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Age and sex differences in levels of proliferation within the zebra finch telencephalic ventricular zone

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Age and sex differences in levels of proliferation within the zebra finch telencephalic ventricular zone

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. U M I films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UM I a complete manuscript and there are missing pages, these w ill be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AGE AND SEX DIFFERENCES IN LEVELS OF PROLIFERATION WITHIN THE ZEBRA FINCH TELENCEPHALIC VENTRICULAR ZONE Copyright 2001 by Valerie DeWulf A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (NEUROBIOLOGY) December 2001 Valerie DeWulf Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U M I Number 3065781 U M I* U M I Microform 3065781 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, M l 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRAOUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by V alerie D. DeWulf under the direction of h. Dissertation Committee, and approved by aU its numbers, has been presented to and accepted by T h e Graduate School, in partial fulfillm ent of r e quirements for the degree of DOCTOR OF PHILOSOPHY Date W .U lim .. DISSERTATION COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Valerie DeWulf Dr. Sarah Bottjer ABSTRACT AGE AND SEX DIFFERENCES IN LEVELS OF PROLIFERATION WITHIN THE ZEBRA FINCH TELENCEPHAUC VENTRICULAR ZONE Brain regions associated with song learning in zebra finches are larger and contain more neurons in males than females. Differences in new neuron production, migration, cell survival, and specification may contribute to the divergent development of the song-control system. We quantified levels of mitotic activity within the telencephalic ventricular zone (VZ) of juvenile and adult birds in order to look for age and sex differences in proliferation that might contribute to the construction of song-control circuits. A single pulse of 3 H- thymidine was administered to juveniles and adults of both sexes and anim als were killed two hours later. Analysis of thymidine labeling within the VZ at the levels of Area X, die Anterior Commissure, and HVC revealed that (1) mitotic activity decreased as a function of age due to a reduction in the number o f dividing cells within die VZ and (2) sex differences in thymidine labeling occurred in small segments of the VZ at the levels of Area X and the Anterior Commissure of juveniles only. Thus, proliferative activity decreases as birds mature, and the incidence of cell division throughout the VZ becomes equivalent in both sexes such that sexually dimorphic proliferation does not occur in adulthood. In a second analysis we constructed a “map" of proliferation throughout the anterior-posterior neuraxis by sub-dividing the telencephalic VZ of juvenile males and females into segments. Our map revealed that proliferation within the VZ of both sexes is spatially differentiated suggesting that differential regulation of neurogenesis occurs throughout die VZ. In addition, sex differences in proliferation were primarily localized to the anterior half of the telencephalon. The most robust sex difference occurred within the ventral VZ at rostral levels of Area X with males having higher levels of proliferation than females. Combined, these data suggest Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that higher rales of neurogenesis in juvenile males may contribute to die growth of song- control nuclei, especially Area X. Therefore, regions of the VZ that contain sexually dimorphic proliferation in juvenile males may contain die precursors that give rise to song- control neurons. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS List of Figures iii General Introduction Vocal Learning in Songbirds I Development of the Song-control System I The Contribution of Cellular Proliferation to the Development of the Song-Control System 3 References 4 Chapter 1. Age and Sex Differences in Mitotic Activity in the Zebra Finch Telencephalon Introduction 6 Materials and Methods g Results 18 Discussion 46 References 52 Chapter 2: A Map of Proliferative Activity throughout the Proliferative Ventricular Zone of Juvenile Zebra Finches. Introduction 57 Materials and Methods 58 Results 64 Discussion 90 References 95 General Discussion Developmental Processes Involved in the Maturation of the Song-Control System 98 Proliferative Zones in the Zebra Finch Telencephalon 99 Age Differences in Levels of Mitotic Activity 100 Sex Differences in Proliferation 101 Levels of Proliferation are Higher within the W Z relative to the DVZ 103 Levels of Proliferation are Spatially Differentiated throughout VZ 105 A Map of Proliferation within the Telencephalic VZ of Zebra Finches 106 References 108 Bibliography 11 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Chapter 1. Figure 1 .1 Schematic sagital diagram of major song-control nuclei in male zebra finches Figure 1.2 Coronal sections through die zebra finch brain Figure 1.3 The area, total number of silver grains, and silver grain density in the VZ (10-pm tissue) Figure 1.4 The area, total number of silver grains, and silver grain density in the SVZ Figure 1.5 Autoradiograms of the VZ in a juvenile and an adult Figure 1.6 The area, total number of silver grains, and silver grain density in the VZ (1-pm tissue) Figure 1.7 Cellular area, total number of cells, and total number of cells labeled cells in the VZ (1-pm tissue) Figure 1.8 Percent cells labeled, and labeled cell density in the VZ (1-jim tissue) Figure 1.9 Histograms of the total number of silver grains in the W Z and DVZ of juveniles and adults at the Anterior Commissure (lO-pm tissue) Figure 1.10 Histograms o f the total number of silver grains in the W Z and DVZ of juveniles and adults at Area X (10-fim tissue) Figure 1.11 The area and total number of silver grains in the W Z and DVZ at die Anterior Commissure and Area X (10-pm tissue) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES (CONT*D) Chapter 2. Figure 2.1 Coronal sections through the zebra finch brain Figure 2.2 The DVZ, IntVZ, and W Z area throughout the telencephalon Figure 2.3 The total number of silver grains within the DVZ, IntVZ, and W Z area throughout the telencephalon Figure 2.4 Histograms of the total number of silver grains at Area X Figure 2.5 Histograms of the total number of silver grains at die Anterior Commissure Figure 2.6 Histogram o f the total number of silver grains at HVC Figure 2.7 Histograms of die total number of silver grains at die near Area X and between Area X and die Anterior Commissure Figure 2.8 Histograms of the total number of silver grains at the Anterior Commissure and HVC Figure 2.9 Wire frame reconstruction of the VZ Figure 2.10 A semi-quantitative map of proliferation in juvenile males Figure 2.11 A semi-quantitative map of proliferation demonstrating regions of sexually dimorphic proliferation Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. General Introduction Vocal Learning in Songbirds Vocal learning in songbirds provides an excellent model system to study the interactions between brain development and behavior during a restricted period of postnatal development. In zebra finches (Taertopygia guttata), males learn to sing a stereotyped song from their fathers to attract females. At the onset of vocal learning, juvenile males begin to team and memorize a tutor’s song during an initial sensory phase (20-40 days) and the song is stored in the form of a memory trace or “template” presumably within the neural substrate involved in vocal learning, the song-control system. During a sensorimotor phase, which overlaps with the sensory phase (25d-90d), juvenile males require auditory feedback to refine and match their own vocalizations to the stored memory of the tutor song. By approximately 90 days males become sexually mature and sing a stereotyped song which does not change thereafter (Immelmann, 1969; Marler and Peters, 1982; Bohner, 1990; Zann, 1990). Development of the Song-Control System The neural substrate that underlies vocal learning in males, the song-control system (Fig. 1.1), develops during a specific sensitive period of postnatal development (Bottjer et ai., 1986; Bottjer, 1997; Bottjer and Arnold, 1997; Bottjer and Johnson, 1997). There are two main pathways within the song-control system. The first consists of a “motor pathway” from high vocal center (HVC within cortex) which projects to the nucleus robustus archistriatalis (RA within motor cortex). RA then sends axons to the hypoglossal nucleus (nXIIts in the brainstem) which innervates the vocal organ (the syrinx)(Nottebohm et al., 1976; Vicario, 1994). A second “anterior forebrain pathway” consists of projections from I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HVC to Area X (within the avian basal ganglia)(Nottebohm et al., 1976). Area X in turn projects to the dorsolateral nucleus of the thalamus (DLM) which then sends projections to the lateral magnocellular nucleus of the anterior forebrain (IMAN) which innervates RA. Sex differences in the development of specific song-control regions (i.e. HVC and Area X) are the result of differential cellular proliferation, cell migration, new cell incorporation and differential cell survival. Neuroblasts that originate in the proliferative epithelium adjacent to the lateral ventricles of the telencephalon, the ventricular zone (VZ)(Goldman and Nottebohm, 1983; Alvarez-Buylla et al., 1988,1990), migrate into the brain along radial glia and become incorporated into functional circuitry (Alvarez-Buylla and Nottebohm, 1988). A larger number of new neurons are incorporated into HVC and Area X of juvenile males compared to juvenile females or adults of either sex (Nordeen and Nordeen, 1988 a, b). Burek et al. (1994) have also shown that sex differences in the number of new HVC cell cohorts are evident several days after their birth and before they finish their migration and differentiation. In addition, the incidence of neuronal death is higher in HVC of juvenile females compared to juvenile males before 20 days (Kim and DeVoogd, 1989; Burek et al., 1994, 1997) but may be equivalent thereafter. In the aggregate, these experiments suggest that differences in cell addition, cell migration, and cell death contribute to the development of the song-control system. However, it is not known if differences in levels of cellular proliferation contribute to the increased addition of new neurons to juvenile male HVC and Area X during vocal development. An increased level of cellular proliferation in juvenile males may result in more new neurons available to be incorporated into song-control regions and thus be permissive of song learning. 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Contribution of Cellular Proliferation to the Development of the Song-Control System Song-control regions of the zebra finch telencephalon continue to grow in volume and neuron number long after other brain regions have reached their adult size (Bottjer et al., 1 985). The studies herein were therefore conceived with the idea that the segments of the VZ in close proximity to song-control regions in 30 day old birds would be labeled by a marker for cell division and thereby indicate potential locations of the progenitors that give rise to song-control neurons. Levels of proliferative activity were measured within the VZ close to two song-control nuclei. Area X and HVC, and at an intermediate level of the telencephalon that does not contain song related nuclei, the Anterior Commissure (Chapter 1). Results from these initial experiments indicated that there are discrete regions of the VZ that contain higher levels of proliferative activity in juvenile males compared to juvenile females at the levels of the Area X and the Anterior Commissure. To further explore the initial finding of developmentally regulated sexually dimorphic proliferation in juveniles, we systematically compared levels of thymidine labeling throughout the telencephalic VZ of 30 day old zebra finches (Chapter 2). These data indicate that the spatial distribution of proliferative activity vary along the rostral- caudal and dorsal-ventral neuraxis. In addition, this “map” of proliferation within the telencephalic VZ indicates the exact locations of sex differences within the VZ of juvenile birds. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Alvarez-Buylla A, Nottebohm F (1988) Migration of young neurons in adult avian brain. Nature 335: 353-354. Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101-109. Bohner J (1990) Early Acquisition of song in the zebra finch. Animal Behavior 39:369- 374. Bottjer SW, Glaessner SL, Arnold AP (1985) Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J Neuroscience 5: 1556-1562. Bottjer SW (1997) Building a bird brain: sculpting neural circuits for a learned behavior. Bioessays 19: 1109-1116. Bottjer SW, Johnson F (1997) Circuits, hormones, and learning: vocal behavior in songbirds. J Neurobio 33: 602-618. Bottjer SW, Arnold A (1997) Circuits, hormones, and learning: vocal behavior in songbirds. J Neurobio 33: 602-618. Burek MJ, Nordeen KW, Nordeen EJ (1994) Ontogeny of sex differences among newly- generated neurons of the juvenile avian brain. Brain Res Dev Brain Res 78: 57-64. Burek MJ, Nordeen KW, Nordeen EJ (1997) Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death. J Neurobiol 33: 61-71. Goldman SA, Nottebohm F(1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc Natl Acad Sci U S A 80: 2390-2394. Immelman K (1969) Song development in the zebra finch and other estrilid finches. In Bird Vocalizations, RA. Hinde (Ed.) New York: Cambridge UP, pp. 61-74. 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Kim JR, DeVoogd TJ (1989) Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J Neurosci 9: 3176-3187. Nordeen KW, Nordeen EJ (1988a) Projection neurons within a vocal motor pathway are bom during song learning in zebra finches. Nature 334: 149-151. Nordeen EJ, Nordeen KW (1988b) Sex and regional differences in the incorporation of neurons bom during song learning in zebra finches. J Neurosci 8:2869-2874. Nottebohm F, Arnold AP (1976) Sexual dimorphism in vocal control areas of the songbird brain. Science 194:211-213. Vicario DS (1994) Motor mechanisms relevant to auditory-vocal interactions in songbirds. Brain Behav Evol 44: 265-278. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1. Age and Sex Differences in Mitotic Activity in the Zebra Finch Telencephalon Young male zebra finches learn to sing a close copy of their father’ s song during a sensitive period of development from -20-90 days after hatching (Bohner, 1990) whereas young females never learn to produce song behavior. The neural substrate underlying bird song, the song-control system (Fig. 1.1), is highly sexually dimorphic (Nottebohm and Arnold, 1976; Nottebohm etal., 1982; Bottjer et al., 1989). However.it is difficult to determine whether neural sex differences are the result of sexually dimorphic proliferation, migration, differentiation, or cell death (see Alvarez-Buylla and Kim, 1997 for review). H V C AREAX Fig. 1.1. Schematic sagital diagram of major song-control nuclei in male zebra finches. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Neuroblasts originate in the proliferative ventricular zone (VZ) adjacent to the lateral ventricles (Goldman and Nottebohm, 1983; Alvarez-Buylla et al., 1988,1990), migrate into the brain along radial glial processes, and become incorporated into various regions of the telencephalon (Alvarez-Buylla and Nottebohm, 1988). A larger number of these new neurons are incorporated into the High Vocal Center (HVC) and Area X of young males compared to young females (Nordeen and Nordeen, 1988a; b). The addition of new neurons to both brain regions continues into adulthood in males, though at a lower rate. Since the volume of HVC in zebra finches does not change in adulthood, it is presumed that the new neurons added to HVC represent the replacement of projection and intemeurons (Paton, et al.. 1985; Nordeen and Nordeen, 1988a). Experiments by Kim and DeVoogd (1989) and Burek et al. (1994; 1997) indicate that the incidence of pyknotic cells within HVC is greater in females than males before 20 days but may be equivalent thereafter. Burek et al. (1994) also demonstrated that sex differences in the number of new HVC cell cohorts are evident several days after their birth and before they finish their migration and differentiation. Taken together, these experiments suggest that differences in cell addition, cell death, and cell migration contribute to the development of the song-control system. The present experiment directly tested whether regions of the VZ contain higher levels of proliferation in juveniles compared to adults, and in males compared to females. We administered a single pulse of 5 H-thymidine to juvenile (30 d) and adult (>90 d) zebra finches of both sexes and killed the animals two hours later in order to examine proliferative activity uncontaminated by cell death or migration away from the VZ. Increased levels of thymidine labeling were evident at the levels of Area X, the Anterior 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Commissure, and HVC in juveniles relative to adults, and single-cell analysis indicated that higher levels of mitotic activity were due to a larger number of dividing cells in the VZ. In addition, restricted regions of sexually dimorphic cell division were observed at the levels of Area X and the Anterior Commissure in which cellular proliferation was greater in juvenile males than juvenile females. No regions of the VZ demonstrated sex differences in thymidine labeling in adults. This observation suggests that sex differences in cellular proliferation may contribute to the growth of song-control nuclei in males. Moreover, regions of sexually dimorphic proliferation within the VZ may offer clues to the location of the precursor cells that give rise to song-control neurons. MATERIALS AND METHODS Thymidine labeling and tissue preparation. Twenty-eight male and female zebra finches were taken from our breeding colony at either 30 days of age (juveniles, range 28- 32 days), or 90 days or older (adults) to measure levels of mitotic activity adjacent to the lateral ventricles within the telencephalon. Each bird received a single intramuscular injection of 3 H-thymidine (2 J pCi/g dose, specific activity 6.7 Ci/mmol; New England Nuclear or ICN) and was killed two hours later. This post-thymidine interval is short enough to effectively preclude loss of cells due to cell death or migration away from the VZ. Therefore, the incidence of thymidine labeling should provide an uncontaminated estimate of proliferative activity for the period of time that thymidine was available. All birds were overdosed with a barbiturate anesthetic (Equithesin) and transcardially perfused with avian saline followed by 2% paraformaldehyde/2% glutaraldehyde fixative. Brains were removed and bisected along the midline into two hemispheres for embedding 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (the left hemisphere in paraffin and the right hemisphere in the water-soluble plastic LR White; Ted Pella). Prior to embedding, each hemisphere was post-fixed in either buffered formalin (left hemisphere) or 2% paraformaldehyde/2% glutaraldehyde (right hemisphere) for several days. To compensate for possible variation across sex and age groups (e.g. emulsion batches, lots of ^H-thymidine) all procedures included at least one animal from each of the four groups for both the paraffin- and plastic-embedded brains. Paraffin-embedded hemispheres. Paraffin sections were used to measure overall levels of proliferation adjacent to the lateral ventricles at three different levels of the brain: Area X, the Anterior Commissure, and the High Vocal Center (HVC) in juveniles (males n = 5, females n = 6) and adults (males n = 5, females n = 5)(Fig. 1.2). Briefly, coronal sections were cut on a rotary microtome at a thickness of 10 pm, placed on chrome-alum subbed slides, and immersed in a series of xylenes and graded alcohols for paraffin removal. The slides were then dipped in nuclear track emulsion (Kodak NTB2, Eastman Kodak Co.), stored at 4°C for 3-8 weeks, developed (Kodak D19), and counterstained with thionin. Plastic-embedded hemispheres. Observation of the 10-pm tissue sections in pilot studies had revealed that the density of cells within the VZ was extremely high, making it impossible to visualize individual cells. Therefore to analyze thymidine labeling within single cells we cut serial I-pm sections from plastic-embedded hemibrains at the level of the Anterior Commissure (juvenile males n = 8, juvenile females n = 8, adult males n = 7, adult females n = 3). Approximately 2 mm of the lateral lobe of the telencephalon was trimmed and the remaining tissue was then bisected into anterior and posterior halves at the optic tectum and each brain quarter was embedded in separate capsules. Plastic- 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. embedded sections were cut in the coronal plane on a JB-4 retracting rotary microtome and processed for tissue autoradiography following the same methods used for the paraffin-embedded tissue. Analysis of autoradiograms Proliferative zones o f the zebra finch telencephalon. All analyses of cellular proliferation were performed using a computer-aided imaging system (Analytical Imaging Concepts) yoked to a microscope. Observation of thymidine-labeled cells adjacent to the lateral ventricles revealed three different proliferative zones within the zebra finch telencephalon (Fig. 1.2). The proliferative epithelium described here is what has been classically referred to as the Ventricular Zone (VZ) in mammalian brain and consists of an epithelial cell layer adjacent to the lateral ventricles (Boulder Committee, 1970). Immediately adjacent to the ventral VZ (within the striatum), a population of small, round, closely packed cells tended to include a large number of silver grains. This Sub-VZ (SVZ) appeared to be morphologically identical to the SVZ described within the developing mammalian brain (Boulder Committee, 1970). The SVZ was only apparent ventral to the dorsal medullary lamina (LMD fiber tract, which separates striatum from overlying cortex), at the levels of Area X and the Anterior Commissure (Fig. 1.2a, b) and the SVZ was never observed at the level of HVC (at this caudal level of the telencephalon the striatum and ventral VZ are gone). In addition to the VZ and SVZ, groups of thymidine-labeled cells were seen adjacent to the VZ and SVZ in the brain parenchyma. These accumulations of thymidine-labeled cells extended up to 185 pm lateral to the VZ, were more abundant within striatum than cortex, and were not associated with blood vessels. Thymidine-labeled cells in the brain parenchyma were excluded from analysis if 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. they were more than 185 iun from the VZ or if they were clearly endothelial cells associated with blood vessels. