadow voles have higher rates o f cell proliferation, establish greater territory sizes in their natural habitats and exhibit better performance across Morris water maze training sessions during the non-breeding compared to the breeding season (Sheridan and Tamarin, 1988; Galea et al., 1995; Galea and M c E w e n , 1999; Ormerod and Galea, 2001). Combined, this evidence suggests high-level baseline neurogenesis is associated with good performance across training trials i n the Morris water maze and low-level baseline neurogenesis is associated with poorer performance i n the task. Here, we show that an increase i n young neuron number does not influence performance over acquisition trials but does improve performance i n the probe trial in the Morris water maze. Voles injected with estradiol over Days 6-10 post-BrdU (but were trained and tested hormone free) exhibit better memory for the maze location o f the maze that previously housed the platform during probe trials than vehicle-treated males, despite similar rates o f learning on training trials. Importantly, both groups o f animals exhibited asymptotic performance across training trials indicating that they had learned the task to the same degree, before the probe test was administered validating the effect on retention that we observed. Many interpretations o f probe trial performance have been proposed. For example, the probe test has been proposed to measure either perseveration (or behavioural inflexibility; Eichenbaum et al., 1990; 1992) or strength o f spatial learning (or retention; Whishaw, 1985).  130 Because we observed no difference between groups on reversal training trials, the idea that the estradiol-induced increased number o f young granule neurons is associated with improved spatial memory is supported. Our observation that performance between estradiol- and vehicletreated meadow voles across training trials complements the work o f Shors and colleagues (2002) showing unimpaired performance across training trials i n the Morris water maze i n rats when the number o f young neurons is drastically reduced. Both lines o f evidence suggest that new granule neurons are not necessary for successful performance across acquisition trials o f the Morris water maze. However, our data demonstrate that an increased number o f young neurons can improve performance on water maze probe trials. These data are interesting given that granule neurons appear necessary for precise spatial search strategy in the Morris water maze (Xavier et al., 1999) and young granule neurons appear to be especially plastic (Wang et al, 1999). In summary, we show that relative to vehicle, estradiol increases the survival o f new neurons and the estradiol-induced increase in new neuron number are related to improved memory in the Morris water maze.  5.5 I M P L I C A T I O N S  We, and others, have shown that estradiol regulates the production (by increasing and then decreasing progenitor cell proliferation) and survival o f new granule neurons i n the rodent dentate gyrus during adulthood. Our understanding o f how to control neurogenesis may be facilitated by discovering the mechanisms by which estradiol dynamically regulates neurogenesis within the adult rodent dentate gyrus. Furthermore, previous work has shown that some forms o f hippocampus-dependent learning requires the presence o f young granule neurons (Shors et al., 2001, 2002) and increases the survival o f young granule neurons (Gould et al., 1999; Ambrogini et al., 2000). This study shows that an increased number o f new neurons improves a facet o f hippocampus-dependent behaviour; memory for a platform  131 position i n the standard Morris water maze task. The present study adds to our understanding o f how estradiol influences components o f neurogenesis in the adult mammalian hippocampus and how changes i n neuron number influence features o f hippocampus-dependent behaviour. B y discovering the mechanisms by which estradiol mediates its influence upon different facets o f adult mammalian neurogenesis, insight about how to control the process may be achieved.  132  CHAPTER 6 G E N E R A L DISCUSSION Perhaps because the field o f adult stem cell biology is young, the effects o f a single factor upon different components o f neurogenesis have been relatively unexplored. However, to argue that a factor influences neurogenesis, its cumulative effects upon progenitor cell proliferation, daughter cell differentiation and survival o f young neurons should be determined to ascertain i f that factor actually does alter net neurogenesis. Combined, the experiments i n this thesis take the approach o f investigating the effects o f estradiol on cell proliferation, the differentiation o f daughter cells and the survival o f young granule neurons i n the dentate gyrus o f adult rodents. Estradiol first increased (within 4 h) and then decreased (within 48 h), partially by stimulating adrenal activity, cell proliferation i n the dentate gyri o f adult female rats and meadow voles. Estradiol did not alter the differentiation o f daughter cells. Estradiol enhanced the survival o f young neurons i n the dentate gyri o f adult male meadow voles and the estradiol-induced increase i n young neuron number was related to improved retention (but not learning) in the spatial Morris water maze task. A general summary o f the experimental results is presented  .  