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 1.2. Schematic coronal sections through the zebra finch brain at the levels of a. Area X, b. the Anterior Commissure, and c. HVC. Proliferation in the telencephalon occurs in the epithelium adjacent to the lateral ventricle, the VZ (black arrows), and the sub-ventricular zone (gray arrows). Asterisks indicate the locations of thymidine labeled cell clusters in the brain parenchyma, d A separate cross section of the telencephalon indicating the regions measured in the 1-pm plastic tissue analysis, e. Photomicrograph of the VZ in a 1-pm thick section demonstrating the out-pocketing of the VZ in juvenile birds. A bbreviations: Cx, cortex; Str, striatum; Cb, cerebellum; V , lateral ventricle; LH, lamina hyperstriatica; LMD, lamina medullaris dorsalis; LAD, lamina archistriatalis dorsalis; AC, Anterior Commissure; DVZ, dorsal VZ; IntVZ, intermediate VZ; WZ, ventral VZ. 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. paraffin-embedded brains. The exact location of the precursor cells that produce song-control neurons is unknown. For this reason we decided to measure levels of proliferation at two levels of the telencephalon that include song-control regions. Area X and HVC, and at one level of the brain not containing song control nuclei, the Anterior Commissure. To ensure that equivalent locations of the VZ were traced, sections taken from the same anterior-posterior level of the telencephalon, based upon their distance from the Anterior Commissure, were used to measure proliferation at Area X and HVC of juveniles and adults of both sexes. The Anterior Commissure was obvious in all animals and thus only sections that contained the Anterior Commissure were used to measure proliferation at this level of the telencephalon. We ensured that all tissue was cut at the same angle by only analyzing brains in which specific nuclei in the telencephalon (dorsally) and tectum (ventraily) were present in individual sections at the levels of both the Anterior Commissure and HVC. Although we could not discern individual VZ cells within the paraffin-sectioned tissue (see above), we could unambiguously count the total number of silver grains in these sections. Thus, we used the 10 pm-sections to measure total, overall levels of mitotic activity in each proliferative zone (VZ SVZ and within the brain parenchyma adjacent to the VZ and SVZ). To estimate the total amount of proliferation within the VZ the perimeter of the entire dorsal-ventral extent of the VZ was outlined in three tissue sections 100-150 pm apart at each level of the brain analyzed (see dark gray area in Fig la, b, c). The average area of the VZ and the total number of silver grains therein were measured at each brain level; the number of silver grains was then divided by area to calculate the silver grain density. To measure total proliferative activity within the SVZ 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the perimeter of the SVZ was outlined where it occurred along the ventral VZ at the levels of Area X and the Anterior Commissure (Fig. 1.2a, b). The area of the SVZ, total number of silver grains, and density of silver grains were measured at each of these brain levels as for the VZ. The overall level of mitotic activity within the proliferative zone adjacent to the VZ was measured by outlining aggregations of labeled cells and counting the total number of silver grains within each aggregate in the brain parenchyma up to 18S pm from the VZ. The total number of silver grains is the only parameter presented for thymidine labeling adjacent to the VZ within the brain parenchyma since these thymidine-labeled clusters of cells were not confined to a specific delimited region and thus it was impossible to determine the area of this proliferative zone. To ensure that our measurements accurately reflected thymidine incorporation into dividing cells distinct from background labeling, the density of silver grains within a large region of brain parenchyma (-14,000 pm2 ) more than 200 pm away from the VZ was measured for each section analyzed, excluding any labeled glial or endothelial cells. This background measure of thymidine labeling was used to estimate the total number of silver grains that would be expected in a given traced area. The expected number of silver grains was then subtracted from the raw silver grain counts in the VZ, SVZ, and proliferation adjacent to the VZ. Since the total number of silver grains is a direct measure of thymidine uptake (and thus an estimate of total mitotic activity), this procedure enabled us to determine the overall incidence of cell division within each proliferative zone above and beyond background thymidine labeling. We compared background levels of thymidine labeling among groups to evaluate whether thymidine was equally available in juvenile and adult brains. There was no difference in the 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. background silver grain density between juvenile or adult males and females, indicating that the availability of thymidine was equivalent among groups. Plastic-embedded brains. Our analysis of proliferative activity in the 10-pm sections demonstrated a robust age difference (mitotic activity was much higher throughout the telencephalon in juveniles than in adults; see below). To determine the cellular parameters underlying this developmental decrease in mitotic activity, we quantified total cell number (both unlabeled and labeled) and cellular area in individual cells within sub-regions of the VZ at the level of the Anterior Commissure in 1-pm sections. These data provided quantitative estimates of the total number of cells as well as the proportion and density of thymidine-labeled cells in different sub-regions of the VZ. In order to see whether counts of single cells corresponded well to our estimates of total mitotic activity based on counting total number of silver grains, we also measured the area and total number and density of silver grains within the same sub-regions of the VZ in which we analyzed single cells. For this analysis, three tissue sections within a 100 pm interval were analyzed for each bird. The VZ within each section was divided into three different sub-regions (Fig. 1.2d); (1) the ventral VZ (W Z) was defined as the proliferative epithelium extending from the ventral tip of the VZ to the LMD fiber tract, (2) an intermediate region of the VZ (IntVZ) was situated between the LMD and LH (hyperstriatal lamina) fiber tracts, and (3) the dorsal VZ (DVZ) extended from the LH fiber tract to the dorsal tip of the VZ. Within each of these sub-regions, segments of the VZ corresponding to the boxed areas shown in Fig. 1.2d were sampled as follows: for each boxed segment we traced the area of the VZ along a linear length of 165 pm (corresponding to three 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. alternating 55-pm segments). We measured two different segments of the VZ within both the DVZ and W Z (e.g., W Z1 and WZ2), and one segment of the VZ within the IntVZ (Fig. 1. 2d). In order to measure numbers of labeled and unlabeled cells within these segments of the VZ, the perimeter of each individual cell was outlined and the total number of silver grains present within each cell was counted. Criteria for tracing cells included the presence of a limiting membrane and some well-stained chromatin within the cellular profile. The intensity of thymidine labeling (number of times background) was calculated for each cell and individual cells were scored as labeled if the density of silver grains over them exceeded 10X the density of silver grains over background. We also counted the total number of silver grains within each segment (silver grain counts were corrected by subtracting levels of background labeling from the raw silver grain counts, as described above). The measures of area and total number of silver grains represent estimates of total proliferative activity within each segment, which are comparable to our previous estimates of overall thymidine labeling made in 10 pm sections. Mapping proliferation within the VZ Our single-cell analysis revealed a sex difference in a small segment of the VZ in juvenile but not adult zebra finches (see below). This serendipitous discovery raised the question of how extensive such sexually dimorphic mitotic activity might be. In addition, the level of proliferative activity within the VZ was highly spatially differentiated in both juvenile and adult brains. For example, qualitative observation at the level of the Anterior Commissure indicated that levels of proliferation were highest in the ventral 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. portion of the VZ within the striatum, raising the question of how much of a quantitative difference would exist between levels of proliferation in cortex versus striatum. As a first step in exploring these questions, we decided to map the incidence of thymidine labeling in progressive segments along the dorsal and ventral VZ at the levels of the Anterior Commissure and Area X in both juvenile and adult birds. We did not include the level of HVC since the ventral VZ is gone by this level. Mapping proliferation in the VZ at the levels o f Area X and the Anterior Commissure. The 10-pm paraffin tissue sections generated for our original measurements of total proliferation were used to measure the incidence of mitotic activity along the DVZ and W Z (separately) in juvenile and adult zebra finches of both sexes. To measure the incidence of mitotic activity in progressive segments along the DVZ, the total linear length of the DVZ (i.e., from the LH fiber tract to the dorsal tip of the VZ, see above and Fig. 1.2) was calculated and divided by 10. The resulting bins represent the incidence of proliferation in 10% increments along the length of the DVZ, thereby allowing us to compare levels of mitotic activity for corresponding locations within the DVZ among animals. The area, total number of silver grains, and silver grain density within each bin was calculated. The same approach was used to measure the spatial distribution of thymidine labeling within the W Z from the ventral tip of the VZ to the LMD fiber tract. Background measurements of silver grain density were made and used to adjust silver grain counts to exclude background labeling within the DVZ and W Z as described above for the 10-p.m tissue analysis. In this way we were able to accurately map levels of proliferation in 10% intervals along the entire length of the DVZ and W Z separately at the level of both Area X and the Anterior Commissure. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All statistical comparisons were made using 2 x 2 analyses of variance with age and sex as between-group factors in our 10- and 1-micron tissue analyses and bins as a within-group factor in our mapping study. Student Mests were used to compare individual group differences. All tests were considered significant if they exceeded the 95% confidence level (2-tailed). RESULTS Overall pattern of thymidine labeling in the brain: qualitative observations Brain and body weight of zebra finches are at their adult levels by 25-30 days of age and therefore we had conceived this study with the idea that ceil division in the telencephalic neuroepithelium of 30-day old zebra finches would likely have achieved adult (i.e., decreased) levels. If so, then we might be able to see isolated pockets of upregulated mitotic activity within the VZ in close proximity to the song-control nuclei HVC and Area X of juvenile males. Both these regions grow substantially in overall volume through neuron addition during song learning in juvenile males after other, non- song regions are fully developed (Bottjer et al., 1986; Herrmann and Bischof, 1986; Nordeen and Nordeen, 1988a). Such pockets of increased mitotic activity might therefore contain the progenitor cells that produce song-control neurons destined for X and HVC. However, qualitative examination of cell proliferation revealed a larger VZ with a higher incidence of thymidine labeling throughout the telencephalon of juvenile birds compared to adults. Although juveniles had higher levels of mitotic activity than did adults, overall levels of proliferation appeared to be equivalent among males and females at each age and no regions of increased proliferative activity were obvious in relation to HVC or X in 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. juveniles or adults of either sex. Proliferative activity at both ages was restricted to the telencephalon, such that no labeled cells were evident in the diencephalon or below (Alvarez-Buylla et al., 1990,1994; Ling et al.. 1997). Mitotic activity within the telencephalon was localized to three distinct proliferative zones in juvenile and adult zebra finches of both sexes: the VZ, the SVZ, and scattered pockets of thymidine labeling adjacent to the VZ and SVZ in the brain parenchyma (Fig. 1.2; see Methods). The SVZ that we observed in the zebra finch telencephalon was localized to the striatum (adjacent to the ventral VZ) in both juveniles and adults, as is true in adult mammals (Garcia-Verdugo et al., 1998). Of the three proliferative zones that we observed, only the VZ and SVZ have been described in the developing mammalian brain (Boulder Committee, 1970). In addition, other studies of cellular proliferation in avian brain using short survival times following thymidine exposure have not described aggregations of labeled cells outside of the VZ and SVZ (Alvarez-Buylla et al., 1990; Ling et al., 1997). The short (two-hour) survival interval following thymidine exposure used in our study seems to rule out the possibility that this group of labeled cells had migrated out from the VZ or SVZ. These labeled cells in the brain parenchyma have similar morphological characteristics to thymidine-labeled cells within the VZ and SVZ, and hence might represent a population of stem cells situated close to the VZ. An alternative possibility is that this zone of proliferation represents a population of cells dividing while en route to their final destination. Although the increased level of mitotic activity seen in juvenile brains was quite ubiquitous along the rostro-caudal axis of the telencephalon, the pattern of labeling was not uniform in either juveniles or adults. The relative thickness of the VZ and SVZ, as 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. well as the incidence of thymidine labeling, varied depending on the dorsal-ventral and anterior-posterior location along the neuraxis. For example, each proliferative zone was thickest along the ventral aspect of the lateral ventricles within medial striatum of the avian basal ganglia (LPO, parolfactory lobe) at the level of the Anterior Commissure (Fig. 1.2) in both juveniles and adults. This region of thicker VZ along the ventral lateral ventricles always included relatively high numbers of silver grains, such that the highest levels of mitotic activity in the telencephalon occurred here. High levels of thymidine labeling were localized not only to this “hot spot” near the ventral horn of the lateral ventricles at the level of the Anterior Commissure, but also to a smaller aggregation of thymidine labeling around the dorsal hom (cf. Alvarez-Buylla et al., 1990). Within the ventral area of increased labeling, we observed a distinct out-pocketing of the proliferative epithelium in juvenile brains (Fig. 1 ,2b, e). This region of the VZ always included a very distinct bulge in juvenile brains, whereas adult brains tended to have a much less pronounced, shallow inclination in the VZ (Fig. 1.5). In addition, this out- pocketing in the proliferative epithelium was significantly larger and appeared to be more proliferatively active in juveniles than in adults. The thickness of the VZ and incidence of silver grains also tended to be slightly higher within the ventral aspect of the VZ more rostrally, at the level of Area X, but this tendency was much less pronounced than at the Anterior Commissure (Fig. 1.2). In addition, the SVZ was much less obvious at this level, particularly in adult brains. The SVZ tended to co-localize with thicker portions of the VZ along the ventral aspect of the lateral ventricles in both juvenile and adult brains, and was First clearly recognizable adjacent to the ventral VZ midway through the rostral-caudal extent of Area X in 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. juveniles. In adults, the SVZ was not apparent until the caudal-most level of Area X. A spatially differentiated pattern of proliferation was much less evident in the caudal telencephalon, at the level of HVC, by which point the basal ganglia are gone. The SVZ was not apparent in either juvenile or adult brains at the level of HVC. In summary, a spatially differentiated pattern of labeling was evident in the telencephalon of both juveniles and adults, despite the higher incidence of proliferative activity in juveniles compared to adults. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Quantitative observations in 10-pm sections Area X: Quantitative analysis at the level of Area X confirmed that the overall area of the VZ was larger in juveniles than in adults (F|. 1 5 = 19.22, p = 0.0005)(Fig. 1.3a), but did not differ between males and females at either age (main effect of sex: F < 1). The total proliferative activity within the VZ at the level of Area X was also significantly greater in juveniles than in adults as indicated by a greater total number of silver grains (F|. 1 5 = 12.66, p = 0.003)(Fig. 13b), but there was no sex difference in number of silver grains within juveniles or adults (main effect: F1 . 1 5 = 1.15, p = 0.3). The age difference in mitotic activity was robust: the total number of silver grains was more than five times higher in juveniles compared to adults at this level of the brain. This greatly increased number of silver grains in juvenile birds contributed to a significantly greater density of silver grains within this portion of the VZ in juveniles compared to adults (Fi. 1 5 = 13.60, p = 0.002), but silver grain density did not differ between males and females (F|.u = 2.07, p = 0.17) (Fig. 1.3c). We did not quantify the incidence of labeling within the SVZ at Area X because this proliferative zone was not consistently represented at this level of the brain (see above). Only three out of nine adult brains had a clearly visible region of SVZ adjacent to the ventral VZ at Area X, whereas seven out of ten juveniles had an observable SVZ. Both juvenile and adult brains did have clusters of thymidine labeling adjacent to the VZ at Area X. The total number of silver grains observed in these clusters within the brain parenchyma was significantly greater in juveniles than in adults (F|. I 4 = 11.96, p = 0.004)(Fig. 1.4d), but mitotic activity did not differ between males and females (F < 1). 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Anterior Commissure: The pattern of proliferation at the level of the Anterior Commissure was similar to that observed at Area X in that the area of the VZ was larger in juveniles than in adults ( F |.i6 = 25.36, p = 0.0001)(Fig. 1.3), and didn’t differ as a function of sex (F < 1). Overall levels of proliferation were also significantly higher in juveniles compared to adults as indicated by the total number of silver grains (Fi. i6 = 13.12, p = 0.002). However the density of silver grains did not differ between juveniles and adults (F|. (6 = 1.22, p = 0.29), since the relative age difference for both VZ area and total number of silver grains was roughly comparable (compare Fig. 1.3a, b). There was no sex difference at either age in the total number of silver grains or the silver grain density within the VZ (both F < 1). The SVZ was also larger and more proliferatively active in juveniles than adults at the level of the Anterior Commissure. Area measurements of the SVZ demonstrated that this proliferative zone was approximately four times larger in juvenile males and females compared to adults at this level of the brain (Ft. t8= 19.56, p = 0.0003; F < I for main effect of sex)(Fig. 1.4a). The total number of silver grains within the SVZ was higher in juveniles than adults but did not differ as a function of sex (FL |g= 9.28. p = 0.007; main effect of sex: F|, 1 8 = 1.84, p = 0.19)(Fig. 1.4b). The silver grain density within the SVZ did not vary as a function of age (F|. is = 1.23, p = 0.28) or sex (F < l)(Fig. 1.4c). The number of silver grains in the brain parenchyma adjacent to the VZ at the level of the Anterior Commissure was approximately twice as high in juveniles as in adults (F|. |g = 19.87, p = 0.0003)(Fig. I.4d) and there was no sex difference in thymidine labeling adjacent to the VZ (F < 1). 