first and then their implications for understanding the role o f neurogenesis i n normal hippocampus-dependent function as well as their potential for neuronal replacement strategies are discussed.  Reproductive status and estradiol influence cell proliferation  Reproductive status influenced cell proliferation i n the dentate gyri o f adult laboratoryreared female meadow voles. Chapter 2 revealed that reproductively inactive females (with low estradiol levels) had significantly more proliferating cells in their dentate gyri than reproductively active females (with high estradiol levels; Tables 1 and 3). This finding  133 replicated Galea and M c E w e n ' s (1999) work showing that cell proliferation is elevated in the dentate gyri o f non-breeding w i l d adult female meadow voles (with l o w estradiol levels) relative to breeding w i l d females (with high estradiol levels). Importantly, the potentially confounding variables o f age and activity level (Kuhn et al., 1996; Seki and A r a i , 1995; Montaron et al., 1998; Cameron and M c K a y , 1999; van Praag 1999a; 1999b) were controlled in the laboratory-reared sample to confirm that the seasonal variable influencing cell proliferation in the dentate gyri o f adult female meadow voles is reproductive status. In both studies, the finding that serum estradiol level was correlated negatively with labelled cell density suggests that estradiol level mediated the effect o f reproductive status on cell proliferation i n both samples (Chapter 2 and Galea and M c E w e n , 1999). In fact, the effect o f shifting reproductive status from inactive to active could be mimicked by exposing reproductively inactive females to estradiol for 48 h (Table 1). In fact, Chapter 2 suggested a novel dynamic effect o f estradiol on cell proliferation i n the adult rodent dentate gyrus. Although the density o f proliferating (BrdU-labelled) cells in the dentate gyri o f adult gonadally intact adult female meadow voles decreased within 48 h, it increased within 4 h o f a single injection o f estradiol, relative to the density o f proliferating cells observed i n the dentate gyri o f reproductively inactive females. The data o f Chapters 3 and 4 verified that estradiol differentially influences cell proliferation i n the dentate gyri o f adult female rodents. The total number o f proliferating (BrdU-labelled cells) cells i n the dentate gyri o f ovariectomized adult female rats (Chapter 3; Figure 4) and meadow voles (Chapter 4; Figure 9) increases within 4 h but decreases within 48 h o f a single injection o f estradiol versus vehicle. Chapter 4 demonstrated that estradiol-induced increases i n other ovarian hormones such as progesterone (see Nubbemeyer, 1999) did not contribute to the changes in labelled cell number observed after 4 versus 48 h o f exposure to estradiol i n intact voles, because the voles in Chapter 4 were ovariectomized. Importantly, Chapters 2, 3 and 4 combined demonstrate that  134 the differential effects o f estradiol on dentate cell proliferation is robust across species (voles and rats) despite different methods used to analyse cell proliferation (labelled cell density versus stereological estimate o f total labelled cell number). The differential effects o f estradiol on cell proliferation i n the adult rodent dentate gyrus appeared to be duration- rather than dose-dependent. Serum estradiol levels i n rats exposed to estradiol for 48 h were intermediate to those o f rats exposed to estradiol for 4 h and to vehicle (Chapter 2; Table 6). In addition, intact female voles exposed to estradiol for 48 h had serum estradiol levels intermediate to females exposed to estradiol for 4 h and reproductively inactive females (Chapter 2; Table 3). If the differential effect o f estradiol were dose-dependent, then I would expect that the number/density o f proliferating cells i n the dentate gyri o f female rats and voles exposed to estradiol for 48 h to be intermediate between those found in the dentate gyri o f females exposed to estradiol for 4 h and to vehicle/no injection. O f course, this hypothesis could be directly tested i n an experiment designed to keep estradiol dose constant while varying duration. For example, i f BrdU-labelled cell number increased following one but then decreased following two daily injections o f estradiol versus vehicle, then effect could clearly be interpreted as duration-dependent. In fact, a recent study i n our laboratory demonstrated that prolonged exposure to estradiol suppresses cell proliferation in the dentate gyri o f adult female rats. Regardless, the differential effects o f estradiol on cell proliferation reported in this thesis are consistent with the findings o f other studies. C e l l proliferation increases in the dentate gyri o f adult female rats and voles exposed to high-level estradiol for short durations (2-4 h) before B r d U is injected (Tanapat et al., 1999; Banasr et al., 2001; Chapters 2, 3 and 4). Similarly, estradiol levels rise on the afternoon o f proestrus i n rats (Buckingham et al., 1978) and cell proliferation increases in the dentate gyri o f rats that are injected with B r d U on the afternoon o f their proestrous phase (Tanapat et al., 1999). C e l l proliferation decreases i n the dentate gyri o f  135 adult female rats and voles exposed to high-level estradiol for longer durations (48 h) before B r d U is injected (Chapters 2, 3 and 4). Most adult female meadow voles trapped during the breeding season exhibit signs o f behavioural estrus (perforated vagina and pregnancy; Galea and M c E w e n , 1999) indicating that their estradiol levels must have risen at least 12-16 h before capture (Lee et al., 1970). These voles have reduced rates o f cell proliferation in their dentate gyri relative to females trapped during the breeding season (Galea and M c E w e n , 1999). Taken together, published studies investigating the relationship between estradiol and neurogenesis support the findings o f this thesis that short-term exposure to estradiol increases and longerterm exposure to estradiol decreases cell proliferation i n the dentate gyri o f adult rodents.  Estradiol partially suppresses cell proliferation  by stimulating adrenal  activity  Estradiol increases cell proliferation i n the dentate gyri o f adult female rats by increasing serotonin synthesis (Banasr et al., 2001) and Chapter 3 demonstrated that estradiol stimulates adrenal activity to suppress cell proliferation in the dentate gyri o f adult female rats, because adrenalectomy eliminated the suppression in cell proliferation observed 48 h after estradiol was administered (Figure 5). Adrenalectomy would also likely eliminate or reverse the estradiolinduced suppression i n cell proliferation in the dentate gyri o f adult female meadow voles because estradiol treatment for 48 h (but not 4 h) increases their adrenal masses (Table 2) and suppresses cell proliferation (Table 1), and future work could examine this possibility. Because the estradiol-induced suppression in cell proliferation i n the dentate gyri o f adult rats was eliminated but not reversed by adrenalectomy, I hypothesized that estradiol may work through a factor in addition to adrenal steroids to suppress cell proliferation within 48 h. The hypothesis that estradiol may stimulate N M D A receptors to influence cell proliferation within either 4 or 48 h was formulated from evidence showing that N M D A receptors work downstream from adrenal steroids to suppress cell proliferation (Cameron et al.,  136 1998) and that estradiol can increase both the sensitivity and number o f N M D A receptors (Weiland, 1992; Gazzaley et al., 1996). In essence, estradiol should potentiate the effect o f an N M D A receptor agonist-induced decrease and antagonist-induced increase on cell proliferation in the rodent dentate gyrus, i f this hypothesis was correct. To expand upon the findings o f previous work showing that N M D A receptor activation suppresses and N M D A receptor blockade enhances cell proliferation in the dentate gyri o f adult rats and tree shrews (Cameron et al, 1994; Cameron et a l , 1995; Gould et al., 1997; Bernabau and Sharp, 2000; Nacher et al., 2001; Nacher et al., 2003), I tested this hypothesis using adult ovariectomized female meadow voles as subjects. A s expected, estradiol enhanced cell proliferation within 4 h and suppressed cell proliferation within 48 h i n the dentate gyri o f adult ovariectomized voles. Consistent with the effects reported in other species, N M D A receptor activation suppressed and N M D A receptor blockade enhanced cell proliferation i n the dentate gyri o f adult female meadow voles. However, estradiol did not appear to interact with N M D A receptors either following 4 or 48 h of exposure to influence cell proliferation because estradiol enhanced cell proliferation and then suppressed cell proliferation despite the presence of N M D A or M K - 8 0 1 , respectively. This result was surprising because the results o f Experiment 4 (Chapter 3) had shown that estradiol stimulated adrenal activity to suppress cell proliferation i n the adult rat dentate gyrus and previous work had shown that N M D A receptor activation works downstream o f adrenal steroids to suppress cell proliferation in the dentate gyri o f adult rats (Cameron et al., 1998). In the Cameron et al. (1998) study the effects o f low-level (via adrenalectomy) or high-level adrenal steroids (corticosterone injection) were blocked by N M D A receptor activation or inactivation, respectively (Cameron et al., 1998). To m y knowledge, the distributions o f N M D A receptors and N M D A receptor subunit sequences have not been investigated in the meadow vole and the genes encoding N M D A receptor subunits are known to vary between species (Eriksson et al., 2002; Andersson et al., 2001 for example). The expression patterns o f  137 arginine vasopressin and oxytocin receptors are known to vary drastically vole species (Insel and Shapiro, 1992; Insel, W a n g & Ferris, 1994) and N M D A receptor distribution patterns could differ between rats and voles. Nonetheless, the finding that N M D A and M K - 8 0 1 decreased and increased cell proliferation i n the dentate gyri o f adult meadow voles similarly to what has been reported i n adult rats, suggests that N M D A receptor distribution and phenotype is similar between the two species and that estradiol simply does not stimulate N M D A receptor activity to suppress cell proliferation within 48 h. Interestingly, in the paraventricular nucleus o f the hypothalamus, estradiol-induced downregulation o f ER« expression returns to control levels 48 h after estradiol is injected (Paul Shughrue, personal communication). W e are currently investigating whether estradiol mediates effects on cell proliferation in the adult rodent dentate gyrus v i a E R s .  