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HVC: The area of the VZ at the level of HVC did not differ among the four groups (age: F < 1; sex: F < l)(Fig. 1.3). However, the level of proliferation within the VZ, as measured by total number of silver grains, was higher in juveniles than adults (F|. i6 = S.28, p = 0.04) and did not differ between the sexes (F < 1). The density of silver grains within the VZ at this level of the telencephalon was relatively low and did not vary as a function of either age (F|. i6= 3.41, p = 0.08) or sex (F < 1). The SVZ was not present at the level of HVC, and clusters of thymidine labeling near the VZ were observed within the brain parenchyma in nine out of ten juveniles, but only a few adults. These scattered clusters of thymidine labeling were not localized to a specific region along the dorsal-ventral axis, but were randomly distributed along the entire extent of the VZ (Fig. 1.2c). In addition to overall measurements of proliferation in the VZ at the level of HVC, we also measured the incidence of thymidine labeling immediately dorsal to HVC since it seemed possible that precursor cells for HVC neurons might reside in the segment of VZ directly overlying HVC. If so, then there might be a higher level of thymidine incorporation in this region of the VZ Measurements of the size of this segment of the VZ demonstrated an age difference (Ft. )0 = 3.44, p = 0.04) but no sex difference (Fi.io = I -17, p = 0.3)(Fig. 1.3). The age difference was due to the fact that the area of VZ overlying HVC was slightly larger in adult birds than in juveniles, which presumably reflects the much larger size of HVC in adults (and hence a greater linear stretch of VZ was measured in adults). Despite this difference, there were significantly more silver grains within this small region of VZ in juveniles than in adults (F|. |0= 13.90, p = 0.003), but no difference between males and females at either age (Fuo= 3.40, p = 0.1). The 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. silver grain density within the VZ above HVC was also higher in juvenile zebra finches than in adults (Ft. 1 0 = 14.18, p = 0.004), and there was no sex difference (F < 1). 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120000 i 80000 40000 c S 0.04 JuvMale Juv Female Ad Male Ad Female g - 0.00 Area X Anterior H V C V Z Above Commissure H V C Fig. 1 J . Ten-micron tissue analysis demonstrating a. the area of the VZ, b. total number o f silver grains, and c. silver grain density within the VZ at the levels o f Area X, the Anterior Commissure, HVC, and the VZ immediately above HVC in juveniles and adults of both sexes (mean ± SE). 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15000 10000 JuvMale Juv Female Ad Male Ad Female m g | J-0.06 e n 0.04 O ^ 0.02 | 1 0.00 < /> c I - u . : h i Anterior Commissure o £2000 fe A J £*1500 1000 Area X Anterior Commissure Fig. 1.4. Ten-micron tissue analysis demonstrating a. the area of the SVZ, b. total number o f silver grains, and c. silver grain density within the SVZ at the level of the Anterior Commissure in juveniles and adults (mean ± SE). d. The total number of silver grains overlying labeled cell clusters adjacent to the VZ within the brain parenchyma in 10-pm thick tissue at the levels of Area X and the Anterior Commissure in juveniles and adults of both sexes (mean ± SE). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. / 250um Fig. 1.5. Autoradiograms showing thymidine labeling within the ventral portion of the VZ at die level of die Anterior Commissure in a. a juvenile male and b. an adult female. Thymidine labeling in the SVZ (arrowheads) and clusters of thymidine labeled cells (arrows) adjacent to the VZ are apparent A large out-pocketing in the VZ is apparent in juveniles while the out-pocketing is much reduced in adults. Abbreviation: V, lateral ventricle. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M s 500 £ 250 40 i s I S 20 o <3 N S > n 04 0 o U U4 s .£ C D 0.00 a. i i It ■ ■ Juv Males Juv Females Ad Males 1 I Ad Femlales h i b. Ill 4 4 L II l l L WZ1 WZ2 IntVZ D V Z 1 D V Z2 Fig. 1.6. One-micron tissue analysis demonstrating a. VZ area, b. total number of silver grains, and c. silver grain density (segments shown in Fig. Id) at the level o f the Anterior Commissure in juveniles and adults of both sexes (mean ± SE). Asterisk indicates a main effect of sex in the total number of silver grains in DVZ2. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Q uantitative observations in 1-pm sections The increased area of die juvenile VZ compared to that of adults indicates that VZ cells may be larger and/or more numerous in juveniles than in adults. In addition, the heightened incidence of thymidine labeling in juvenile birds suggests that many more VZ cells are actively dividing in juvenile telencephalon. In order to determine which cellular parameters change with age, we performed a single-cell analysis at the level of the Anterior Commissure within 3 sub-regions o f the VZ: the ventral VZ (WZ), the intermediate VZ (IntVZ), and the dorsal VZ (DVZXsee Methods; Fig. 1.2d). In order to determine the degree of correspondence between our measurements of total proliferative activity in 10-pm sections and this single-cell analysis, we also measured the area and total number of silver grains within each subregion of the VZ measured. In the following description these estimates of total proliferative activity within each sub-region are given first, followed by the cellular analyses. W Z: Visual inspection at the level o f the Anterior Commissure had revealed that the ventral VZ was thicker in juveniles than in adults (see above). Quantitative analysis confirmed that die area of each segment measured within the ventral VZ was significantly larger in juveniles than in adults (W Z1: F1 .2 2 . = 36.87, p < 0.0001; W Z2 Fi.22= 22.56, p < O.OOOlXFig. 1.6a), but VZ area did not differ as a function of sex (p > 0.25 for both W Z 1 and WZ2). Levels of proliferation within die W Z , as measured by the total number o f silver grains, were also significantly higher in juveniles than adults (WZ1: F|.22= 7.92, p = 0.01; WZ2: F|j2= 5.35, p = 0.03), but showed no sex difference (p > 0.3 for both VVZ1 and WZ2)(Fig. I.6b). The silver grain density 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was approximately equivalent between juveniles and adults within WZ1 (F|. n = 3.63, p = 0.07) but was significantly higher in juveniles than adults in W Z2 (FL 22= 5.36, p = 0.03)(Fig. 1.6c). There was no sex difference in silver grain density within either segment of the W Z (p > 0.25 for both segments). The area of individual cells within both segments of the W Z was the same among juveniles and adults of both sexes (age: W Z l, F < I; W Z2, Ft. 2 2 = 1.69, p = 0.21 ; sex: F < 1 for both W Z l and WZ2)(Fig. 1.7a). The total number of cells (both labeled and unlabeled) was significantly greater in juveniles compared to adults (W Zl Fi.22= 18.33, p = 0.0003; W Z2 Ft,2 2= 11.56, p = 0.003), and there was no sex difference in cell number (p > 0.15 for both segments)(Fig. 1.7b). The total number of thymidine-labeled cells was also significantly greater in juveniles than in adults (W Zl: F,.2 2= 7.65, p = 0.01; WZ2: F,.2 2= 19.48, p = 0.0002: sex: both F< 1 ) (Fig. 1.7c). In summary, these data indicate that the increased incidence of mitotic activity in juvenile birds revealed by total silver grain counts is due to a larger number of dividing cells in juveniles than adults. The proportion and density of labeled cells were significantly higher in juveniles than adults only in W Z 2 (% labeled: W Z l, F,.n = 2.78, p = 0.11; WZ2, = 23.13, p <0.000l)(density labeled: W Z l, F< 1 ; W Z2, F,jq= 12.57, p = 0.002)(Fig. 1.8). There was no sex difference in percent cells labeled or the labeled cell density at either age (all p’s > 0.20 for both % labeled and labeled cell density in W Zl and W Z2). W Z l lies in the more ventral aspect of the W Z which includes the area of out- pocketing in the VZ (Fig. 1.2), and this segment tended to be relatively larger and more proliferatively active than the segment of W Z just above it (i.e., the number of total cells 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and of labeled cells were higher overall within W Z l than W Z2; Fig. 1.7). It is interesting that there was no selective increase in proliferation within W Z l of juvenile birds, as evidenced by the fact that the proportion and density of labeled cells were not different between juveniles and adults (Fig. 1.8). In contrast, within W Z2 we observed not only an absolute increase in total number of cells and labeled cells in juveniles relative to adults, but also an increase in the proportion and density of dividing cells in young birds. IntVZ: The area of the segment measured within IntVZ was significantly greater in juveniles than in adults (Fi. n =7.89, p = 0.01), and there was no sex difference at either age (F < l)(Fig. 1.6). The total number and density of silver grains in the IntVZ were also significantly higher in juveniles than in adults (number of silver grains: Fi. 22= 8.74, p = 0.007; silver grain density: F < i.2 2 )= 7.94, p = 0.01), and did not vary as a function of sex (both number and density: F < 1). Measurements of cell area within the IntVZ revealed that the size of individual cells was the same among all groups (age: Fi.2 2= 1.14, p = 0.3; sex: F < l)(Fig. 1.7). The total number of IntVZ cells (both unlabeled and labeled) was greater in juveniles than adults, and did not differ between males and females (age: Fi. 2 2 = 5.44, p = 0.03; sex: F < 1). The total number of thymidine-labeled cells in the IntVZ was also greater in juveniles than adults and showed no sex difference (age: Fi. 2 2=9.00, p = 0.007; sex: F < 1). The age difference in incidence of thymidine-labeled cells was also significant for the proportion of cells labeled (age: F,. 2 2 = 6.77, p = 0.02; sex: F1 . 2 2 = 1 -25, p = 0.28)(Fig. 1.8) and approached significance for labeled cell density (age: F|.2 2 = 4.08, p = 0.06; sex: F1 . 2 2 = 1.83, p = 0.19). 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DVZ: Both segments measured within the DVZ (Fig. 1.2d) demonstrated an age but not a sex difference in VZ area (age: DVZI. Fu7 = 8.69, p = 0.009; DVZ2, Fiji = 9.19, p = 0.006Xsex: both F < IXFig. 1.6). The total number and density of silver grains were greater in juveniles relative to adults within both DVZI and DVZ2 (number DVZI, F,. ,7 = 23.14, p = 0.0002; DVZ2, F,. 2 i = 12.52, p = 0.002)(density: DVZI, F|.i7= 19.77, p = 0.0004; DVZ2, Fiji = 9.67, p = 0.005). There was no effect of sex on the number or density of silver grains within DVZI (both F < 1), but there was a significant main effect of sex on both of these measures within DVZ2 (number Fi. 2 1 = 7.53, p = 0.01 ;density: Fi. 2 i = 4.36, p = 0.05). Individual t-tests revealed that juvenile males had an increased total number of silver grains in DVZ2 (immediately above the LH fiber tract) compared to juvenile females (t)( = 2.56, p = 0.03), but the incidence of thymidine labeling was equivalent among adults in this region (t|0 = 1.23, p = 0.25). Individual t-tests for silver grain density revealed no differences in either juveniles <tu = 1.84, p = 0.09) or adults (tt0 = 1.13, p = 0.28). Measurements of individual cells in the DVZ revealed no difference in cellular area among juveniles or adults of either sex (age; DVZI, Fi. i7= 1.18, p = 0.29; DVZ2, F < I)(sex: DVZI, F|.i7= 2.33, p = 0.15; DVZ2, F < l)(Fig. 1.7). The total number of cells was greater in juveniles than in adults in both segments of the DVZ (DVZI; Ft. |7 = 10.04, p = 0.006; DVZ2: Fiji = 5.40, p = 0.03), but did not differ between males and females (both F < I). The number of thymidine-labeled cells was significantly greater in juveniles than adults within both segments of the DVZ (DVZI: F|. i7 = 30.03, p < 0.0001; DVZ2: Fi. 2 1 = 8.74, p = 0.008), but there was no main effect of sex in number of labeled cells (DVZI: F|, |7 = 1.3, p = 0.27; DVZ2: Fiji = 2.27, p = 0.15). 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Juvenile males tended to have a higher incidence of labeled cells than juvenile females in DVZ2, however this difference was not significant (tn = 1.74, p = 0.11). Both the proportion and density of labeled cells were higher in juveniles than adults in both DVZ segments measured (% labeled: DVZI, F|. n = 12.68, p = 0.002; DVZ2, F, j, = 10.83, p = 0.004)(labeled cell density: DVZI, F,.,? = 12.93, p = 0.002; DVZ2, Fiji = 7.87, p = 0.01). There was no main effect of sex within DVZI (% labeled: Fi. 1 7 = 3.0, p = 0.10; labeled cell density: F|. n = 2.78, p = 0. 11), but there was a main effect of sex on the proportion of cells labeled in DVZ2 {% labeled: Fiji = 5.10, p = 0.03; labeled cell density: Fui = 2.30; p = 0.14)(Fig. 1.8). This sex difference was attributable to a larger proportion of labeled cells in males than females for juveniles (tM = 2.96, p = 0.01) but not adults (t < I). Individual t-tests also revealed a marginal sex difference in labeled cell density for juveniles but not adults (juveniles: tu = 2. 11, p = 0.06; adults: t < 1). The increased incidence of labeled cells in juvenile males compared to juvenile females corresponds to the same segment of the DVZ in which we observed a sex difference in number and density of silver grains (Fig. 1.6b, c). Despite the fact that the total number of labeled cells tended to be greater in juvenile males than juvenile females in DVZ2 (Fig. 1.7) this measure was not statistically different, presumably because juvenile females had a greater total number of cells (labeled and unlabeled), thereby offsetting a sex difference in the absolute number of labeled cells. However, the proportion of labeled cells was significantly greater in juvenile males than juvenile females in DVZ2 (Fig. 1.8). Thus, the sex difference in proliferation in the DVZ just above the LH fiber tract (DVZ2) is the result of a selective increase in the incidence of dividing cells in juvenile males compared to juvenile females. 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Juv Males Juv Females Ad Males Ad Females S S = 2 WZ1 W Z2 IntVZ D V Z 1 DVZ2 Fig. 1.7. One-micron tissue analysis demonstrating a. cellular area, b. total number of cells (labeled and unlabeled), and c. total number of labeled cells within each VZ segment measured at the level o f the Anterior Commissure in juveniles and adults of both sexes (mean ± SE). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Juv Males Juv Females Ad Males Ad Females WZ1 WZ2 IntV Z D V Z1 DVZ2 Fig, 1.8. One-micron tissue analysis demonstrating a. percent cells labeled and b. labeled cell density within each VZ segment measured at the level of the Anterior Commissure in juveniles and adults o f both sexes (mean ± SE). Asterisk indicates a significant difference in the proportion of labeled cells between juvenile males and females in DVZ2. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mapping proliferation in the VZ at the levels of the Anterior Commissure and Area X Our initial studies had revealed the existence of a sex difference in mitotic activity in a small segment of the VZ, as well as a spatially differentiated pattern of cell division across the VZ. In order to search for other examples of sex differences in proliferation, and to compare levels of proliferative activity between different sub-regions of the VZ, we decided to map levels of thymidine labeling in 10% increments along the W Z (from the ventral horn to the LMD fiber tract) and DVZ (from the LH fiber tract to the dorsal horn) at the levels of the Anterior Commissure and Area X in IO-pm tissue sections (see Methods). These data allowed us to compare overall levels of proliferation (total number of silver grains) between the DVZ (within cortex) and the W Z (within striatum), and to construct a map of mitotic activity along the dorsal-ventral axis for both the DVZ and W Z at these two levels of the telencephalon. The Anterior Commissure DVZ: Histograms of the total number of silver grains within each 10% increment of the DVZ at the level of the Anterior Commissure indicated no effect of age or sex (age: F|. 1 7 = 3.34, p = 0.09; sex: F < l)(Ftg. 1.9a, b). However, there was a main effect of bins (F9 . 1 5 3 = 4.47, p < 0.0001) indicating that the level of proliferation varied along the dorsal-ventral axis of the DVZ. The incidence of cell division was highest near the dorsal horn in juvenile males and females but not adults (compare Fig. 1,9a and b, bins 8-10). These three bins at the tip of the DVZ are adjacent to the dorsal horn of the lateral ventricles and correspond to a region of increased mitotic activity described by Alvarez- Buylla et al. (1990) in adult canaries at the same location. Comparisons of the total 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. number of silver grains within individual bins revealed a sex difference in thymidine labeling in bin I, just dorsal to LH, between juvenile males and females (but not adults). That is, juvenile males had significantly more silver grains in the segment immediately above LH than did juvenile females (t9= 2.42, p = 0.04). This sex difference in proliferation is located in the same segment of the DVZ as the sex difference in the proportion of thymidine-labeled cells we observed in the 1-pm tissue analysis (see above). Bins 2-10 did not reveal other regions of sexually dimorphic proliferation in either juveniles or adults. WZ: The pattern of thymidine labeling along the dorsal-ventral axis of the W Z at the level of the Anterior Commissure was similar between juveniles and adults of both sexes, with the highest levels of proliferation occurring near the ventral hom (in bins 2,3, and 4; Fig. 1.9c, d). This area resides within the region of W Z that bulges out into the brain parenchyma as an out-pocketing of the VZ (Fig. 1.5), which corresponds to a region of increased mitotic activity referred to as a proliferative “hot spot" close to the ventral hom of the lateral ventricles (Alvarez-Buylla et al., 1990). Total silver grain counts revealed that proliferation was higher throughout the W Z in juveniles than in adults (Fi.i7= 11.44, p = 0.004), but there was no main effect of sex (Fi.n= 1.17, p = 0.30). There was also a main effect of bins and an age by bins interaction (bins: F9 . ^ = 8.76, p < 0.0001; age by bins: F9 .is3= 2.55, p = 0.009), reflecting the higher levels of proliferation near the ventral hom, particularly in juvenile animals. A sex difference in the total number of silver grains within individual bins was seen in bin 8 for juvenile animals with males having higher levels of proliferative activity than females (bin 8: t < > = 2.58, p = 0.03), and this difference approached significance in 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bin 9 (t9= 2.12, p = 0.06). The total number of silver grains in individual bins within the W Z of adults did not reveal sexually diraoiphic mitotic activity. This pattern of results indicates that a segment of the W Z lying ventral to the LMD fiber tract in the striatum contains a region of sexually dimorphic proliferative activity in juveniles but not adults. In addition, there is a consistent trend towards higher levels of proliferation in juvenile males compared to juvenile females throughout the dorsal-ventral extent of the W Z at the level of the Anterior Commissure (i.e., all bins have more silver grains in juvenile males than females, although this difference was significant only in bin 8 and approached significance in bin 9; Fig. 1.9c). Area X DVZ: Thymidine labeling in the DVZ at the level of Area X did not demonstrate a strong pattern of spatially differentiated proliferation in juveniles or adults (Fig. 1.10a, b). The total number of silver grains across bins demonstrated an age difference in the incidence of thymidine labeling (F|. |g= 7.63, p = 0.01), reflecting a greater than two-fold increase in overall levels of mitotic activity in juveniles compared to adults. There was no main effect of sex (Fi. is = 2.14, p = 0.16) or bins (F9 ! 6 2 = 1.83, p = 0.07). The total number of silver grains in bin 1 , immediately dorsal to LH, of juvenile males and females revealed no sex difference (t|0 = 1.50, p = 0.17). Thus, the pattern of sexually dimorphic labeling we observed just dorsal to LH at the level of the Anterior Commissure (more silver grains in bin 1 of juvenile males; Figs. 5b, 8a), did not extend rostrally to this level of the telencephalon. WZ: The spatial distribution of thymidine labeling along the dorsal-ventral axis of the W Z at the level of Area X was different among males and females at both ages 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Fig. 1.10). Juvenile males demonstrated a slight increase in the total number of silver grains in bins 1-6 followed by lower levels between bins 7 and 10 while juvenile females demonstrated relatively consistent levels of proliferative activity in bins 1-10. Adults did not demonstrate as much change in the spatial distribution of thymidine labeling, and both males and females demonstrated low levels of proliferation throughout the W Z at this level. Statistical analysis revealed an overall effect of age, sex, and an age by sex interaction (age: F,. ,7 = 17.35, p = 0.0006; sex: FU 7 = 5.40, p = 0.03; age by sex: Fi.n = 4.84, p = 0.04), but no effect of bins (F < 1). Overall, juvenile males had higher levels of proliferation throughout the W Z than juvenile females (F|. |7= 10.70, p = 0.005) but levels of thymidine labeling were equivalent in adults (F < 1). When we compared thymidine labeling within individual bins between juvenile males and females, bins 1 , 2,4, and 5 demonstrated sex differences in proliferation and a tendency towards a sex difference in bins 3,6, 7, and 8 (bin 1 : t»= 2.46, p = 0.04; bin 2: tq= 2.40, p = 0.04; bin 3: t9= 1.89, p = 0.09; bin 4: 1?= 2.50, p = 0.04; bin 5: t?= 3.02, p = 0.02, bin 6: t9= 2.07; p = 0.07; bin 7: U= 2.13; p = 0.06; bin 8:1,= 2.12; p = 0.06). Interestingly, the segment of the W Z containing bins 4 and 5 is located approximately adjacent to Area X, raising the possibility that this region of the VZ (and adjacent regions at this level) might contain precursor cells that produce Area X neurons. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. £ ..60 0 Juv Male Juv Female Z <»400 200 - 8 9 10 .= 200 600 400 200 Ad Male Ad Female 1 2 3 4 5 6 7 8 9 10 ^L M D V H ^LM D 600 400 200 2 3 4 5 6 7 8 9 10 ---------------------- ^LM D V H 2 3 4 5 6 7 8 9 10 ----------------------^LMD Fig. 1.9. Histograms of the total number of silver grains within each bin of the DVZ o f a. juveniles and b. adults and in the W Z of c. juveniles and d. adults at the level of the Anterior Commissure (mean ± SE). Asterisks indicate significant differences in thymidine labeling between juvenile males and females in DVZ bin 1 (a.) and W Z bin 8 (c.). Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Juv Male Juv Female Ad Male Ad Female 8 9 10 ^LMD 8 9 10 ^LM D 8 9 10 ^ L M D 8 9 10 ^ L M D Fig. 1.10. Histograms of the total number of silver grains within each bin of the DVZ of a juveniles and b. adults and in the W Z of c. juveniles and d. adults at rostral levels of Area X (mean ± SE). Asterisks in c. indicate significant differences in thymidine labeling between juvenile males and females in W Z bins 1,2,4, and S. Comparing levels of proliferation between the W Z and the DVZ In order to directly compare both area and levels of proliferation between the DVZ and the W Z , we collapsed the binned data from our mapping study within each of these regions at the levels of the Anterior Commissure and Area X (Fig. 1.11). Anterior Commissure: The area of the entire DVZ was greater than that of the W Z at the level of the Anterior Commissure (Ft. is = 72.85, p < 0.0001). This increased area of the DVZ was due to the fact that the linear length of the DVZ was much greater than that of the W Z at this level of the brain (not shown). Thus, the enlarged area of the ventral VZ close to the ventral horn (see above) was offset by the greater length of the DVZ at the Anterior Commissure. As expected, there was also a main effect of age, reflecting the larger area of both W Z and DVZ in juveniles (Fug = 25.01 , p < 0.0001). The total number of silver grains was higher in the W Z than the DVZ at this level, despite the smaller area of the W Z (Fi.|g= 19.29, p = 0.0004). There was also a main effect of age for number of silver grains (Fug = 10.70, p = 0.004), no effect of sex (Fug= 1.22, p = 0.28), and an age by sex by region interaction (Fug = 5.95, p = 0.03). This interaction was due to a sex difference in the W Z but not the DVZ of juvenile birds (juvenile males versus females in W Z: Fug = 4.37, p = 0.05), whereas adult males and females were not different in either region. There was also an age by region interaction (Fug = 8.53, p = 0.009), reflecting the fact that the number of silver grains was greater in the W Z than the DVZ for juveniles (Fug = 29.42, p < 0.0001), but not for adults (F < 1). This pattern indicates that the higher level of proliferative activity in the W Z than the DVZ is unique to juveniles, and is presumably due primarily to the presence of the hot spot of labeling in the pronounced out-pocketing of the W Z seen in juveniles along the 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ventral-most aspect of the lateral ventricles at this level (Fig. 13). In summary, levels of proliferation were significantly higher within the W Z than the DVZ at the level of the Anterior Commissure in juvenile, but not adult brains with the highest levels of mitotic activity occurring within the W Z of juvenile males compared to juvenile females or adults. Area X : Collapsing across bins from dorsal versus ventral VZ at the level of Area X indicated a difference in area between the W Z and DVZ (Fug = 26.25, p < 0.0001) but no difference in levels of thymidine labeling (FU 8 = 3.66, p = 0.07; Fig. 1 .1 Ic, d). Thus, the higher levels of proliferation evident in the ventral VZ of juveniles at the level of the Anterior Commissure were not evident at this more rostral level of the brain. (However, it was true that the incidence of mitotic activity at this level tended to be higher within a restricted region of the W Z close to the ventral horn, despite the fact that this pattern was much less pronounced at Area X than at the Anterior Commissure; see qualitative description above.) The area of both the DVZ and W Z were larger in juveniles than adults (main effect of age: F|. |8 = 57.91 , p < 0.0001), and did not vary as a function of sex (F|. i8 = 1.60, p = 0.22). In addition, juveniles had significantly greater numbers of silver grains in both the DVZ and W Z (main effect of age: FU 8 = 16.83, p = 0.0007) and there was an effect of sex on levels of proliferation (Fi. is = 5.04, p = 0.04) which was attributable to higher levels of proliferation in juvenile males than females within the W Z (F|. |8 = 12.46, p = 0.002) but not the DVZ (Fi. is = 3.14, p = 0.09). Thus, juvenile males demonstrate higher overall levels of proliferative activity throughout the W Z compared to juvenile females at this level of the brain. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ( Ultl) M IV Z A ( uni) M JV ZA 40000 30000 - 20000 - 10000 - Anterior Commissure § 3000 f i o k 1 2000 C O 1000 - WZ D V Z 40000 30000 - 20000 10000 - 2 E 3 Z I AreaX §3000 f i o W Z D V Z d. Juv Males Juv Females Ad Males l I Ad Females £2000 WZ D V Z W Z D V Z Fig. 1.11. The area and total number o f silver grains within the DVZ and W Z at the levels of the Anterior Commissure a & by and Area X c & d (mean ± SE). 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION The vast majority of studies investigating neurogenesis in the telencephalon of songbirds have focused on observing new neuron addition and/or seasonal neuronal turnover within song-control nuclei (Goldman and Nottebohm, 1983; Nordeen and Nordeen, 1988a, b; Kim et al.. 1994). Because new neurons require approximately one to three weeks to arrive in song-control nuclei following their generation within the VZ (Burd and Nottebohm, 1985; AIvarez-Buylla and Nottebohm, 1988; Burek et al., 1994; Kim et al., 1999), the long post-thymidine intervals required in such studies make it impossible to determine the relative contribution of proliferation, migration, differentiation, and cell death. Until now there have been no systematic, quantitative studies investigating the incidence of cell proliferation throughout the telencephalic VZ of songbirds. Our data directly assess the contribution that different levels of cell division can potentially make to neuronal addition and replacement in both male and female zebra Finches during song development and adulthood. The enhanced incidence of cell division seen in juveniles relative to adults correlates with rapid growth and neuron addition to the song-control nuclei HVC and Area X in young males, indicating that the higher levels of proliferation seen during vocal learning could contribute directly to the growth of song-control nuclei. In particular, we observed restricted regions of developmentally regulated sexually dimorphic proliferation in which the incidence of cell division was higher in juvenile males than juvenile females, especially at the level of Area X. Cells bom within such regions might contribute to the sexually dimorphic growth of song-control nuclei that occurs during the sensitive period for vocal learning. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mitotic activity is spatially differentiated throughout the telencephalic VZ Mitotic activity was ubiquitous throughout the telencephalon of both juveniles and adults with the highest levels at the Anterior Commissure and lower levels at Area X and HVC. This result seems to contradict our hypothesis that high levels of proliferation, at least in 30 day old male birds, would occur close to song-control regions that incorporate a large number of new neurons over the course of song development (Bottjer et al., 1986; Nordeen and Nordeen, 1988a, b; Alvarez-Buylla et al., 1992). This result suggests that the location of precursor cells that give rise to song-control neurons may be spatially remote from their target. However, a region with consistently higher levels of mitotic activity in juvenile males than females occurs in close proximity to Area X, suggesting that this region may give rise to Area X neurons. There was also a difference in the total amount of proliferative activity between the dorsal and ventral VZ in juveniles (but not adults) at the level of the Anterior Commissure, where overall levels of proliferation were substantially higher within the W Z than within the DVZ of young birds (Fig. 11). The ventral VZ of juveniles contained a prominent out-pocketing of the neuroepithelium (Fig. 1 .5), that might represent a remnant of a sulcus such as the one that divides the medial and lateral ganglionic eminences, which are thought to generate cells destined for the striatum and pallidum, respectively, in embryonic mammals (Smart and Sturrock, 1979). The enhanced proliferative activity within the W Z (relative to the DVZ) of juveniles is interesting, because it correlates with a large addition of new neurons to striatum of birds during the first several weeks post-hatch, whereas neighboring cortex receives many fewer new neurons (Alvarez-Buylla et al., 1994). 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We do not know whether cells generated in the dorsal versus ventral proliferative zones are restricted to the cortex and basal ganglia, respectively. However, this seems possible as studies of telencephalic development have provided evidence for migrational boundaries that prevent movement across the corticostriatal sulcus of the VZ or from postmitotic cortex to striatum (e.g., Fishell et al., 1993; Neyt et al., 1997; Striedter et al., 1998). Since the SVZ generates both neurons and glia (Smart, 1961; Lois and Alvarez- Buylla, 1993; Szele and Chesselet, 1996; Doetsch et al., 1999), it is possible that this proliferative zone in conjunction with the ventral VZ generates both cell types within the striatum whereas the dorsal VZ might be a source of new neurons and glia in the cortex (glia dividing in situ presumably contribute to glial turnover throughout the telencephalon; Cameron and Rakic, 1991). However, numerous studies have also provided evidence for extensive tangential migration of neurons both within and between striatum and cortex (e.g., O’Rourke et al., 1995; Anderson et al., 1997; Lavdas et al., 1999). Proliferation is higher in juveniles than in adults. Overall levels of telencephalic proliferation are much higher in 30d juvenile birds compared to adults, despite the fact that brain size and mass have achieved adult levels by 30 days. These results are consistent with previous work in birds showing that neurogenesis decreases with age, but continues into adulthood (Alvarez-Buylla et al., 1994; Ling et al., 1997). We found that the area of the VZ was significantly larger in juveniles than in adults throughout the telencephalon. Measurements of the total number of silver grains to estimate overall levels of mitotic activity revealed significantly higher levels of cell division within the VZ of juvenile zebra finches compared to adults at the levels of Area X, the Anterior 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Commissure, and HVC (including the VZ immediately above HVC). Our single-cell analysis at the level of the Anterior Commissure showed that the enlarged VZ of juveniles was attributable to a larger number of cells, such that the total number of cells in the VZ decreases over the course of development. The VZ of juveniles also had a larger number of thymidine-labeled cells compared to adults, indicating that the increased incidence of mitotic activity in juvenile birds as revealed by total silver grain counts is due to a larger number of dividing cells in juveniles than in adults. The decrease in VZ cell number may involve either a terminal division of stem cells and their migration away from the VZ and/or the death of stem cells within the VZ. Telencephalic regions not involved with song learning appear to achieve their adult size by approximately 30 days of age in zebra finches, whereas song-control regions continue to show large-scale morphological changes in size and neuron number thereafter, during the period of vocal learning (Bottjer et al., 1985). Because the majority of brain development is complete by 30 days, it is surprising that relatively high rates of proliferative activity within the VZ are maintained in juveniles. In fact, more new cells are generated than could presumably be incorporated into the brain. Therefore, the majority of new cells must die en route to their final destination, or after they have reached their targets. Alternatively, a high level of cell turnover throughout the brain must occur in order to accommodate the arrival and subsequent incorporation of new neurons (i.e., older cells must die, at least within certain phenotypic populations; Alvarez- Buylla et al., 1988). Approximately half of newly generated neurons die between the second and third weeks after their arrival in HVC (Kim et al., 1999), suggesting that newly generated cells from the VZ undergo cell death if they are not incorporated into 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. functional circuitry within some finite period following the end of migration. This idea suggests that there is a discrete window of time in which young neurons produced in the avian telencephalon must receive appropriate trophic support (i.e. growth or activity- dependent factors) if they are to survive (Johnson et al., 2000; Rasika et al., 1999). Sex differences in proliferation. A major finding of the present study is that specific regions of the VZ demonstrate sexually dimorphic cell division in juvenile birds. Previous studies examining cellular proliferation in various sexually dimorphic systems have reported no evidence for differential cell division between males and females (Jacobson and Gorski, 1981; Breedlove et al., 1983; Gorlick and Kelley, 1987), suggesting that the development of sexually dimorphic brain regions may result primarily from sex differences in migration, differentiation, and/or neuron death (Kim and DeVoogd, 1989; Burek et al., 1997). The incidence of newly generated neurons within HVC is sexually dimorphic in juvenile zebra finches nine days after thymidine administration, before most new neurons in a thymidine-labeled cohort have migrated into HVC (Burek et al., 1994). However, it is impossible to know whether this difference results at least in part from sex differences in migration, differentiation, or cell death (for example, many labeled pyknotic cells are seen in areas of migrating neurons in adult canaries (Alvarez-Buylla and Nottebohm, 1988). In general, it is extremely difficult to resolve mechanisms of sexually dimorphic neuron addition since the location of precursor cells within the VZ that give rise to neurons destined for highly specific brain regions such as HVC and Area X is not known. By dividing the VZ into discrete segments we discovered regions of sexually dimorphic proliferation. In particular, we found higher levels of cell division in juvenile males than females within segments of the dorsal and 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ventral VZ at the level of the Anterior Commissure, as well as the ventral VZ at the level of Area X. In general, sexually dimorphic proliferation in the VZ represents what appears to be a rare example of regulation at the level of cell division (cf. van Praag et al., 1999). A large-scale mapping study of the VZ across different ages seems likely to reveal additional areas of sexually dimorphic proliferation. Identifying regions of sexually dimorphic mitotic activity would permit a more sensitive test of the role of steroid hormones on neuronal production, and might alter the conclusions of past studies indicating that sex hormones have little or no influence on cellular proliferation (Goldman and Nottebohm, 1983; Brown et al., 1993; Rasikaet al., 1994). Any selective increase in genesis of neuroblasts fated to become song-control neurons in juvenile males could contribute to the construction of sexually dimorphic song-control regions. Thus, our observation of increased proliferation within restricted regions of the VZ of juvenile males may indicate potential sites of stem cells that give rise to song-control neurons during the sensitive period for vocal learning. This idea is supported by the finding that the incorporation of new neurons into HVC is greater in juvenile males than females (Nordeen and Nordeen, 1988a; to our knowledge it is not known whether the low level of new neuron incorporation in adult zebra finches is dimorphic). Our data indicate that the number of cells (both labeled and unlabeled) in the VZ decreases as zebra finches mature. Therefore we conclude that the number of progenitor cells that are sexually dimorphic in young birds decrease over the course of development. By adulthood, there are no longer regions of sexually dimorphic proliferative activity within the VZ. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Alvarez-Buylla A, Nottebohm F (1988) Migration of young neurons in adult avian brain. Nature 335: 353-354. Alvarez-Buylla A, Theelen M, Nottebohm F (1988) Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning. Proc Natl Acad Sci U S A 85: 8722-8726. Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101-109. Alvarez-Buylla A, Ling CY, Nottebohm F (1992) High vocal center growth and its relation to neurogenesis, neuronal replacement and song acquisition in juvenile canaries. J Neurobiol 23: 396-406. Alvarez-Buylla A, Ling CY, Yu WS (1994) Contribution of neurons bom during embryonic, juvenile, and adult life to the brain of adult canaries: regional specificity and delayed birth of neurons in the song-control nuclei. J Comp Neurol 347: 233-248. Alvarez-Buylla A, Kim JR (1997) Birth, migration, incorporation, and death of vocal control neurons in adult songbirds. J Neurobiol 33: 585-601. Anderson SA, Eisenstat DD, Shi L, Rubenstein JL (1997) Intemeuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278: 474-476. Bohner J (1990) Early acquisition of song in the zebra finch. Animal Behavior 39: 369- 374. Bottjer SW, Glaessner SL, Arnold AP (1985) Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J Neuroscience 5: 1556-1562. Bottjer SW, Miesner EA, Arnold AP (1986) Changes in neuronal number, density and size account for increases in volume of song-control nuclei during song development in zebra finches. Neurosci Lett 67: 263-268. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bottjer SW, Halsema KA, Brown SA, Miesner EA (1989) Axonal connections of a forebrain nucleus involved with vocal learning in zebra finches. J Comp Neurol 279: 312-326. Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anat Rec 166:257-261. Breedlove SM, Jordan CL, Arnold AP (1983) Neurogenesis of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavemosus in rats. Brain Res 285: 39- 43. Brown SD, Johnson F, Bottjer SW (1993) Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels. J Neurosci 13: 2024-2032. Burd GD, Nottebohm F(1985) Ultrastructural characterization of synaptic terminals formed on newly generated neurons in a song control nucleus of the adult canary forebrain. J Comp Neurol 240: 143-152. Burek MJ, Nordeen KW, Nordeen EJ (1994) Ontogeny of sex differences among newly- generated neurons of the juvenile avian brain. Brain Res Dev Brain Res 78: 57-64. Burek MJ, Nordeen KW, Nordeen EJ (1997) Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death. J Neurobiol 33: 61-71. Cameron RS, Rakic P (1991) Glial cell lineage in the cerebral cortex: a review and synthesis. Glia 4: 124-137. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97: 703-716. Fishell G, Mason CA, Hatten ME (1993) Dispersion of neural progenitors within the germinal zones of the forebrain. Nature 362: 636-638. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Garcia-Venfugo JM, Doetsch F, Wichterie Ht Lim DA, Alvarez-Buylla A (1998) Architecture and cell types of the adult subventricular zone: in search of the stem cells. J Neurobiol 36: 234-248. Goldman SA, Nottebohm F(1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc Natl Acad Sci U S A 80: 2390-2394. Gorlick DL, Kelley DB (1987) Neurogenesis in the vocalization pathway of Xenopus laevis. J Comp Neurol 257:614-627. Herrmann K, Bischof HJ (1986) Delayed development of song control nuclei in the zebra Finch is related to behavioral development. J Comp Neurol 245: 167-175. Jacobson CD, Gorski RA (1981) Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat. J Comp Neurol 196:519-529. Johnson F, Norstrom E, Soderstrom K (2000) Increased expression of endogenous biotin, but not BDNF, in telencephalic song regions during zebra finch vocal learning. Brain Res Dev Brain Res 120: 113-123. Kim J, O’ Loughlin Kasparian BS, and Nottebohm F (1994) Cell death and neuronal recruitment in the high vocal center of adult male canaries are temporally related to changes in song [see comments]. Proc Natl Acad Sci U S A 91: 7844-7848. Kim JR, DeVoogd TJ (1989) Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J Neurosci 9: 3176-3187. Kim JR, Fishman Y, Sasportas K, Alvarez-Buylla A, Nottebohm F (1999) Fate of new neurons in adult canary high vocal center during the first 30 days after their formation. J Comp Neurol 411: 487-494. Lavdas AA, Grigoriou M, Pachnis V, Pamavelas JG (1999) The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex. J Neurosci 19: 7881-7888. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ling C, Zuo M, Alvarez-Buylla A, Cheng MF (1997) Neurogenesis in juvenile and adult ring doves J Comp Neurol 379:300-312. Lois C, Alvarez-Buylla A (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A 90: 2074-2077. Neyt C, Welch M, Langston A, Kohtz J, Fishell G (1997) A short-range signal restricts cell movement between telencephalic proliferative zones. J Neurosci 17:9194-9203. Nordeen KW, Nordeen EJ (1988a) Projection neurons within a vocal motor pathway are bom during song learning in zebra finches. Nature 334: 149-151. Nordeen EJ. Nordeen KW ( 1988b) Sex and regional differences in the incorporation of neurons bom during song learning in zebra Finches. J Neurosci 8:2869-2874. Nottebohm F, Arnold AP (1976) Sexual dimorphism in vocal control areas of the songbird brain. Science 194: 211-213. Nottebohm F, Kelley DB, Paton JA (1982) Connections of vocal control nuclei in the canary telencephalon. J Comp Neurol 207:344-357. O'Rourke NA, Sullivan DP, Kaznowski CE, Jacobs AA, McConnell SK (1995) Tangential migration of neurons in the developing cerebral cortex. Development 121: 2165-2176. Paton JA, OLoughlin BE, Nottebohm F (1985) Cells bom in adult canary forebrain are local intemeurons. J Neurosci 5: 3088-3093. Rasika S, Nottebohm F, Alvarez-Buylla A (1994) Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries. Proc Natl Acad Sci U S A 91: 7854-7858. Rasika S, Alvarez-Buylla A. and Nottebohm F (1999) BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain. Neuron 22: 53-62. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Smart I (1961) The subependymal layer in the mouse brain and its cell production as shown by autoradiography after thymidine-3 H injection. J Comp Neurol 116: 325-347. Smart I, Sturrock RR (1979) Ontogeny of the neostriatum. In: Divac I, Oberg RGE. (The Neostriatum, ed), pp 127-146. Oxford: Pergamon Press. Striedter GF, Marchant TA, Beydler S (1998) The "neostriatum" develops as part of the lateral pallium in birds. J Neurosci 18:5839-5849. Szele FG, Chesselet MF (1996) Cortical lesions induce an increase in cell number and PSA-NCAM expression in the subventricular zone of adult rats. J Comp Neurol 368: 439-454. van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2: 266-270. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2: A map of proliferative activity throughout the proliferative ventricular zone of juvenile zebra (Inches The sensitive period for vocal learning in zebra finches extends from -20-90 days after hatching during which time young males learn to sing a close copy of their father’ s song (Bdhner, 1990). Young females never leam to produce song behavior. The neural substrate underlying vocal learning in songbirds, the song-control system, is highly sexually dimorphic (Nottebohm and Arnold, 1976; Nottebohm et al. 1982; Bottjer et al., 1989). However, the exact contribution that cell proliferation (DeWulf and Bottjer, 2001), neuron addition (Nordeen and Nordeen, 1988 a; b), differential cell death (Kim and DeVoogd, 1989; Burek et al., 1994; 1997), and cell migration (Burek et al.,1994) makes the development of the sexually dimorphic song-control system is unclear. Our original quantitative analysis of mitotic activity in zebra finches revealed small regions of the VZ containing sexually dimorphic proliferation in juveniles but not adults (DeWulf and Bottjer, 2001). Specifically, higher levels of thymidine labeling in juvenile males compared to juvenile females were observed in restricted regions of the W Z and DVZ at the levels of Area X and the Anterior Commissure. Identifying sexually dimorphic mitotic activity in juveniles led us to conceive this large-scale mapping study in which cellular proliferation was measured along the rostral-caudal axis of the telencephalic VZ in 30 day old zebra finches. In this experiment we systematically compared levels of thymidine labeling in juvenile males and females to construct a map which indicated relative levels mitotic activity throughout the anterior-posterior and dorsal-ventral axis of the telencephalic VZ. Increased levels of proliferation within the VZ of males may contribute to the 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. construction of the song-control system during the period of song learning. Our map revealed that small segments of the VZ had higher levels of thymidine labeling in males than females that were primarily localized to the anterior half of the telencephalon, particularly near Area X. These regions of increased proliferation are presumably due to a larger number of dividing progenitors in males than females. This observation suggests that sex differences in cellular proliferation within the VZ may contribute to the development of the song-control nuclei in males and thus, regions of sexually dimorphic proliferation may offer clues to the location of the progenitors that give rise to song- control neurons. Additionally, our data indicate that relative levels of proliferation throughout the telencephalic VZ differ along the anterior-posterior axis suggesting that different mechanisms may regulate neurogenesis throughout the telencephalon. Materials and Methods Thymidine labeling and tissue preparation. The tissue used in this study was originally prepared as part of a previous study, DeWulf and Bottjer (2001). Briefly, seven male and six female juvenile zebra finches were taken from our breeding colony at 30 days of age (range 28-32 days) to measure levels of mitotic activity adjacent to the lateral ventricles within the telencephalon. Each bird received a single intramuscular injection of 'H-thymidine (2.5 p.Ci/g dose, specific activity 6.7 Ci/mmol; New England Nuclear or ICN) and was killed two hours later. All birds were overdosed with a barbiturate anesthetic (Equithesin) and transcardially perfused with avian saline followed by 2% paraformaldehyde/2% glutaraldehyde fixative. Brains were removed, bisected along the midline, and the left hemisphere was post-fixed in buffered formalin for several 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. days and then embedded in paraffin. To compensate for possible variation during tissue processing (e.g. emulsion batches, lots of ^-thymidine) all procedures included at least one animal from each group. Coronal sections were cut on a rotary microtome at a thickness of 10 pm, placed on chrome-aium subbed slides, and immersed in a series of xylenes and graded alcohols for paraffin removal. Every third series of five consecutive sections was mounted; thus the sampling interval between slides was 150 pm. The slides were dipped in nuclear track emulsion (Kodak NTB2, Eastman Kodak Co.), stored at 4°C for 3-8 weeks, developed (Kodak D19), and counterstained with thionin. We ensured that all tissue was cut at the same angle by only analyzing brains in which specific nuclei in the dorsal telencephalon and in the ventral portion of the tectum were present in individual sections at the levels of both the Anterior Commissure and HVC. Analysis of autoradiograms AH analyses of cellular proliferation were performed using a computer-aided imaging system (Analytical Imaging Concepts) yoked to a microscope. The proliferative epithelium adjacent to the lateral ventricles within the telencephalon, the Ventricular Zone (VZ), contains mitotically active progenitors (Boulder Committee. 1970; Goldman and Nottebohm, 1983). In our previous study we presented two major findings. First, regions of sexually dimorphic proliferation occur within the VZ of juvenile but not adult zebra finches at the levels of Area X and the Anterior Commissure. This discovery raised the question of whether sex differences in proliferation might also occur within other restricted regions of the VZ that were not observed in our initial study. Second, mitotic activity within the VZ was highly spatially differentiated. For example, levels of 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. proliferation at the Anterior Commissure were highest in the ventral portion of the VZ within the striatum. We wanted to analyze proliferative activity throughout the VZ in order to determine how much of a quantitative difference in proliferative activity exists between cortex and striatum and where along the rostral-caudal axis this difference first arises? As a first step in exploring these questions, we mapped the incidence of thymidine labeling within the VZ along the lateral wall of the lateral telencephalic ventricle of juvenile birds. Measuring levels of proliferation throughout the telencephalic VZ. Proliferation within the telencephalic VZ spanning a distance of approximately 5 mm along the rostral- caudal axis was measured in 2-3 sections per slide starting anterior to Area X and ending at rostral to HVC with a 150-jun sampling interval occurring between slides. We divided the VZ into three different sub-regions: (1) the ventral VZ (WZ) extended from the ventral tip of the VZ to the dorsal medullary lamina (LMD), (2) an intermediate region of the VZ (IntVZ) was situated between the LMD and the lamina hyperstriatal (LH), and (3) the dorsal VZ (DVZ) extended from the LH fiber tract to the dorsal tip of the VZ (Fig. 2.1). LMD and LH contact the VZ at different dorsal-ventral locations depending upon the anterior-posterior location within the brain and thus the linear length of each sub- region changes along the anterior-posterior axis (see Fig. 2.1). At posterior levels of the telencephalon, immediately before HVC, it was difficult to determine whether the distal tips of the VZ were DVZ and W Z because the locations where LH and LMD contacted the VZ were ambiguous. We therefore excluded the DVZ and W Z from our analysis in the two slides immediately anterior to HVC to avoid including parts of the DVZ and W Z in our IntVZ measurements at these posterior levels of the telencephalon. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HVC LH LH - - V Str AC Cx Fig. 2. 1. Schematic coronal sections through the zebra finch brain at the levels of a. Area X, b. the Anterior Commissure, and c. HVC. Proliferation in the telencephalon occurs in the epithelium adjacent to the lateral ventricle, the VZ (black arrows), and the sub-ventricular zone (gray arrows). Asterisks indicate the locations of thymidine labeled cell clusters in the brain parenchyma. Abbreviations: Cx, cortex; Str, striatum; Archi, archistriatum; Cb, cerebellum; V , lateral ventricle; LH, lamina hyperstriatica; LMD, lamina meduUaris dorsalis; LAD, lamina archistriatalis dorsalis; AC, Anterior Commissure; DH, dorsal hom; Int, intermediate; V H , ventral hom. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To measure the incidence of mitotic activity along the DVZ and W Z , the linear length of each sub-region was divided into 10 bins. The total number of silver grains in the resulting 10 bins represented the incidence of proliferation in 10% increments along the length of each VZ sub-region, thereby allowing us to compare levels of mitotic activity for corresponding locations within the VZ among animals. The linear length of the IntVZ was shorter in the anterior half of the telencephalon than the posterior half. We therefore divided the IntVZ into 5 bins from Area X to the Anterior Commissure and into 10 bins posterior to the Anterior Commissure. Occasionally a segment of VZ was damaged during tissue processing making it impossible to accurately measure levels of proliferation within the sub-region that had sustained damage. As a result we only included animals in which we could trace two (minimum) to three (maximum) sections per VZ sub-region in our analysis. Therefore, the number of animals in which data was collected for each VZ sub-region differed along the rorstral-caudal axis. To ensure that the silver grain counts accurately represented cellular proliferation uncontaminated by background labeling, the density of silver grains within a large region of brain parenchyma (- 14,000 pm2 ) more than 200 pm away from the VZ was measured for each section analyzed, excluding any labeled glial or endothelial cells. Separate background measurements were taken within the brain parenchyma associated with each VZ-subregion. That is, the background measurement associated with the W Z was taken within the striatum while background measurements for the IntVZ and the DVZ were taken within cortex. The background measures were then used to estimate the total number of silver grains that would be expected within a traced area. The expected number of silver grains was then subtracted from the raw silver grain counts within the 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VZ. Since the total number of silver grains is a direct measure of thymidine uptake (and thus an estimate of mitotic activity), this procedure enabled us to determine the overall incidence of cell division within each VZ sub-region above and beyond background thymidine labeling. A visual representation of proliferation throughout the zebra finch telencephalon Wire Frame Reconstruction of the VZ: In addition to measuring levels of proliferation throughout the telencephalon, we were interested in observing the anatomical changes that occur in the VZ across the rostral-caudal axis. To do this, we outlined each VZ sub-region in one section per slide throughout the anterior-posterior axis in two males and two females using the Neurolucida image analysis program (MicroBrightfield. Inc.). The outline was then filled with different colors to delineate each VZ sub-region. Aggregations of silver grains within the W Z, DVZ, and IntVZ were also outlined and filled with a different color. In this way we produced a wire frame reconstruction of the VZ that allowed us to visualize the shape of the VZ as well as highlighted areas containing high densities of silver grains along the anterior-posterior neuraxis. A semiquantitative representation o f mitotic activity in the VZ: While our wire frame reconstruction of the VZ enabled us to visualize gross changes in proliferative activity across the anterior-posterior axis in single animals, relative differences in cell division were not easily recognized. We therefore constructed a semiquantitative representation of mitotic activity within each sub-region of the VZ throughout the anterior-posterior axis to facilitate the comparison of levels of proliferation within specific segments of the VZ between males and females. In this semi-quantitative map, 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. relative levels of proliferation for each bin were represented as gray scale values. The highest level of mitotic activity, which occurred within a single bin of the W Z of males, was used as the baseline measurement to which all other measures of mitotic activity in individual bins of both males and females were compared. Each bin was assigned a gray value, from a scale of 100 gray levels, based upon the percent of baseline labeling with black representing the highest level of thymidine labeling, white representing the lowest levels, and different shades of gray representing levels in between. This semi-quantitative map of proliferative activity in the VZ allowed us to observe how levels of proliferation change across the dorsal-ventral and anterior-posterior neuraxis, the location of proliferative hot spots, and locations of the VZ containing sexually dimorphic cell division. Results Area X, the Anterior Commissure, and HVC were used as anatomical markers along the rostral-caudal axis (Fig. 2.1), and every slide that occurred anterior to a particular marker level was designated minus the number of slides anterior to that level. For example, one slide anterior to the Anterior Commissure was considered AC-1 whereas one slide posterior to the Anterior Commissure was designated HVC-12 (e.g.. Fig. 2.2). Slides within marker regions were numbered in an anterior-posterior order such that the slides at Area X, the Anterior Commissure, and HVC were designated AX 1-3, AC 1-2, and HVC 1-3 respectively. In this paper we characterize levels of mitotic activity throughout the telencephalic VZ in three ways. In the first section we describe the overall pattern of 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thymidine labeling within each VZ sub-region across the anterior-posterior axis. The second section describes levels of mitotic activity within discrete segments or bins in each VZ sub-region collapsed across the levels of Area X. the Anterior Commissure, and HVC. Additionally, we report the occurrences of sexually dimorphic proliferation at individual levels of the telencephalon. In the third section we present two reconstructions of the VZ across the anterior-posterior axis of the telencephalon that highlight regions of thymidine labeling. The first reconstruction qualitatively demonstrates thymidine labeling within the VZ of a single male while the second reconstruction presents a semi quantitative representation of mitotic activity within the VZ of both males and females. Overall pattern of thymidine labeling throughout the brain: VZ Area: The area of the DVZ was similar between males and females throughout the anterior-posterior axis of the telencephalon with a slight increase occurring at the level of the Anterior Commissure (Fig. 2.2). Posterior to the level of the Anterior Commissure the DVZ area remained stable and then decreased until the DVZ was no longer evident. The IntVZ was relatively small at anterior to mid levels of the telencephalon in both males and females (Fig. 2.2). Posterior to the Anterior Commissure the area of the IntVZ increased along the anterior-posterior axis reaching maximum values at the level of HVC, at which point the entire VZ was made up of the IntVZ. The area of the W Z was roughly equivalent between males and females with maximum levels occurring between Area X and the Anterior Commissure (Fig. 2.2). Starting slightly before the Anterior Commissure the area of the W Z decreased along the rostral-caudal axis until it was no longer evident anterior to HVC. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. 0 4 E 6. S s < 2 0 4 E 3 2 < 2 90000 - 60000 - 30000 0 90000 - 60000 - 30000 DVZ A nterior Commissure Female Area X -3-2-1 1 2 3 -8 -7 -6 -5 -4 -3 -2-1 1 2 -12-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 IntVZ H V C AreaX J i l J U L A nterior Commissure -4 -3 -2 -1 1 2 3 -8 -7 -6 -5 -4 -3 -2 -1 1 2 -12-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 CM E 3 90000 - 60000 - 30000 - w z Area X Anterior Commissure o O' -4 -3 -2 -1 1 2 3 -8 -7 -6 -5 -4 -3 - 2- 11 2 -12-11 -10 -9 -8 -7 -8 -5 -4 -3 -2 -1 1 2 3 Fig. 2.2. The overall VZ area within the DVZ, IntVZ, and VVZ throughout the anterior-posterior axis o f the telencephalon (mean ± SE). Levels ofThvmidine Labeling: Overall levels of proliferation within the DVZ varied as a function of the anterior-posterior level with the highest levels of proliferation occurring near the level of the Anterior Commissure (Fig. 2.3). There was a bias in favor of males having higher levels of mitotic activity than females at some levels of the DVZ For example, males frequently demonstrated a 2-fold increase proliferative activity in the anterior half of the telencephalon (particularly near Area X). Thymidine labeling within the IntVZ was low throughout most of the telencephalon with the lowest values occurring posterior to the Anterior Commissure (Fig. 2.3). Starting several slides anterior to HVC the total number of silver grains in the IntVZ at each level increased with maximum levels of proliferation occurring at HVC (at which point the entire VZ was made up of IntVZ). Males had slightly higher levels of thymidine labeling in the anterior half of the telencephalon but at posterior levels, near HVC, there was a bias in favor of females demonstrating higher levels of mitotic activity. The VVZ contained the highest levels of thymidine labeling among the three VZ sub-regions (Fig. 2.3). Thymidine labeling increased within the W Z from anterior levels of the telencephalon to Area X, remained fairly stable from Area X to the Anterior Commissure, and then decreased precipitously posterior to the Anterior Commissure. Silver grain counts indicated a strong bias in favor of males demonstrating higher levels of mitotic activity than females within the W Z from anterior to mid levels of the telencephalon. The 2-fold increase in thymidine labeling within the VVZ of males at Area X is consistent with the significantly higher levels of proliferative activity in juvenile males relative to juvenile females at rostral levels of Area X (DeWulf and Bottjer, 2001). 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. • 4000 0 3 0 0 0 Female Anterior Commissure >2000 - A r ea X «4000 § 3 0 0 0 1 2000 2 1000 - 3 - 2 - 1 1 2 3 -8 -7 -6 -5 -4 -3 - 2 - 1 12 -12-11-10 -9 -8 -7 -8 -5 -4 -3 -2 -1 1 2 3 IntVZ Area X I fc fe f t asi Anterior Commissure H V C i i i •>4000 c -4 -3 -2 -1 1 2 3 - 8 -7 -6 -S -4 -3 -2 -1 1 2 -12-11 -10 -9 -8 -7 -6 -S -4 -3 -2 -1 1 Anterior Commissure AreaX >2000 75 1000 o 0 0 -4 -3 -2 -1 1 2 3 -8 -7 -6 -5 -4 -3 -2 -1 1 2 -12-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 Fig. 2 J . The total number of silver grains within the DVZ, IntVZ, and VVZ throughout the anterior-posterior axis of the telencephalon (mean ± SE). Proliferation at the levels of Area X, the Anterior Commissure, and HVC The overall measures of thymidine labeling indicate that males have higher levels of proliferation than females throughout much the W Z , particularly at the level of Area X and at the rostral level of the Anterior Commissure (see Fig. 2.3). This is consistent with our earlier work that demonstrated sexually dimorphic proliferation within the W Z at Area X and at the Anterior Commissure (DeWulf and Bottjer, 2001). Our overall measures of proliferation within the DVZ at the Anterior Commissure indicated that levels of proliferation are roughly equivalent between males and females (see Fig. 2.3). However, in our previous work, when we sub-divided the DVZ into 10 bins sexually dimorphic proliferation was observed in bin 1 immediately above LH (DeWulf and Bottjer, 2001 ). Thus the other nine bins within the DVZ at the Anterior Commissure which did not exhibit sexually dimorphic proliferation swamped out the true sex difference above LH. Within the IntVZ, females had higher levels of thymidine labeling than males at posterior levels of the telencephalon at HVC suggesting a female biased sex difference in proliferation at the level of HVC. Based upon previous findings (DeWulf and Bottjer, 2001) and our current pattern of data and we were curious if sexually dimorphic proliferation occurred at the levels of Area X, the Anterior Commissure, and HVC. We therefore divided the DVZ, IntVZ, and W Z into individual bins and collapsed our bin measurements across slides at Area X (AX1-AX3), the Anterior Commissure (AC1-AC2), and HVC (HVC1-HVC3). This procedure allowed us to compare levels of mitotic activity within individual bins across the anterior-posterior axis at each marker level among birds. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Area X: Although the total number of silver grains within the DVZ was higher in males than females (Fig. 2.3), when we divided the DVZ into bins there was no effect of sex or a sex by bins interaction (sex: FUo = 1.37, p = 0.27; sex by bins: F < l)(Fig. 2.4). However, there was an effect of bins (F9 . 