Estradiol did not influence the differentiation of daughter cells  Estradiol did not influence the differentiation o f daughter cells when administered prior to pulse o f B r d U . In Experiment 3 (Chapter 3), regardless o f whether ovariectomized female rats were exposed to estradiol or vehicle, 60-70% o f the BrdU-labelled cells expressed the neuronal marker D C X , 25-30% expressed the neuronal marker T U C - 4 and 20-25% expressed the glial marker G F A P by 4 d B r d U injection (Table 8). T U C - 4 - i r or G F A P - i r was observed in more 4-day old versus 24-h old BrdU-labelled cells but the percentages did not vary with treatment (either estradiol or vehicle exposure for 4 or 48 h; Table 5). These data are consistent with those o f Tanapat and her colleagues (1999) who found that 60-70% o f the labelled cells in the dentate gyri o f adult rats injected with B r d U i n either proestrus or estrus expressed T U C - 4 or calbindin and approximately 15-20% expressed G F A P between 4 d and 3 weeks after birth.  138 There are differences i n T U C - 4 - i r BrdU-labelled cells reported i n Chapter 3 and by Tanapat et al. (1999) and these differences likely reflect the use o f a commercially available (Chemicon) versus homemade (Hockfield laboratory) antibody raised against T U C - 4 in the respective studies. The commercially available antibody appears lower i n affinity for either T U C - 4 or the fluorescent probe-conjugated secondary antibody used to detect it under confocal microscopy than the homemade antibody (Brandi Ormerod, unpublished data; Clive Svendsson personal communication; Martin Wojtowicz, personal communication). The important point is that the percentage o f BrdU-labelled cells either expressing a neuronal ( D B X and T U C - 4 ) or glial marker ( G F A P ) was unaffected by treatment i n both studies (hormone treatment, duration of hormone treatment, estrus cycle stage). Estradiol also did not influence the differentiation o f labelled cells when administered over either Days 1-5, 6-10 or 11-15 after a pulse o f B r d U . Chapter 5 demonstrated that 55-60% o f BrdU-labelled cells expressed the neuronal marker N S E , 60-70% expressed the neuronal marker N e u N and 12-20% expressed G F A P (Table 11) in the dentate gyri o f adult males meadow voles treated with estradiol or vehicle. The finding that estradiol did not influence the differentiation o f daughter cells in the adult rodent hippocampus is interesting because estradiol influences differentiation during development, particularly o f cells located in sexually dimorphic nucleii (see Kawata, 1995; Toran-Allerand, 1996; Beyer, 1999 for review). In line with this observation, in vitro evidence suggests that estradiol may influence the differentiation o f cells derived from the developing versus adult rodent nervous system differently. Brannvall and her colleagues (2002) found that the proportion o f adult neural progenitor derived BrdU-labelled cells that expressed either the neuronal marker p-tubulin versus G F A P did not differ i n media containing estradiol versus vehicle but the proportion o f embryonic neural progenitor derived BrdU-labelled cells expressed p-tubulin versus G F A P increased upon exposure to estadiol. In their culture system,  139 estradiol promoted a neuronal fate among daughter cells 'developmentally' but not during 'adulthood'. The potential and phenotype o f stem cells is known to change developmentally, likely because o f intrinsic changes in the responsiveness o f stem cells to growth factors (Spradling et al. 2001, Weissman et al., 2001) and adult stem/progenitor cells could become insensitive to estradiol as a differentiative cue in this manner. Interestingly, adrenal steroids did not appear to influence the differentiation o f daughter cells (Chapter 3). The percentage o f BrdU-labelled cells that expressed T U C - 4 and G F A P did not differ in the dentate gyrus o f adrenal intact (Table 5) and adrenalectomized (Table 9) adult female rats despite differences i n circulating corticosterone levels (Table 6). This result is incongruent with the findings o f Cameron and M c K a y (1999) who reported that a significantly higher percentage o f BrdU-labelled cells i n the dentate gyri of aged adrenalectomized male rats expressed T U C - 4 than i n the dentate gyri o f aged adrenal intact male rats. The same low dose of corticosterone was added to the drinking water o f adrenalectomized rats i n both studies to prevent cell death and the rats were adrenalectomized one week prior to B r d U injection in both studies, suggesting that the age or sex o f the rats used may account for the differences reported. Basal corticosterone levels do increase in aged rats (Sapolsky, 1992) and ligand-bound glucocorticoid and mineralocorticoid receptors are known transcription factors (see Meijer, 2002 for review). Determining the changes in potential and phenotype o f adult neural progenitor cells not only between development and adulthood, but across adulthood would be interesting given that adrenal steroids appear to influence the differentiation o f cells in the aged but not young adult rodent dentate gyrus. In theory, prolonged exposure to elevated glucocorticoid levels could alter transcription o f factors that influence the intrinsic responsiveness o f progenitor cells to extrinsic cues guiding differentiation i n aged or even between male and female rats.  140 Estradiol enhances the survival of young granule neurons  The experiments described i n Chapter 2 suggested that estradiol enhances the number o f new cells that survive i n the dentate gyri o f adult female meadow voles. The proportion o f [ H]thymidine-labelled cells that survived 5 weeks relative to the number o f BrdU-labelled 3  cells observed at 2 h was higher i n the dentate gyri o f reproductively active versus inactive females (Table 4 versus Table 1). Because reproductive status and therefore serum estradiol level differed between the groups prior to and after the administration o f cell synthesis marker in each experiment and serum estradiol remains elevated for at least 22 days i n reproductively active voles (Seabloom, 1985), both cell proliferation and the survival o f new cells were likely affected i n Chapter 2. Therefore, Experiment 7 (Chapter 5) was designed to specifically investigate the effect o f estradiol on the survival o f young granule neurons by injecting B r d U at least 24 h (the time required for adult progenitor cells to complete 1 mitotic division; Cameron and M c K a y , 1999) before treating the animals with estradiol or vehicle. This design revealed that estradiol approximately doubles the number o f 16 day-old neurons i n the dentate gyri o f adult male meadow voles but only when administered over Days 6-10 (but not Days 1-5 or Days 11-15) post B r d U label. I have since replicated this finding i n adult female meadow voles, although the effect appears more robust and not as temporally discrete i n females versus males (Figure 17). The more robust and temporally less discrete estradiol-induced enhancement o f survival among young granule neurons i n the dentate gyri o f adult female versus male meadow voles could reflect dimorphisms i n the expression o f estrogen or other receptors in the adult male and female rodent central nervous system. The number o f ERct-ir cells is similar i n the hippocampus o f gonadectomized adult male and female rats (Weiland et al., 1997). However, sex differences i n E R p expression that have not yet been investigated in the adult rodent hippocampus could account for the differential effect o f estradiol on granule neuron survival  141  25000 "i  ** j»  20000  E Z  O  15000  "O JD  CD  -O  ro i  ZD TD  10000  m ro o  5000  Condition  Figure 17. Stereological estimates of BrdU-labelled cells in the dentate gyrus of adult female meadow voles following the administration of estradiol or vehicle. The white bar depicts the data of female voles treated with vehicle over Days 1-5, 6-10 or 11-15 after BrdU (data were collapsed as the number of BrdU-labelled cells in vehicle groups did not statistically differ). The gray bars depict the data of voles treated with estradiol either over either Days 1-5, 6-10 or 11-15 after BrdU was injected. Relative to vehicle, estradiol increased the number of BrdU-labelled cells that expressed a neuronal protein (NSE or NeuN) in the dentate gyrus of adult female meadow voles when administered over Days 11-15 post BrdU and tended to increase neuron number when administered over Days 1-5 and Days 6-10. **denotes p<0.05  observed in the dentate gyri o f adult male versus female meadow voles. Estrogen increases the survival o f migrating neurons i n the adult avian songcircuit and in vitro work suggests that estradiol mediates this effect by increasing B D N F expression i n endothelial cells (Hidalgo et al., 1995; Loissant et al., 2002). Interestingly, neurogenesis i n the adult rodent dentate gyrus is associated with angiogenisis (Palmer et al., 2000). Whether angiogenesis is specifically related to increased proliferation or the survival o f young neurons i n the adult rat dentate gyrus has not been investigated but estradiol could promote cell survival similarly i n the adult rat and avian C N S . Estradiol enhances B D N F expression in the hippocampus o f adult female rats, most prominently in the dentate gyrus and in the olfactory bulbs o f adult female prairie voles and (Gibbs, 1998; Smith et al., 2001). B D N F increases the survival o f adult subventricular zonederived cells both in vivo and in vitro (Kirschenbaum and Goldman, 1995; Zigova et al., 1996).  142 Future work could investigate whether estradiol enhances the expression o f B D N F to promote the survival o f young granule neurons and whether differences i n either E R p \ B D N F and/or the B D N F receptor trkA in the dentate gyri o f female versus male rodents is related to the robustness and temporal discreteness o f this effect. Estradiol could increase the number o f 16-day old neurons i n the dentate gyri o f adult meadow voles by either providing trophic support or by inducing the expression o f antiapoptotic factors to reduce cell death. Potential mechanisms by which estradiol could mediate either effect are described in detail i n Chapter 5.  