9 0 = 2.85, p = 0.005). Levels of thymidine labeling in 6 out of 10 bins were higher in males compared to females and, while these differences were not significant, bin 2 demonstrated a marginal sex difference in thymidine labeling (t|0 = 2.02, p = 0.07). Proliferation within the IntVZ demonstrated no main effects of sex, bins, or a sex by bins interaction (sex: F < 1; bins: F4 j 2 = 1.98, p = 0.12; sex by bins: F4 J2 = 1.24, p = 0.32)(Fig. 2.4). Despite an approximate 2-fold increase in mitotic activity in bins I, 2, and 4 in males compared to females, these differences were not significant. Thymidine labeling within the VVZ showed no effect of sex, bins, or a sex by bins interaction (sex: F|.7 = 2.50, p = 0.16; bins: F 9 . 6 3 = 1.75, p = 0.10; sex by bins: F < 1) and bins I, 2, and 5 demonstrated marginally higher levels of thymidine labeling in males compared to females (bin 1 : t7 = 2.28, p = 0.06; bin 2: t7 = 2.09, p = 0.08; bin 5: t7 = 2.00, p = 0.09). In our previous study, sex differences occured in the ventral half of the VVZ (i.e. bins l-5)(DeWulf and Bottjer, 2001). However, these data were collected within the VVZ at rostral levels of Area X while our current data represent proliferation in the W Z throughout Area X. Thus, the tendency towards sexually dimorphic proliferation within the VVZ must be due to the measurements taken from rostral levels of Area X. When we collapse our W Z data across the rostral two levels at Area X (i.e. AX1-AX2), we observed sexually dimorphic proliferation in bins 1 , 2,4, and 5 and a tendency towards a sex difference in bins 3,6,7, and 8 (bin 1 : t9 = 2.46, p = 0.04; bin 2: t9= 2.40, p = 0.04; 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bin 3: tq= 1.89, p = 0.09; bin 4: t*= 2.50, p = 0.04; bin 5: t*= 3.02, p = 0.02, bin 6: t, = 2.07; p = 0.07; bin 7: t, = 2.13; p = 0.06; bin 8: t, = 2.12; p = 0.06)(Fig. 2.4b). 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a. Area X 0 g 3 0 0 - 1 O 2 7 .200 - Female 100 - B in 1 Bin 2 B in 3 B in 4 B in 5 B in 6 Bin 7 B in 8 Bin9Bin10 w fi £ • § 3 0 0 J ?200 - 5 J100 - o C O 0 IntVZ *8 §0200 - . ■ t ® H a s . B in 1 Bin 2 Bin 3 B in 4 BinS 300 - r 2 1 0 0 - fc § 3 0 0 e fi I < 5 200 - B in 1 Bin 2 B in 3 B in 4 B in 5 B in 6 B in 7 B in 8 Bin 9 B in 10 b . Rostral Area X * = >100 - B in 1 Bin2 Bin3 Bin4 BinS Bin6 Bin7 Bin8 Bin9 B in 10 Fig. 2.4. Histograms of the total number of silver grains within each bin of the a. DVZ, IntVZ, and W Z at the level of the Area X (mean ± SE). b. Demonstrates levels of thymidine labeling collapsed across the levels o f AX1-AX2 (mean ± SE). Asterisks indicate significant differences in thymidine labeling between juvenile males and females in W Z bins 1,2,4, and 5 (b.). 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Anterior Commissure: Thymidine labeling within the DVZ at the level of the Anterior Commissure revealed no main effect of sex or a sex by bins interaction (sex: F < 1 ; sex by bins: FW| = 1.04, p = 0.42) but there was an effect of bins (bins: F9 . 8i = 4.20, p = 0.0002)(Fig. 2.5). A 4-fold increase in proliferation in males compared to females occurred in bin I immediately dorsal to LH (bin 1 : = 2.42, p = 0.04). This sex difference is consistent with our previous work in which the proportion of dividing cells immediately above LH was higher in juvenile males than juvenile females (DeWulf and Bottjer, 2001). The pattern of mitotic activity throughout the IntVZ at the level of the Anterior Commissure demonstrated no effect of sex, bins, or a sex by bins interaction (sex: F1 . 1 0 = 1 -64, p = 0.230; bins: F < 1; sex by bins: F < l)(Fig. 2.5). No bin within the IntVZ demonstrated sexually dimorphic proliferation. Although there was a 2-fold increase in levels of thymidine labeling at the rostral-most level of the Anterior Commissure (AC1, Fig. 2.3), proliferation was roughly equivalent between males and females at AC2. Therefore, when we collapsed across the two slides there was no difference in mitotic activity between males and females. Thymidine labeling within the W Z at the level of the Anterior Commissure revealed a main effect of bins (F9 . 8 1 = 6.21, p < 0.0001) but no effect of sex or a sex by bins interaction (sex: F1 . 9 = 2.34, p = 0.16; sex by bins: F < I)(Fig. 2.5). When we divided the VVZ into segments, we observed a bias towards males having higher levels of mitotic activity compared to females in all bins, with a significant sex difference occurring in bin 8 (t9 = 2.58, p = 0.03). The increased levels of thymidine labeling in all bins of males is consistent with the observation that higher overall levels of proliferation 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. occur in males compared to females at the rostral level of the Anterior Commissure in AC1 (Fig. 2.3). ft 2600 | C § 0 4 0 0 - Anterior Commissure Female m £ 200 - Bin 1 B in 2 B in 3 B in 4 Bin 5 Bin 6 B in 7 B in 8 B in 9 Bin 10 ft 2600 c 2 § 0 4 0 0 1 1 200 - li» • “ *5 o ft 2600 IntVZ Bin 1 Bin 2 Bin 3 B in 4 Bin 5 § 0 4 0 0 W Z 5 * 2 0 0 H o <0 0 w Bin 1 B in 2 B in 3 B in 4 Bin 5 Bin 6 Bin 7 Bin 8 B in 9 Bin 10 Fig. 2.5. Histograms of the total number o f silver grains within each bin o f the DVZ, IntVZ, and W Z at the level of the Anterior Commissure (mean ± SE). 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HVC: The pattern of mitotic activity at the level of HVC demonstrated main effects of bins and a sex by bins interaction (bins: F96i = 7.80, p < 0.0001; sex by bins: F9 - 6 3 = 3.71, p = 0.0009) indicating that sex differences in proliferation are dependent upon location within the IntVZ (Fig. 2.6). There was no main effect of sex (F|. 7 = 1.17, p = 0.32). Although there was a consistent trend for increased proliferation in females compared to males in bins 7-10, only bin 10 demonstrated significantly higher levels of proliferation in females (t7 = 2.48, p = 0.04). fe e 450 E o 300 3 Z. 2 | 150 is o _ _ _______ ____________ B in 1 B in 2 Bin 3 B in 4 B in 5 B in 6 B in 7 B in 8 B in 9 B in 10 Fig. 2.6. Histograms of the total number o f silver grains within each bin of the IntVZ at HVC (mean ± SE). M ale Female IntVZ 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sex Differences in Thymidine Labeling We were curious if sex differences in thymidine labeling could be localized to individual levels of the telencephalon since sexually dimorphic proliferation seems to be highly dependent upon the anterior-posterior level observed. We therefore divided each VZ sub-region into bins and compared levels of mitotic activity within each bin of each sub-region between males and females across the anterior-posterior axis. To our surprise, we only found a handful of bins that contained sexually dimorphic proliferation throughout the entire VZ (4 in the DVZ, 3 in the IntVZ, and 3 in the W Z). Nine out of the ten sex differences that we observed at individual levels occurred in the anterior half of the telencephalon while one was located posteriorly at HVC suggesting that sexually dimorphic proliferation is primarily localized to the anterior half of the telencephalon. Sex differences near Area X: Sex differences in levels of thymidine labeling were observed within the DVZ at the levels of AX-2 and AX1. There was no effect of sex, bins or a sex by bins interaction at either AX-2 or AX1 (AX-2 sex: F < I; bins: F < 1; sex by bins: F9 . 7 2 = 1 -59, p = 0.14)(AX 1 sex: Fu = 3.78, p = 0.09; bins: F < 1; sex by bins: F < l)(Fig. 2.7a). However, bin 5 at AX-2 and bin 8 at AX1 demonstrated higher levels of thymidine labeling in males compared to females (AX-2 bin 5: t8 = 2.29, p = 0.05; AX1 bin 8: t7= 2.61, p = 0.04). The W Z at AX-1 demonstrated no effect of sex or a sex by bin interaction (sex: F1 . 6 = 1 .04, p = 0.35; sex by bin: F 9 . 5 4 = 1.20, p = 0.32) but there was an effect of bins (F9 . 3 4 = 2.31, p = 0.03) and a sex difference in proliferation in bin 8 (t7 = 2.79, p = 0.03)(Fig. 2.7b). Thymidine labeling within the VVZ at AX2 revealed no effect of sex, bins, or a sex by bins interaction (sex: Fi. 7 = 2.15, p = 0.19; bins: F < 1 ; 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sex by bins: F < 1). Although proliferation was two times higher in males than females across the length of the VVZ at AX2, only bin 5 demonstrated a significant sex difference (ts= 2.85, p = 0.03)(Fig. 2.7b). Sex differences between Area X and the Anterior Commissure: When we subdivided the DVZ at AC-4 into 10 bins there was no overall effect of sex, bins, or a sex by bins interaction (sex: Ft.|0 = 2.77, p = 0.13; bins F9 . 9 0 = 1.41, p = 0.19; sex by bins: F < I)(Fig. 2.7c). Despite an overall increase in levels of proliferation throughout the DVZ of males at AC-5, only bin 10 demonstrated higher levels of proliferation in males compared to females (tio= 3.03, p = 0.01). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. w Bird Bin 2 Bin 3 B in 4 Bin 5 Bin 6 B in 7 Bin 8 B in 9 Bin 10 Fig. 2.7. Histograms o f the total number of silver grains within each bin of the a. DVZ near the level o f Area X, b. W Z near the level of Area X, and c. the DVZ between Area X and the Anterior Commissure at AC-5 (mean ± SE). Asterisks indicate significant differences in thymidine labeling between juvenile males and females in DVZ AX-2 bin 5, DVZ AX1 bin 8 (a.), W Z AX-l bin 8, W Z AX2 bin 5 (b.), and DVZ AC-5 bin 10 (c.). 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sex differences at the Anterior Commissure: When we collapsed our binned DVZ data across slides at the levels of the Anterior Commissure, we found a sex difference in proliferation immediately above LH in bin I (see above). We therefore subdivided the DVZ at the levels of AC1 and AC2 into 10 bins to determine if the sex difference above LH occurred at one or both levels at the Anterior Commissure. Our binned data indicated that a significant sex difference in proliferation above LH was primarily localized to AC1 (Fig. 2.8a). Although proliferation within the DVZ at AC1 revealed no main effect of sex or a sex by bins interaction (sex: F < 1; sex by bins F < I), there was a main effect of bins ( F 9 . 7 2 = 2.89, p = 0.006) and bin 1 immediately above the LH fiber tract contained more silver grains in males than females (t8= 3.04, p = 0.02)(Fig. 2.8a). Sexually dimorphic proliferation within the IntVZ was observed at AC1 only. The overall pattern of thymidine labeling revealed a marginal effect of sex but no effect of bins, or a sex by bins interaction (sex: F| 9 = 4.20, p = 0.07; bins F < 1; sex by bins F»j6 = LI 1 , p = 0.37)(Fig. 2.8a). Bins I and 2, immediately above LMD, demonstrated a 3-fold increase in the total number of silver grains in males compared to females (bin 1 : ^ = 2.30, p = 0.05; bin 2: W = 2.46, p = 0.04). When we divided the W Z at AC1 and AC2 into individual bins, we saw sexually dimorphic proliferation at AC1 only. The pattern of thymidine labeling within VVZ revealed an effect of bins ( F 9 . 8 1 = 7.20, p < 0.0001) but there was no effect of sex or a sex by bins interaction (sex: F1 . 9 = 2.29, p = 0.16; sex by bins: F < l)(Fig. 2.8a). Although there was a bias in favor of males having higher levels of proliferation throughout the VVZ at ACL only bin 8 demonstrated a sex difference in thymidine labeling (t9= 2.38, p = 0.04). This sex difference is consistent with results from our 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. collapsed W Z data at the level of the Anterior Commissure (see above) and with results from our previous paper (DeWulf and Bottjer, 2001). Sex differences at HVC: Silver grain counts within the IntVZ at HVC 1 demonstrated no effect of sex but there was an effect of bins and a sex by bins interaction (sex: Fw = 2.43, p = 0.16; bins: F9 . 7 2 = 7.10, p < 0.0001; sex by bins: F9 . 7 2 = 3.36, p = 0.002) (Ftg. 2.8b) indicating that sexually dimorphic proliferation is related to the location along the VZ. Increased levels of proliferation in females extend from bins 7-10, and bin 10 demonstrated a significant sex difference with females having a higher level of mitotic activity than males (tg=2.50, p = 0.04)(Fig. 2.8b). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bin 1 Bin 2 Bin 3 Bin 4 Bin 5 Bin 6 Bin 7 Bin 8 Bin 9 Bin 10 * §600 IntVZ i $ 4 0 0 - ■ £200 c 600 E 0 4 0 0 - 200 - Bin1 B in 2 Bin 3 B in 4 B in 5 B in 6 Bin 7 B in 8 Bin 9 B in 10 s g 600 *>Jfl£C1 E 0 400 = § 200 - B in 1 Bin2 Bin 3 Bin4 BinS Bin6 Bin7 Bin8 Bin9 B in 10 Fig. 2.8. Histograms o f the total number of silver grains within each bin of the a. DVZ, IntVZ, and W Z at the levels of the Anterior Commissure and b. HVC (mean ± SE). Asterisks indicate significant differences in in thymidine labeling between juvenile males and females in DVZ bin 1, IntVZ bins I and 2, W Z bin 8 at the level of the Anterior Commissure (a.), and significantly more silver grains in IntVZ bin 10 o f females compared to males HVC (b.). 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A Visual Representation of Proliferation within the VZ We created a wire frame reconstruction of the VZ throughout the telencephalon to visualize the relative levels of mitotic activity across the anterior-posterior axis. We therefore traced the perimeter of each VZ sub-region in four birds (males n = 2, females n = 2) and separately outlined aggregations of silver grains within the VZ to demonstrate locations of proliferative activity. VZ Unear Leneth: The general appearance of the VZ and distribution of silver grains within the VZ were similar among all birds analyzed. One striking feature of our VZ reconstruction was the variation in linear length of each VZ sub-region across the anterior-posterior neuraxis (Fig. 2.9). For example, the length of the DVZ increased from anterior to mid levels of the telencephalon. The DVZ length then decreased at posterior levels until it was absent posterior to HVC. Changes in the W Z linear length were similar to those seen in the DVZ. That is, the VVZ length increased from anterior to mid levels of the telencephalon where the length remained fairly constant and then decreased rapidly until the W Z was no longer present at posterior levels (Fig. 2.9). The prominent out-pocketing that we observed in the W Z at the level of the Anterior Commissure (DeWulf and Bottjer, 2001) was apparent in our reconstruction. Changes in the linear length of the IntVZ complemented those in the W Z and DVZ. The IntVZ was small at anterior levels of the telencephalon but the length increased at mid levels and by posterior levels of the telencephalon the IntVZ was the only proliferative zone present (Fig. 2.9). Thymidine Labeling in the VZ: The overall measures of mitotic activity within the VZ demonstrated that the highest levels of thymidine labeling within the DVZ and VVZ occurred in the anterior half of the telencephalon (Fig. 2.3). This pattern of results 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was also evident in our reconstruction such that the amount of thymidine labeling within the DVZ and W Z appeared to be highest at anterior levels (Fig. 2.9). Thymidine labeling within the IntVZ of our reconstruction indicated moderate levels of proliferation at anterior levels and lower at posterior levels of the telencephalon (Fig. 2.7). However, our overall silver grain counts indicate that proliferation was in fact fairly low at anterior levels of the telencephalon relative to posterior levels (Fig. 2.3). This discrepancy in results is presumably due to the different sampling methods used in the two analyses. That is, two to three sections per slide were measured to determine overall levels of proliferation at a given brain level in our quantitative analysis while only one section per telencephalic level was traced in our visual representation of the VZ. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. V’. \ ' * 1 ’ • \ N \ \ \ V v I A n t I Comm □ = DVZ ■ = IntVZ ■ = VVZ H = Silver Grains Fig. 2.9. A wire frame reconstruction of the VZ across the anterior-posterior axis in a single juvenile male. Sections at the levels of Area X, the Anterior Commissure, and HVC are indicated in brackets. 0 0 A semi-quantitative map o f proliferation within the V Z- While our initial wire frame reconstruction allowed us to observe the anatomical changes that occurred within the VZ across the anterior-posterior axis for an individual bird, it did not accurately reflect differences in levels of proliferative activity within the IntVZ (see above). In addition, it was impossible to compare levels of thymidine labeling between males and females in order to identify regions of the VZ that contained sexually dimorphic mitotic activity. To better visualize relative differences in thymidine labeling throughout the VZ we constructed a semi-quantitative map of proliferation. This map represents levels of thymidine labeling within each bin of the DVZ, IntVZ, and W Z as a gray scale value, using 100 gray levels across males and females, with black being the highest level of mitotic activity and white the lowest. In this way it was possible to directly observe sex differences in levels of proliferation in specific segments of the VZ and also allowed us to see gradients in thymidine labeling along the anterior-posterior and dorsal-ventral axis in both males and females. The variable levels of mitotic activity that we observed in our overall measures of proliferation (Fig. 2.3) and in our binned data (Figs. 2.4-2.8) appeared as proliferative gradients in our semi-quantitative representation of proliferation within the VZ (Figs. 2.3 and 2.10). Starting at anterior levels of the telencephalon, levels of thymidine labeling increased along the rostral-caudal axis such that the highest levels of proliferation were observed within the W Z between the levels of Area X and the Anterior Commissure. Posterior to the Anterior Commissure levels of thymidine labeling within the W Z were greatly reduced. Levels of thymidine labeling within the DVZ were considerably lower throughout the anterior-posterior axis compared to the W Z (Figs. 2.3 and 2.10). The 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. highest level of proliferative activity within the DVZ occurred at the dorsal tip of the DVZ in the posterior half of the telencephalon and there were no clear gradients in mitotic activity within the DVZ. Within the IntVZ levels of thymidine labeling were intermediate at anterior levels of the telencephalon, low at mid levels, and highest near HVC (Rgs.2.3 and 2.10). Observing levels of thymidine labeling in our semi-quantitative map made it possible to visualize proliferative hot spots within the VZ. The most robust proliferative hot spot was apparent within the VVZ between Area X and the Anterior Commissure (Fig. 2.10). Other areas of the VZ in which we were able to visualize proliferative hot spots were the dorsal tip of the DVZ in the posterior half of the telencephalon and the tip of the IntVZ at HVC (Fig. 2.10). These observations agree with previous reports identifying the location of proliferative hot spots in birds (Alvarez-Buylla et al., 1990; Alvarez-Buylla et al., 1994). An interesting feature associated with proliferative hot spots were gradients in levels of thymidine labeling. That is, each hot spot was flanked on all sides by lower levels of thymidine labeling. This pattern of proliferation suggests that neurogenesis is differentially regulated within the VZ along the anterior-posterior and dorsal-ventral axes. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. I AraaX AM. HVC I I I I 12-1 >10 00 -J Fig. 2.10. A semi-quantitative representation of the VZ across the anterior-posterior axis of males which indicates the relative levels of proliferation within each bin of the DVZ, IntVZ, and W Z , The highest level of thymidine labeling appears in black and the lowest levels are represented in white. A schematic sagital representation if the zebra finch brain (inset) highlights the locations of Area X, the Anterior Commissure, and HVC along the anterior-posterior axis. By comparing levels of proliferative activity in females to the highest level of thymidine labeling in males we were able to visualize regions of the VZ containing sexually dimorphic proliferation (Fig. 2.11). For example, the W Z near Area X contained higher levels of mitotic activity in males compared to females with sex differences occurring in bin 8 at AX-1 and bin 5 at AX2 (Fig. 2.7). These same segments of the VZ in our semi-quantitative map demonstrate lower gray scale values in females than in males (Fig. 2.11). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. ArtaX Art. HVC Comm. C o m m IntVZ VVZ -4-3-2 m ■ ■ ■ ■ ■ H ■ I ■ ■ ■ ■ ■ I M j M H • ; Comm. S Comm. 1 2 U)^9 3f-7 -5 -5*4 -3 -2*1 1 2 3 00 N O > p Fig. 2.11. A semi-quantitative representation of the VZ across the anterior-posterior axis of females, which indicates the relative levels of proliferation within each bin of the DVZ, IntVZ, and W Z , compared to males. d*= A male biased sex difference. 