Net neurogenesis and behaviour in adult voles  Chapter 2 showed that although a greater proportion o f new cells survived 5 weeks versus 2 h in the dentate gyri o f adult reproductively active (with high estradiol levels) versus inactive (with l o w estradiol levels) meadow voles, the number cells at both time frames was higher i n the dentate gyri o f inactive versus active females. This finding suggests that net neurogenesis is higher i n the dentate gyri o f reproductively inactive versus active females. Kempermann and his colleagues (Kempermann et al., 1997; Kempermann and Gage, 2002) have shown that strains o f mice with high net neurogenesis (C57BL/6) outperform strains with low net neurogenesis (129/SvJ and D B A / 2 , for example) on standard Morris water maze trials. Interestingly, Galea and her colleagues (1995) have shown that reproductively inactive female meadow voles (with high net neurogenesis; Chapter 2) outperform reproductively active females (with low net neurogenesis; Chapter 2) on standard Morris water maze training trials. Seasonal changes in net neurogenesis could influence performance on this hippocampusdependent task.  143 Conclusive evidence that changes i n net neurogenesis influence the performance o f adult female meadow voles would require dissociating estradiol's effects on neurogenesis from its effects on learning (Galea et al., 2001; Holmes et al., 2002). Nevertheless, because the reproductive status o f adult voles is seasonally regulated and their behaviour i n the field has been studied relatively thoroughly, speculation about the influence o f new neurons i n the dentate gyri o f adult meadow voles on normal hippocampus function is tempting/Evidence from previous studies suggests reproductive status-induced changes i n net neurogenesis could alter spatial performance i n adult female meadow voles despite reproductive status-induced changes in estradiol. M a l e exposure increases serum estradiol and induces ovulation i n another vole species, the prairie vole (Cohen-Parsons and Carter, 1987; Smith et al., 2001). Interestingly two studies have shown that, when trapped during the breeding season, adult male meadow voles outperform females (with high-level estradiol) on a spatial land maze task but adult male and female (with high-level estradiol) prairie voles exhibit similar performance on the same task (Gaulin and Fitzgerald, 1989; Gaulin et al., 1990). Interestingly, net neurogenesis is diminished i n the dentate gyri o f reproductively active adult female meadow voles (Chapter 2) but Fowler and her colleagues (2002) have shown that cell proliferation and the survival o f new cells is similar i n the dentate gyri o f reproductively active and inactive adult female prairie voles. Combined, these studies suggest that net neurogenesis in the dentate gyri o f adult female voles could influence spatial performance independent o f changing estradiol levels. Chapter 5 demonstrated that estradiol-induced increases i n young dentate granule neuron number i n the dentate gyri o f adult male meadow voles are related to improved retention (but not learning) in the Morris water maze. In these voles, cell proliferation was similar in estradiol- and vehicle-treated voles because B r d U was administered prior to the onset o f treatment. Thus, net neurogenesis was enhanced in estradiol- versus vehicle-treated voles because estradiol enhanced the survival o f young neurons. These results are consistent with  144 previous work showing that net neurogenesis is higher i n the dentate gyri o f adult reproductively active versus inactive male meadow voles and the effect is due to enhanced survival (Ormerod and Galea, i n press). Male meadow voles increase their home range sizes during the breeding season to encompass many female ranges within their own (Madison, 1980, 1985; Gaulin and Fitzgerald, 1989). Whether reproductively active males have better retention i n the field has not been tested, but a seemingly good strategy for a polygamous breeder like the meadow vole would be to remember where he's been so that he finds a receptive rather than pregnant female. In addition, male meadow voles compete aggressively for females during the breeding season and remembering where another dominant male's territory is could prevent wounding that would diminish reproductive fitness (Clarke, 1956; Turner and Iverson, 1978). Future work could verify that the seasonal changes in net neurogenesis that occur in the dentate gyri o f male and female meadow voles are related to seasonal changes i n spatial ability measured i n the laboratory and space use in the field. O f course, theories about the functional role o f neurogenesis constitutive to the adult C N S are currently speculative. O n one hand, Alvarado and Newmark (1998) believe the phenomenon to be a "remnant o f evolution from more primitive organisms like planaria or fish in which organ renewal affords an advantage i n adverse environments". O n the other hand, Gage (2000) has argued that specific C N S regions retained the capacity to produce neurons because new neurons assist normal functioning, like learning and memory in the case o f the hippocampus. Structural similarities among the C N S areas i n which neurogenesis occurs postnatally could be taken as evidence that adult neurogenesis is a vestigial phenomenon. For example, new neurons are only added in significant numbers postnatally to the granule cell layers the olfactory bulbs, dentate gyrus and for a short time the cerebellum (2-3 weeks; Lois and Alvarez-Buylla, 1993; Cameron et al., 1993; Cameron and M c K a y , 2001; Altman and Bayer, 1997). These areas are archicortical, or the evolutionarily oldest mammalian cortex  145 (Reiner, 1993). Nonetheless, new granule neurons produced i n the dentate gyri o f adult rodent electrophysiologically resemble mature granule neurons (Wang et al., 2000; Snyder et al., 2002; van Praag et al., 2002), are influenced by hippocampus-dependent learning (Gould et al., 1999) and participate i n some forms o f hippocampus-dependent learning (Shors et al., 2001, 2002; Chapter 5), suggesting that they assist normal hippocampus function. In fact, the theories described by Alvarado and Newmark and by Gage are not mutually exclusive. A trait can evolve and persist after speciation because it is still adaptive in the descendent species. For example, penguins are birds that cannot fly but that swim exceedingly well because o f their wings.  Estradiol-induced  changes in components of neurogenesis and their implications for neuronal  replacement  Neurogenesis occurs constitutively in the dentate gyri o f both humans and rodents (Eriksson et al., 1998; Cameron et al., 1993; Experiments 1-7). Therefore, discovering the mechanisms by which neurogenesis is controlled i n the adult rodent dentate gyrus may profoundly and imminently influence neuronal replacement strategies for human neurodegenerative disease or neurotrauma. Bjorkland and Lindvall (2000) have proposed that the symptoms o f neurodegenerative disease or neurotrauma could be alleviated by replacing lost neurons with new neurons that establish the appropriate efferent and afferent connections and/or by introducing cells genetically engineered to secrete growth factors or neurotransmitters to promote the survival or regeneration o f existing neurons. The experimental data o f this thesis suggest that estradiol treatment could improve the success o f both approaches. Estradiol treatment could increase net neurogenesis by enhancing both the proliferation of progenitor cells and the survival o f young neurons i f the suppressive effect o f estradiol on  146 cell proliferation is antagonized. Excessive glucocorticoid levels i n Cushing's disease patients have been successfully decreased with adrenolytic compounds such as metyrapone and ketoconazole (see Morris and Grossman, 2002 for review). Treatment that combines estradiol with metyrapone or ketoconazole could therefore sustain elevated rates o f progenitor cell proliferation in the dentate gyrus and enhance the survival o f new granule neurons. This approach would most imminently improve the outcome o f neuronal replacement strategies that seek to manipulate neuron production i n the dentate gyrus. Combined estradiol/adrenolytic treatment could be used to increase net neurogenesis in extradentate C N S areas as well. Cells produced in the subventricular zone are known to migrate several m m to the olfactory bulbs (Lois and Alvarez-Buylla, 1993; Rousselot et al., 1995), demonstrating that the potential for directed long-distance migration i n the adult mammalian C N S exists. If the cues directing the migration o f neuroblasts were discovered, then estradiol/andrenolytic-induced increased numbers o f neuroblasts produced i n the dentate gyrus or other areas could potentially be directed to migrate into affected C N S areas and their rate o f survival could be improved. Interestingly, recent evidence suggests that following ischemic injury, cells that differentiate into neurons migrate from the hippocampal periventricular zone into damaged the C A I region and dentate gyrus (Nakatomi et al., 2002) and estradiol/adrenolytic treatment could enhance this process. Targeted cell death induces neurogenesis i n nonneurogenic regions o f the C N S (Magavi et al., 2001) and progenitor cells are located in non-neurogenic regions o f the adult rodent and human C N S (Palmer et al., 1995,1997; Shihabuddin et al., 1997; Kukekov et al., 1999; R o y et al., 2000; Aresenijevic et al., 2001), suggesting that neurogenesis could be induced throughout adult mammalian C N S . Targeted cell death is not a reasonable method o f increasing neuron number in affected C N S areas, but when those mechanisms mediating the effect o f targeted cell death on neurogenesis are discovered, combined estradiol/adrenolytic treatment could  147 potentially enhance both the proliferation o f progenitor cells i n normally quiescent C N S regions and promote the survival o f their progeny. Combined estradiol/adrenolytic treatment could also improve the success o f transplant technology. For example, increasing the proliferation cultured progenitor cells that are transplanted in the adult C N S as well as enhancing the survival o f their progeny would presumably increase the net number o f neurons produced. When cultured adult hippocampal progenitor cells are transplanted into various brain regions, a greater percentage o f the progeny die than survive (Suhonen et al., 1996). Estradiol/adrenolytic treatment could increase net neurogenesis by increasing the proliferation o f these cells in situ and by enhancing the number of their progeny that survive. Because the progeny o f cultured progenitor cells only differentiate into neurons when transplanted into neurogenic regions (Gage et al., 1995; Suhonen et al., 1996), this treatment would need to be combined with a factor that targets the differentiation o f progeny into a neuronal phenotype. Alternatively, estradiol could be administered to enhance the survival o f more differentiated cells that are transplanted into nonneurogenic C N S regions. O f course, the effects o f estradiol on the proliferation o f progenitor cells derived from different C N S areas and on the survival o f different neuronal phenotypes would need to be determined to ascertain whether any o f these approaches would be feasible.  