9 = A female biased sex difference. A schematic sagital representation if the zebra finch brain (inset) highlights the locations of Area X, the Anterior Commissure, and HVC along the anterior-posterior axis. DISCUSSION In a previous study we measured ceil proliferation juvenile and adult zebra finches and found spatially restricted regions of the VZ that contained higher levels of mitotic activity in juvenile males compared to juvenile females at the levels of Area X and the Anterior Commissure (DeWulf and Bottjer, 2001). Higher levels of cell proliferation within the VZ of juvenile males might contribute to the sexually dimorphic growth of song-control nuclei that occurs during the sensitive period for vocal learning. However, in this original study we measured mitotic activity at 3 levels of the telencephalon and thus may have inadvertently missed other regions of the VZ containing sexually dimorphic proliferation. In the current study we therefore systematically measured of levels of thymidine labeling throughout the VZ and generated a map that represents relative levels of proliferation across the anterior-posterior axis. This map allowed us to visualize the spatial distribution of proliferation throughout the entire VZ and identify locations of sexually dimorphic mitotic proliferation. The spatial distribution of mitotic activity throughout the telencephalon: While mitotic activity is ubiquitous throughout the telencephalic VZ of songbirds (Goldman and Nottebohm, 1983; Alvarez-Buylla et al., 1988; DeWulf and Bottjer, 2001), levels of proliferation differed along the dorsal-ventral and anterior-posterior axis. The highest levels of cell division in both males and females were within the W Z while lower levels occurred in the DVZ and IntVZ The difference in levels of mitotic activity along the dorsal-ventral axis correlates with the large addition of new neurons to the striatum of birds during the first few weeks of after hatching while the cortex receives many fewer neurons (Alvarez-Buylla et al., 1994). The anterior-posterior distribution of proliferation 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. differed among the three VZ sub-regions. The DVZ demonstrated little variability in thymidine labeling throughout the telencephalon while proliferation within the IntVZ was low at anterior levels but increased at posterior levels near HVC and mitotic activity within the W Z was highest between Area X and the Anterior Commissure. These data suggest that the rate of new neuron production is relatively consistent within the DVZ across the anterior-posterior axis while the highest levels of neurogenesis within the IntVZ occur near HVC, and between Area X and the Anterior Commissure within the W Z. Currently it is not clear if higher rates of neurogenesis in the W Z (within the striatum) or the IntVZ (near HVC) contribute to the development to song-control regions. In addition, it is not known if the migration of new neurons produced within the DVZ, IntVZ or W Z are restricted to specific locations of the telencephalon and thus it is difficult to speculate where the ultimate destination for the new neurons produced within each VZ sub-region will be. Gradients in thvmidine labeling and proliferative hot spots within the VZ: Graded levels of thymidine labeling within each VZ sub-division were associated with segments of the VZ that contained particularly high levels of thymidine labeling referred to as hot spots (Alvarez-Buylla et al., 1988; Ling et ai., 1997). These gradients in proliferative activity may reflect the distribution of diffuseable factors, such as growth factors and neurotransmitters, which regulate mitotic activity within the VZ. Proliferation of neocortical VZ cells in vivo and in vitro has been shown to be modulated by several extracellular molecules that have been implicated in the regulation of cell proliferation in the developing telencephalon (for review see Cameron et al., 1998; Haydar et al., 2000). Jiang et al. (1998) have demonstrated that IGF-I, a growth factor 9 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that is a potent stimulus for the production and survival of neural precursors (Lenoir and Honegger, 1983; Drago et al., 1991; Lin and Bulleit, 1997), is expressed heavily by radial cells and their fibers within the adult zebra finch brain. Interestingly, proliferative hot spots coincide with regions rich in radial glia (Alvarez-Buylla et al., 1990) and thus it is possible that the presence of growth factors, specifically IGFs, my regulate levels of proliferation within the VZ of songbirds. Future experiments will be necessary to determine what specific extracellular molecules modulate proliferation within the zebra finch VZ and whether graded levels of mitotic activity throughout the telencephalon are the result of differential expression of these molecules and or the presence of radial glia. Sex differences in proliferation: In our previous study we found that a large span of the W Z at the level of Area X that contained higher levels of proliferation in juvenile males than juvenile females presumably due to a larger population of dividing cells in males (DeWulf and Bottjer, 2001). Our current findings indicate that when we observed thymidine labeling at individual levels of the telencephalon, sexually dimorphic proliferation within the W Z at the level of Area X is restricted to a few bins. However, when we collapse our measurements across rostral levels of Area X, the strong pattern of sexually dimorphic proliferation that was evident in our previous findings becomes apparent (DeWulf and Bottjer, 2001). It is possible that in younger birds, sexually dimorphic proliferation occurs within the W Z throughout Area X and that by 30 days, sex differences are only evident in the anterior half of the nucleus. Previous work has demonstrated that levels neurogenesis decrease over the course of development (Alvarez-Buylla et al., 1994; Ling et al., 1997) presumably due to a decrease in VZ cell number (DeWulf and Bottjer, 2001). Therefore, as birds develop the total number of 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells within regions of sexually dimorphic proliferation gradually decrease until levels of proliferation are equivalent between males and females. Our current analysis also demonstrated sexually dimorphic proliferation within the DVZ (immediately above LH), IntVZ (immediately above the LMD), and W Z (below LMD) at the level of the Anterior Commissure. These sex differences are surprising because they occur at a level of the telencephalon that does not contain song- control nuclei. Thus it is unclear what the implication of sexually dimorphic proliferation at this level of the telencephalon may be. Further work will be required to determine where neuroblasts generated in specific sexually dimorphic regions of the VZ migrate to within the telencephalon and whether they become incorporated into functional circuitry. A map of mitotic activity within the V Z- Previous work has demonstrated that new neurons produced within the VZ of birds migrate into the telencephalon and a subset of these new neurons become incorporated into functional circuitry (for review see Alvarez-Buylla and Kim, 1997). Since neurogenesis is ubiquitous throughout the telencephalic VZ of songbirds (Alvarez-Buylla and Nottebohm, 1988; DeWulf and Bottjer, 2001) it is difficult to determine where the stem cells that give rise to song- control neurons reside within the VZ. This is the first study in which levels of mitotic activity have been systematically analyzed throughout the telencephalic neuraxis. By measuring levels of mitotic activity in small segments of the VZ we were able to (1) determine where high levels of proliferation occur within the VZ, (2) determine where sex differences in proliferation occur within the VZ, and (3) observe graded levels of mitotic activity across the anterior-posterior and dorsal-ventral axis. When we compared the relative levels of mitotic activity throughout the VZ, and represented the data as a 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. semi-quantitative map of proliferation, we were able to visualize the overall pattern of mitotic activity throughout the VZ of males and females (see Figs. 2.10 and 2.11). Using our map of proliferation, it is now possible start testing whether a specific locations of the VZ, for example regions containing sexually dimorphic proliferation, give rise to song- control neurons. In addition, it is now possible to test whether steroid hormones effect the proliferation of progenitors within sexually dimorphic regions of the VZ. Thus, our map of mitotic activity will be a valuable tool in future experiments testing the contribution of proliferation to the postnatal development songbird telencephalon and, more specifically, to the development of the song-controi system. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Alvarez-Buylla A, Theelen M, Nottebohm F (1988) Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning. Proc.Natl.Acad.Sci.U.SA 85:8722-8726. Alvarez-Buylla A, Nottebohm F (1988) Migration of young neurons in adult avian brain. Nature 335: 353-354. Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101-109. Alvarez-Buylla A, Ling CY, Yu WS (1994) Contribution of neurons bom during embryonic, juvenile, and adult life to the brain of adult canaries: regional specificity and delayed birth of neurons in the song-control nuclei. J.Comp Neurol. 347: 233-248. Bohner J (1990) Early acquisition of song in the zebra finch. Animal Behavior 39: 369-374. Bottjer SW, Halsema KA, Brown SA, Miesner EA (1989) Axonal connections of a forebrain nucleus involved with vocal learning in zebra finches. J.Comp Neurol. 279: 312-326. Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology Anat.Rec. 166:257-261. Burek MJ, Nordeen KW, Nordeen E J (1994) Ontogeny of sex differences among newly- generated neurons of the juvenile avian brain. Brain Res.Dev.Brain Res. 78:57-64. Burek MJ, Nordeen KW, Nordeen E J (1997) Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death. J.Neurobiol. 33: 61-71. Cameron HA, Hazel TG, McKay RD (1998) Regulation of neurogenesis by growth factors and neurotransmitters. J.Neurobiol. 36: 287-306. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DeWulf V, Bottjer SW (2001) Age and sex differences in mitotic activity in the zebra finch telencephalon. J.Neurosci. Under review. Drago J, Murphy M, Carroll SM, Harvey RP, Bartlett PF (1991) Fibroblast growth factor- mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I. Proc.Natl.Acad.Sci.U.S.A 88:2199-2203. Goldman SA and Nottebohm F (1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc.Natl.Acad.Sci.U.S.A 80: 2390-2394. HaydarTF, Wang F, Schwartz ML, Rakic P (2000) Differential modulation of proliferation in the neocortical ventricular and subventricular zones. J.Neurosci. 20: 5764-5774. Jiang J, McMurtry J, Niedzwiecki D, Goldman SA (1998) Insulin-like growth factor-1 is a radial cell-associated neurotrophin that promotes neuronal recruitment from the adult songbird edpendyma/subependyma. J.Neurobiol. 36: 1-15. Kim JR. DeVoogd TJ (1989) Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J.Neurosci. 9: 3176-3187. Lenoir D, Honegger P (1983) Insulin-like growth factor I (IGF-I) stimulates DNA synthesis in fetal rat brain cell cultures. Brain Res. 283: 205-213. Lin X, Bulleit RF, (1997) Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells. Brain Res.Dev.Brain Res. 99: 234-242. Ling C, Zuo M, Alvarez-Buylla A, Cheng MF (1997) Neurogenesis in juvenile and adult ring doves. J.Comp Neurol. 379: 300-312. Nordeen KW , Nordeen EJ ( 1988a) Projection neurons within a vocal motor pathway are bom during song learning in zebra finches. Nature 334: 149-151. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nordeen EJ, Nordeen KW (1988b) Sex and regional differences in the incorporation of neurons bom during song learning in zebra finches. J.Neurosci. 8: 2869-2874. Nottebohm F, Arnold AP (1976) Sexual dimorphism in vocal control areas of the songbird brain. Science 194:211-213. Nottebohm F, Kelley DB, Paton JA (1982) Connections of vocal control nuclei in the canary telencephalon. J.Comp Neurol. 207: 344-357. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GENERAL DISCUSSION Developmental processes involved in the maturation of the Song-Control System Results presented herein demonstrate that (1) cell division occurs within three distinct proliferative zones within the telencephalon of both juvenile and adult zebra finches, (2) proliferation is higher in juveniles than adults, (3) sexually dimorphic proliferation occurs within the VZ of 30 day old juvenile zebra finches and (4) thymidine labeling is highly spatially differentiated along the rostral-caudal and dorsal-ventral axes of the VZ. Most studies investigating neurogenesis in the telencephalon of songbirds have focused on observing new neuron addition and/or seasonal neuronal turnover within song-control nuclei (Goldman and Nottebohm, 1983; Burd and Nottebohm, 1985; Nordeen and Nordeen, 1988a, b; Alvarez-Buylla and Nottebohm, 1988; Kim et al., 1994; Burek et al., 1994; Kim et al., 1999). New neurons require approximately one to three weeks to arrive in song-control regions following their generation within the ventricular zone (VZ)(Nordeen and Nordeen, 1988b). This relatively long period of time makes it extremely difficult to determine how many new neurons are generated by the VZ, observe the migrational pattern of newly generated cells, and ascertain what proportion of new cells eventually differentiate as song-control neurons. Because we were interested in determining the contribution cellular proliferation makes to the developing song-control system, it was important to observe the incidence of thymidine labeling shortly after 3 H- thymidine is administered. In this way we were confident that cell death or migration away from the VZ would not contaminate our measurements of mitotic activity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Proliferative zones in the zebra finch telencephalon Thymidine labeling was observed within the zebra finch telencephalon in three distinct zones: (1) a ventricular zone, VZ, that is adjacent to the lateral telencephalic ventricles, (2) a sub-ventricular zone (SVZ) which consists of small, round, mitotically active cells that are adjacent to the VZ, and (3) proliferation that occurres adjacent to the VZ and SVZ within the brain parenchyma. The VZ of zebra finches looks identical to what has been described in developing mammalian brain (Boulder Committee, 1970). Evidence from mammalian brain suggests that during early development, newly generated cells from the VZ migrate along radial glia and eventually differentiate as neurons (Caviness, 1982; Bayer and Altman, 1991; Goldman et al., 1996; Rakic, 1981), while later in development the SVZ becomes apparent and cells originating in the SVZ become both neurons and glia (Smart, 1961; Bayer and Altman, 1991;SzeleandChesselet, 1996). The SVZ that we observed was localized to the striatum in both juveniles and adults. Since the SVZ generates both neurons and glia (Louis and Alvarez-Buylla, 1993). it is possible that this proliferative zone in conjunction with the VZ generates both cell types within the striatum while in the cortex the VZ is the sole source of new neurons and glia (with the exception of glia dividing in situ). We report here an apparently novel class of thymidine labeling adjacent to the VZ and SVZ. This population of labeled cells in the brain parenchyma has similar morphological characteristics to thymidine-labeled cells within the VZ and SVZ. To our knowledge this area of proliferation within the brain parenchyma has not been reported previously (cf. Alvarez-Buylla et al., 1990; Ling et al., 1997). The short (two-hour) survival interval following thymidine exposure used in our study seems to rule out the 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. possibility that this group of labeled cells has migrated out from the VZ or SVZ. Rather, this zone of proliferation may represent either a population of stem cells dividing within the brain parenchyma or cells dividing while en route to their final destination. Age differences in levels of mitotic activity The area of the VZ was significantly larger and more proliferatively active in juveniles than in adults at the levels of Area X, the Anterior Commissure, and HVC (including the VZ immediately above HVQ(Chapter 1). Single cell measurements at the level of the Anterior Commissure revealed that the enlarged VZ of juveniles was attributable to a greater number of cells, such that the total number of cells in the VZ decreases over the course of development. The VZ of juveniles also had a larger number of thymidine-labeled cells compared to adults, indicating that the increased incidence of mitotic activity in juvenile birds is due to a larger number of dividing cells. The decrease in VZ cell number may involve either a terminal division of precursor cells and their migration away from the VZ and/or the death of progenitors within the VZ The SVZ and thymidine labeled cells adjacent to the VZ and SVZ also demonstrated significantly higher levels of proliferative activity in juveniles compared to adults (Chapter 1). Although single cells were not counted within the SVZ (or groups of thymidine labeled cells within the brain parenchyma), visual inspection of 1-fim tissue indicates that the SVZ appears to contain fewer cells in adults suggesting that similar mechanisms might regulate progenitor cell number in at least in the VZ and SVZ over the course of vocal development. It is surprising that relatively high rates of proliferative activity occur within the VZ of juveniles since the majority of brain development is complete by 30 days (Bottjer 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al., 1985). The high rate of mitotic activity in juveniles implies that more new cells are generated than could presumably be incorporated into the brain thus the majority of new cells must either die en route to their final destination, or after they have reached their targets. An alternate hypothesis is that a high level of cell turnover throughout the brain must occur in order to accommodate the arrival and subsequent incorporation of new neurons (i.e., older cells must die, at least within certain phenotypic populations; Alvarez- Buylla et al., 1988). Kim et al. (1999) demonstrated that approximately half of newly generated neurons die between the second and third weeks after their arrival in HVC, suggesting that newly generated cells from the VZ undergo cell death if they are not incorporated into functional circuitry within some finite period following the end of migration. This idea suggests that there is a discrete window of time in which young neurons produced in the avian telencephalon must receive appropriate trophic support (i.e. growth or activity-dependent factors) if they are to survive (Johnson et al., 2000; Rasikaetal., 1999). Sex differences in proliferation A major finding of the studies herein is that restricted regions of the VZ demonstrate developmentally regulated sexually dimorphic mitotic activity in juvenile birds, particularly at the level of Area X (Chapters 1 and 2). Previous studies examining cellular proliferation in various sexually dimorphic systems have reported no evidence for differential cell division between males and females (Jacobson and Gorski, 1981; Breedlove et al., 1983; Gorlick and Kelley, 1987), suggesting that the development of sexually dimorphic brain regions may result primarily from sex differences in migration, differentiation, and/or neuron death (Kim and DeVoogd, 1989; Burek et al., 1997). 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, more recent work demonstrating gender differences in neurogenesis within the dentate gyrus of young rats suggests that sexually dimorphic proliferation in post-natal brain may contribute to learning (Perfilieva et al., 2001). It is possible that the increased cell production in the VZ of 30 day old males might contribute to the growth of song- control nuclei thereby influencing the ability of young males to learn birdsong. At this time it is unclear what the general contribution of sexually dimorphic proliferation is to the development of the song-control system. The selective increases in proliferation within the VZ of juvenile males may indicate potential sites of progenitor cells that give rise to song-control neurons during the sensitive period for vocal learning. This idea is supported by the finding that the incorporation of new neurons into HVC is greater in juvenile males than females (Nordeen and Nordeen, 1988a). It would be interesting to measure levels of thymidine labeling in the VZ of birds younger than 30 days to determine if regions sexually dimorphic proliferation within the VZ area more common and larger in younger birds. Sexually dimorphic proliferation occurs in the rostral half of the ventral VZ (W Z) at Area X in 30 day old birds (Chapters 1 and 2). Previous work has demonstrated that levels neurogenesis decrease over the course of development (Alvarez-Buylla et al., 1994; Ling et al., 1997) presumably due to a decrease in VZ cell number (DeWulf and Bottjer, 2001). Therefore, as birds develop the total number of cells within sexually dimorphic regions of the VZ gradually decrease until levels of mitotic activity are equivalent between males and females. If this is the case, then the higher levels of thymidine labeling might be seen throughout the W Z at Area X of males younger than 30 days old. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Levels of Proliferation are higher within the WZ relative to the DVZ The pattern of proliferation within the VZ of juvenile and adult birds demonstrated that the highest levels of mitotic activity occurred within the W Z at the Anterior Commissure of juveniles (Chapter I). This result contradicted the original hypothesis that spatially restricted regions of upregulated proliferation, at least in juvenile male birds, would be in close proximity to the song-control regions which incorporate a large number of new neurons over the course of vocal development (Bottjer et al., 1986; Nordeen and Nordeen, 1988a, b; Alvarez-Buylla et al., 1992). In addition, these data suggest that the location of precursor cells that give rise to song-control neurons may be spatially remote from their target. However, the selective increase in levels of thymidine labeling in juvenile males adjacent to Area X imply that this region may give rise to Area X neurons. Further experiments testing the fate of new cells generated in sexually dimorphic regions of the VZ are needed to resolve whether or not sexually dimorphic proliferation indicates the location of song-control system progenitors in the VZ The total amount of proliferative activity was higher in the W Z than the DVZ in juveniles (but not adults) at the levels of the Anterior Commissure and Area X. That is, overall levels of thymidine labeling were substantially higher within the W Z than within the DVZ of young birds even though the DVZ had a larger overall area (at least at the level of the Anterior Commissure). The enhanced levels of proliferative activity within the W Z of juveniles is interesting, because it correlates with a large addition of new neurons to striatum of birds during the first several weeks post-hatch, whereas neighboring cortex receives many fewer new neurons (Alvarez-Buylla et al., 1994). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Increased levels of proliferation within the W Z , at least at the level of the Anterior Commissure in juveniles, may be due to the prominent out-pocketing of the neuroepithelium that contained high levels of proliferative activity and has been referred to as a hot spot in proliferation in canaries (Alvarez-Buylla et al., 1990)(Chapter 1). This bulge might represent a remnant of the sulcus that divides the medial and lateral ganglionic eminences, which are thought to generate cells destined for the striatum and pallidum, respectively, in embryonic mammals (Smart and Sturrock, 1979). The increased levels of mitotic activity in the W Z relative to the DVZ is that is unique to juveniles is presumably due primarily to the presence of the hot spot of labeling in the pronounced out-pocketing of the W Z seen in juveniles along the ventral-most aspect of the lateral ventricles at this level. We do not know whether cells generated in the dorsal versus ventral proliferative zones are restricted to the cortex and basal ganglia, respectively. However, this seems possible as studies of telencephalic development have provided evidence for migrational boundaries that prevent movement across the corticostriatal sulcus of the VZ or from postmitotic cortex to striatum (e.g., Fishell et al., 1993; Neyt et al., 1997; Striedter et al., 1998). Since the SVZ generates both neurons and glia (Smart, 1961; Lois and Alvarez- Buylla, 1993; Szele and Chesselet, 1996; Doetsch et al., 1999), it is possible that this proliferative zone in conjunction with the ventral VZ generates both ceil types within the striatum whereas the dorsal VZ might be a source of new neurons and glia in the cortex (glia dividing in situ presumably contribute to glial turnover throughout the telencephalon; Cameron and Rakic, 1991). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Levels of proliferation are spatially differentiated throughout the VZ Overall levels of thymidine labeling were highest within the W Z compared to the DVZ or the intermediate VZ (IntVZ) of young birds throughout the anterior-posterior axis (Chapters 1 and 2). hi addition to dorsal-ventral differences in levels of thymidine labeling, there was a spatially differentiated pattern of proliferative activity throughout the anterior-posterior axis among the three VZ sub-regions (Chapter 2). The DVZ demonstrated little variability in thymidine labeling throughout the telencephalon while proliferation within the IntVZ was low at anterior levels but increased at posterior levels near HVC and mitotic activity within the W Z was highest between Area X and the Anterior Commissure. These data suggest that the rate of new neuron production is relatively consistent across the anterior-posterior axis within the DVZ while the highest levels of neurogenesis within the IntVZ occur near HVC. and between Area X and the Anterior Commissure within the W Z Currently it is not clear if higher rates of proliferation in the W Z (within the striatum) or the IntVZ (near HVC) contribute to the development to song-control regions. In addition, it is not known if the migration of new neurons produced within the DVZ IntVZ or W Z are restricted to specific locations of the telencephalon (see above) and thus it is difficult to speculate where the ultimate destination for the new neurons produced within each VZ sub-region will be. Graded levels of thymidine labeling within the W Z IntVZ and DVZ were associated with segments of the VZ that contained particularly high levels of thymidine labeling referred to as hot spots (Alvarez-Buylla et al., 1990; Ling et al., 1997). These gradients in proliferative activity may reflect the distribution of diffuseable factors, such as growth factors and neurotransmitters, which regulate mitotic activity within the VZ. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Proliferation of neocortical VZ cells in vivo and in vitro has been shown to be modulated by several extracellular molecules that have been implicated in the regulation of cell proliferation in the developing telencephalon (for review see Cameron et al., 1998; Haydar et al., 2000). For example, IGF-I, a growth factor that is a potent stimulus for the production and survival of neural precursors (Lenoir and Honegger, 1983; Drago et al., 1991; Lin and Bulleit, 1997), is expressed heavily by radial glia and their fibers within the adult zebra finch brain (Jiang et al., 1998). Interestingly, proliferative hot spots coincide with regions rich in radial glia (Alvarez-Buylla et al., 1990) and thus it is possible that the presence of growth factors my regulate levels of proliferation and cell survival within the VZ of songbirds. A map of proliferation within the telencephalic VZ of zebra finches Previous work has demonstrated that new neurons produced within the VZ of birds migrate into the telencephalon and a subset of these new neurons become incorporated into functional circuitry (for review see Alvarez-Buylla and Kim, 1997). Since neurogenesis is ubiquitous throughout the telencephalic VZ of songbirds (Alvarez- Buylla and Nottebohm, 1988; DeWulf and Bottjer, 2001) it is difficult to determine where the stem cells that give rise to song-control neurons reside within the VZ. This is the first study in which levels of mitotic activity have been systematically analyzed throughout the telencephalic neuraxis. By measuring levels of thymidine labeling in small segments of the VZ we were able to (1) determine where the highest levels of proliferation occur within the VZ, (2) determine where sex differences in proliferation reside within the VZ, and (3) observe graded levels of mitotic activity across the anterior- posterior and dorsal-ventral axis. When we compared the relative levels of mitotic 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity throughout the VZ, and represented the data as a semi-quantitative map of proliferation, we were able to visualize the overall pattern of mitotic activity throughout the VZ of males and females (Chapter 2). Understanding the distribution of thymidine labeling throughout the telencephalic VZ of juvenile birds, in addition to knowing the location of sexually dimorphic proliferation, makes it possible to test the fate of cells bom within specific locations of the VZ. For example, it would be interesting to know if regions containing sexually dimorphic proliferation give rise to song-control neurons. In addition, knowing the location of sexually dimorphic proliferation permits more sensitive experiments testing of the role of steroid hormones on neuronal production, and might alter the conclusions of past studies indicating that sex hormones have little or no influence on cellular proliferation (Goldman and Nottebohm, 1983; Brown et al., 1993; Rasika et al., 1994). Thus, our map of mitotic activity is a valuable tool in determining what the contribution cellular proliferation makes to neural development during the period of song learning in juvenile birds. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Alvarez-Buylla A, Nottebohm F (1988) Migration of young neurons in adult avian brain. Nature 335: 353-354. Alvarez-Buylla A, Theelen M, Nottebohm F (1988) Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning. Proc Natl Acad Sci U S A 85: 8722-8726. Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101-109. Alvarez-Buylla A, Ling CY, Nottebohm F (1992) High vocal center growth and its relation to neurogenesis, neuronal replacement and song acquisition in juvenile canaries. J Neurobiol 23: 396-406. Alvarez-Buylla A, Ling CY, Yu WS (1994) Contribution of neurons bom during embryonic, juvenile, and adult life to the brain of adult canaries: regional specificity and delayed birth of neurons in the song-control nuclei. J Comp Neurol 347: 233-248. Alvarez-Buylla A, Kim JR (1997) Birth, migration, incorporation, and death of vocal control neurons in adult songbirds. J Neurobiol 33:585-601. Anderson SA, Eisenstat DD, Shi L, Rubenstein JL (1997) Intemeuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278: 474-476. Bottjer SW, Glaessner SL, Arnold AP (1985) Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J Neuroscience 5: 1556-1562. Bottjer SW, Miesner EA, Arnold AP (1986) Changes in neuronal number, density and size account for increases in volume of song-control nuclei during song development in zebra finches. Neurosci Lett 67: 263-268. Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anat Rec 166: 257-261. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Breedlove SM, Jordan CL, Amoid AP (1983) Neurogenesis of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavemosus in rats. Brain Res 285: 39-43. Brown SD, Johnson F, Bottjer SW (1993) Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels. J Neurosci 13:2024-2032. Burd GD, Nottebohm F (1985) Ultrastructural characterization of synaptic terminals formed on newly generated neurons in a song control nucleus of the adult canary forebrain. J Comp Neurol 240: 143-152. Burek MJ, Nordeen KW, Nordeen EJ (1994) Ontogeny of sex differences among newly- generated neurons of the juvenile avian brain. Brain Res Dev Brain Res 78: 57-64. Burek MJ, Nordeen KW, Nordeen EJ (1997) Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death. J Neurobiol 33:61-71. Cameron RS, Rakic P (1991) Glial cell lineage in the cerebral cortex: a review and synthesis. Glia 4: 124-137. DeWulf V, Bottjer SW (2001) Age and Sex Differences in Mitotic Activity in the Zebra Finch Telencephalon. J.Neurosci. Under review. Doetsch F, Caille L Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97: 703-716. Drago J, Murphy M, Carroll SM, Harvey RP, Bartlett PF (1991) Fibroblast growth factor- mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I. Proc Natl Acad Sci U S A 88: 2199-2203. Fished G, Mason CA, Hatten ME (1993) Dispersion of neural progenitors within the germinal zones of the forebrain. Nature 362: 636-638. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Goldman SA, Nottebohm F (1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc Natl Acad Sci U S A 80: 2390-2394. Gorlick DL, Kelley DB (1987) Neurogenesis in the vocalization pathway of Xenopus laevis. J Comp Neurol 257:614-627. Immelman K (1969) Song development in the zebra finch and other estrilid finches. In Bird Vocalizations, RA. Hinde (Ed.) New York: Cambridge UP, pp. 61-74. Jacobson CD, Gorski RA (1981) Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat. J Comp Neurol 196: 519-529. Jiang J, McMurtry J, Niedzwiecki D, Goldman SA (1998) Insulin-like growth factor-1 is a radial cell-associated neurotrophin that promotes neuronal recruitment from the adult songbird edpendyma/subependyma. J .Neurobiol. 36: 1-15. Johnson F, Norstrom E, Soderstrom K (2000) Increased expression of endogenous biotin, but not BDNF, in telencephalic song regions during zebra finch vocal learning. Brain Res Dev Brain Res 120: 113-123. Kim J, O’ Loughlin Kasparian BS, and Nottebohm F (1994) Cell death and neuronal recruitment in the high vocal center of adult male canaries are temporally related to changes in song [see comments]. Proc Natl Acad Sci U S A 91: 7844-7848. Kim JR. DeVoogd TJ (1989) Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J Neurosci 9: 3176-3187. Kim JR. Fishman Y, Sasportas K, Alvarez-Buylla A, Nottebohm F (1999) Fate of new neurons in adult canary high vocal center during the first 30 days after their formation. J Comp Neurol 411: 487-494. Lavdas AA, Grigoriou M, Pachnis V, Pamavelas JG (1999) The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex. J Neurosci 19: 7881-7888. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lenoir D, Honegger P (1983) Insulin-like growth factor I (IGF-I) stimulates DNA synthesis in fetal rat brain cell cultures. Brain Res. 283:205-213. Lin X, Bulieit RF (1997) Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells. Brain Res.Dev.Brain Res. 99:234-242. Ling C, Zuo M, Alvarez-Buylla A, Cheng MF (1997) Neurogenesis in juvenile and adult ring doves J Comp Neurol 379:300-312. Lois C, Alvarez-Buylla A (1993) Proliferating subventricuiar zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A 90: 2074-2077. Neyt C, Welch M, Langston A, Kohtz J, Ftshell G (1997) A short-range signal restricts cell movement between telencephalic proliferative zones. J Neurosci 17: 9194-9203. Nordeen KW, Nordeen EJ ( 1988a) Projection neurons within a vocal motor pathway are bom during song learning in zebra finches. Nature 334: 149-151. Nordeen EJ, Nordeen KW (1988b) Sex and regional differences in the incorporation of neurons bom during song learning in zebra finches. J Neurosci 8: 2869-2874. O'Rourke NA, Sullivan DP, Kaznowski CE, Jacobs AA, McConnell SK (1995) Tangential migration of neurons in the developing cerebral cortex. Development 121: 2165-2176. Perfilieva E, Risedal A, Nyberg J, Johansson BB, Eriksson PS (2001) Gender and strain influence on neurogenesis in dentate gyrus of young rats. J.Cereb.Blood Flow Metab. 21:211-217. Rasika S, Nottebohm F, Alvarez-Buylla A (1994) Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries. Proc Natl Acad Sci U S A 91: 7854-7858. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rasika S, Alvarez-Buylla A, and Nottebohm F (1999) BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain. Neuron 22:53-62. Smart I (1961) The subependymal layer in the mouse brain and its cell production as shown by autoradiography after thymidine-3 H injection. J Comp Neurol 116: 325-347. Smart I, Sturrock RR (1979) Ontogeny of the neostriatum. In: Divac I, Oberg RGE, (The Neostriatum, ed), pp 127-146. Oxford: Pergamon Press. Striedter GF, Marchant TA, Beydler S (1998) The "neostriatum" develops as part of the lateral pallium in birds. J Neurosci 18: 5839-5849. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bibliography Alvarez-Buylla A, Nottebohm F (1988) Migration of young neurons in adult avian brain. Nature 335:353-354. Alvarez-Buylla A, Theelen M, Nottebohm F (1988) Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning. Proc Natl Acad Sci U S A 85:8722-8726. Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101-109. Alvarez-Buylla A, Ling CY, Nottebohm F (1992) High vocal center growth and its relation to neurogenesis, neuronal replacement and song acquisition in juvenile canaries. J Neurobiol 23: 396-406. Alvarez-Buylla A, Ling CY, Yu WS (1994) Contribution of neurons bom during embryonic, juvenile, and adult life to the brain of adult canaries: regional specificity and delayed birth of neurons in the song-control nuclei. J Comp Neurol 347: 233-248. Alvarez-Buylla A, Kim JR (1997) Birth, migration, incorporation, and death of vocal control neurons in adult songbirds. J Neurobiol 33: 585-601. Anderson SA, Eisenstat DD, Shi L, Rubenstein JL (1997) Intemeuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278:474-476. Bohner J (1990) Early acquisition of song in the zebra finch. Animal Behavior 39: 369-374. Bottjer SW, Glaessner SL, Arnold AP (1985) Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J Neuroscience 5: 1556-1562. Bottjer SW, Miesner EA, Arnold AP (1986) Changes in neuronal number, density and size account for increases in volume of song-control nuclei during song development in zebra finches. Neurosci Lett 67: 263-268. Bottjer SW, Halsema KA, Brown SA, Miesner EA (1989) Axonal connections of a forebrain nucleus involved with vocal learning in zebra finches. J Comp Neurol 279: 312-326. Bottjer SW (1997) Building a bird brain: sculpting neural circuits for a learned behavior. Bioessays 19: 1109-1116. Bottjer SW, Arnold A (1997) Circuits, hormones, and learning: vocal behavior in songbirds. J Neurobio 33: 602-618. 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anat Rec 166: 257-261. Breedlove SM, Jordan CL, Arnold AP (1983) Neurogenesis of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavemosus in rats. Brain Res 285:39-43. Brown SD, Johnson F, Bottjer SW (1993) Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels. J Neurosci 13:2024-2032. Burd GD, Nottebohm F (1985) Ultrastructural characterization of synaptic terminals formed on newly generated neurons in a song control nucleus of the adult canary forebrain. J Comp Neurol 240: 143-152. Burek MJ, Nordeen KW, Nordeen EJ (1994) Ontogeny of sex differences among newly- generated neurons of the juvenile avian brain. Brain Res Dev Brain Res 78:57-64. Burek MJ, Nordeen KW, Nordeen EJ (1997) Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death. J Neurobiol 33:61-71. Cameron RS, Rakic P (1991) Glial cell lineage in the cerebral cortex: a review and synthesis. Glia 4: 124-137. HA Cameron, TG Hazel, RD McKay (1998) Regulation of neurogenesis by growth factors and neurotransmitters. J.Neurobiol. 36:287-306. DeWulf V, Bottjer SW (2001) Age and sex differences in mitotic activity in the zebra finch telencephalon. J .Neurosci. Under review. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97: 703-716. Drago J, Murphy M, Carroll SM, Harvey RP, Bartlett PF(1991) Fibroblast growth factor- mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I. Proc.Natl.Acad.Sci.U.S.A 88:2199-2203. Fished G, Mason CA, Hatten ME (1993) Dispersion of neural progenitors within the germinal zones of the forebrain. Nature 362:636-638. Garcia-Verdugo JM, Doetsch F, Wichterle H, Lim DA, Alvarez-Buylla A (1998) Architecture and cell types of the adult subventricular zone: in search of the stem cells. J Neurobiol 36: 234-248. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Goldman SA, Nottebohm F (1983) Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proc Natl Acad Sci U S A 80: 2390-2394. Gortick DL, Kelley DB (1987) Neurogenesis in the vocalization pathway of Xenopus laevis. J Comp Neurol 257:614-627. Haydar TF, Wang F, Schwartz ML, Rakic P (2000) Differential modulation of proliferation in the neocortical ventricular and subventricular zones. J .Neurosci. 15: 5764-5774. Herrmann K, Bischof HJ (1986) Delayed development of song control nuclei in the zebra finch is related to behavioral development. J Comp Neurol 245: 167-175. Jacobson CD, Gorski RA (1981) Neurogenesis of the sexually dimorphic nucleus of the preoptic area in the rat. J Comp Neurol 196: 519-529. J Jiang, J McMurtry, D Niedzwiecki, SA Goldman (1998) Insulin-like growth factor-1 is a radial cell-associated neurotrophin that promotes neuronal recruitment from the adult songbird edpendyma/subependyma. J.Neurobiol. 36:1-15. Johnson F, Norstrom E, Soderstrom K (2000) Increased expression of endogenous biotin, but not BDNF, in telencephalic song regions during zebra finch vocal learning. Brain Res Dev Brain Res 120: 113-123. Kim J, OLoughlin Kasparian BS, and Nottebohm F (1994) Cell death and neuronal recruitment in the high vocal center of adult male canaries are temporally related to changes in song [see comments]. Proc Natl Acad Sci U S A 91: 7844-7848. Kim JR, DeVoogd TJ (1989) Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J Neurosci 9: 3176-3187. Kim JR, Fishman Y, Sasportas K, Alvarez-Buylla A, Nottebohm F (1999) Fate of new neurons in adult canary high vocal center during the first 30 days after their formation. J Comp Neurol 411:487-494. Lavdas AA, Grigoriou M, Pachnis V, Pamavelas JG (1999) The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex. J Neurosci 19: 7881-7888. Lenoir D, Honegger P (1983) Insulin-like growth factor I (IGF-I) stimulates DNA synthesis in fetal rat brain cell cultures. Brain Res. 283: 205-213. Lin X, Bulleit RF (1997) Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells. Brain Res.Dev.Brain Res. 99: 234-242. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ling C, Zuo M, Alvarez-Buylla A, Cheng MF (1997) Neurogenesis in juvenile and adult ring doves J Comp Neurol 379:300-312- Lois C, Alvarez-Buylla A (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A 90: 2074-2077. Neyt C, Welch M, Langston A, Kohtz J, Fishell G (1997) A short-range signal restricts cell movement between telencephalic proliferative zones. J Neurosci 17: 9194-9203. Nordeen KW, Nordeen EJ (1988a) Projection neurons within a vocal motor pathway are born during song learning in zebra finches. Nature 334:149-151. Nordeen EJ, Nordeen KW (1988b) Sex and regional differences in the incorporation of neurons bom during song learning in zebra finches. J Neurosci 8:2869-2874. Nottebohm F, Arnold AP (1976) Sexual dimorphism in vocal control areas of the songbird brain. Science 194:211-213. Nottebohm F, Kelley DB, Paton JA (1982) Connections of vocal control nuclei in the canary telencephalon. J Comp Neurol 207: 344-357. O'Rourke NA, Sullivan DP, Kaznowski CE, Jacobs AA, McConnell SK (1995) Tangential migration of neurons in the developing cerebral cortex. Development 121: 2165-2176. Paton JA, OT-oughlin BE Nottebohm F (1985) Cells bom in adult canary forebrain are local intemeurons. J Neurosci 5:3088-3093. Perfilieva E Risedal A, Nyberg J, Johansson BB, Eriksson PS (2001) Gender and strain influence on neurogenesis in dentate gyrus of young rats. J.Cereb.Blood Flow Metab. 21:211-217. Rasika S, Nottebohm F, Alvarez-Buylla A (1994) Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries. Proc Natl Acad Sci U S A 91: 7854-7858. Rasika S, Alvarez-Buylla A, and Nottebohm F (1999) BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain. Neuron 22: 53-62. Smart I (1961) The subependymal layer in the mouse brain and its cell production as shown by autoradiography after thymidine-3 H injection. J Comp Neurol 116: 325-347. Smart I, Sturrock RR (1979) Ontogeny of the neostriatum. In: Divac I, Oberg RGE (The Neostriatum, ed), pp 127-146. Oxford: Pergamon Press. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Striedter GF, Marchant TA, Beydler S (1998) The "neostriatum" develops as part of the lateral pallium in birds. J Neurosci 18:5839-5849. Szele FG, Chesselet MF (1996) Cortical lesions induce an increase in cell number and PSA-NCAM expression in the subventricular zone of adult rats. J Comp Neurol 368:439-454. Van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2:266-270. Vicario DS (1994) Motor mechanisms relevant to auditory-vocal interactions in songbirds. Brain Bebav Evol 44:265-278. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator DeWulf, Valerie Dawn (author)

Core Title Age and sex differences in levels of proliferation within the zebra finch telencephalic ventricular zone

School Graduate School

Degree Doctor of Philosophy

Degree Program Neurobiology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, neuroscience,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Bottjer, Sarah (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-191171

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