Summary and overall  implications  The purpose o f this chapter was to integrate the experimental data o f this thesis with previous findings to discuss the influence o f estradiol over components o f neurogenesis in the adult rodent dentate gyrus, discuss the potential consequences o f estradiol-induced changes in neurogenesis for normal hippocampus function and to discuss the implications o f the experimental findings for potential neuronal replacement strategies. To this end, the overall conclusions o f the discussion are as follows.  148 1) Estradiol influences some but not all components o f neurogenesis constitutive to the adult rodent dentate gyrus. Estradiol first increases and then decreases progenitor cell proliferation in the dentate gyri o f adult female rats and meadow voles, demonstrating that the effect is robust across species with different reproductive strategies. Estradiol does not influence the differentiation o f daughter cell in the dentate gyri o f young adult female rats. However, the potential and phenotype o f neuronal progenitors may change across lifespan because estradiol influences the differentiation o f cells i n the developing C N S . Further evidence to support this notion is that adrenal steroids appear to influence the differentiation o f daughter cells in the dentate gyri o f aged rats but do not influence the differentiation o f daughter cells i n young adult rats. In part, estradiol stimulates adrenal activity to suppress cell proliferation because the suppression is reversed in the dentate gyri o f adult female rats. Although estradiol increases the affinity and upregulates the expression o f N M D A receptors and N M D A receptor activation decreases cell proliferation, estradiol does not stimulate N M D A receptors to influence cell proliferation i n the dentate gyri o f adult female meadow voles. However, N M D A receptor activation and N M D A receptor blockade respectively increases and decreases cell proliferation i n the dentate gyri o f adult female meadow voles, similar to the effects reported i n the dentate gyri o f adult rats and tree shrews. Estradiol enhances the survival of young granule neurons but the temporal specificity and robustness o f the effect appear different in the dentate gyri o f adult female versus male meadow voles.  2) Seasonally regulated changes i n net neurogenesis, spatial ability and space use may be related i n adult meadow voles. Net neurogenesis decreases (because cell proliferation decreases) in the dentate gyri o f laboratory-reared reproductively active versus inactive adult female meadow voles but is similar in the dentate gyri o f laboratory-reared reproductively active and inactive adult female prairie voles. Serum estradiol level is similarly elevated i n  149 reproductively active adult female meadow and prairie voles. Whereas a sex difference in spatial ability occurs between reproductively active male and female meadow voles, reproductively active male and female prairie voles exhibit similar spatial ability, suggesting that diminished neurogenesis i n the dentate gyri o f adult breeding female meadow voles is related to their diminished spatial ability. In contrast, net neurogenesis increases (because cell survival increases) in the dentate gyri o f laboratory-reared reproductively active versus inactive adult male meadow voles. W i l d adult male meadow voles increase their territory size during the breeding season to encompass the territories o f females, suggesting that increased net neurogenesis could be related to increased space use. Perhaps analogously, adult male meadow voles with more surviving young granule neurons in their dentate gyri (and therefore higher net neurogenesis) outperform males with lower young neuron number on a retention trial in the Morris water maze. A t least in the meadow vole, seasonal changes in net neurogenesis could be related to seasonal changes in spatial ability and space use.  3) B y eliminating estradiol's influence over adrenal activity, estradiol's stimulatory effect on progenitor cell proliferation could be prolonged. Cumulatively, estradiol-induced increases in progenitor cell proliferation and the survival o f young neurons could increase net neurogenesis i n a number o f different approaches for replacing neurons lost in the diseased or damaged adult C N S . Estradiol/adrenolytic treatment treatment could improve neuronal replacement strategies by a) enticing neuroblasts to migrate from the dentate gyrus into affected C N S regions and increasing their survival over time, b) enhancing cell proliferation induced by another factor (perhaps associated with targeted cell death) and the survival o f progeny and/or c) increasing the survival o f relatively differentiated neuroblasts that have been transplanted into non-neurogenic regions o f the adult C N S .  150 Overall, the experiments described i n this thesis explored the role o f estradiol on multiple components o f neurogenesis in the adult rodent dentate gyrus and have provided general insight about the effect o f estradiol on net neurogenesis. 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