Original Paper Brain Behav Evol 2014;84:181–196 DOI: 10.1159/000364778

Received: November 18, 2013 Returned for revision: December 13, 2013 Accepted after revision: May 20, 2014 Published online: October 7, 2014

Seasonal Variation in Cell Proliferation and Cell Migration in the Brain of Adult Red-Sided Garter Snakes (Thamnophis sirtalis parietalis) Ashley R. Maine Sean D. Powers Deborah I. Lutterschmidt Department of Biology, Portland State University, Portland, Oreg., USA

Key Words Neurogenesis · Cell proliferation · Cell migration · Bromodeoxyuridine · Immunohistochemistry · Migration · Reproduction · Snake · Reptile

Abstract Plasticity in the adult central nervous system has been described in all vertebrate classes as well as in some invertebrate groups. However, the limited taxonomic diversity represented in the current neurogenesis literature limits our ability to assess the functional significance of adult neurogenesis for natural behaviors as well as the evolution of its regulatory mechanisms. In the present study, we used freeranging red-sided garter snakes (Thamnophis sirtalis parietalis) to test the hypothesis that seasonal shifts in physiology and behavior are associated with seasonal variation in postembryonic neurogenesis. Specifically, we used the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) to determine if the rates of cell proliferation in the adult brain vary between male snakes collected during spring and fall at 1, 5, and 10 days post-BrdU treatment. To assess rates of cell migration within the brain, we further categorized BrdU-labeled cells

© 2014 S. Karger AG, Basel 0006–8977/14/0843–0181$39.50/0 E-Mail [email protected] www.karger.com/bbe

according to their location within the ventricular zone or parenchymal region. BrdU-labeled cells were localized mainly within the lateral, dorsal, and medial cortex, septal nucleus, nucleus sphericus, preoptic area, and hypothalamus. In all regions, the number of BrdU-labeled cells in the ventricular zone was higher in the fall compared to spring. In the parenchymal region, a significantly higher number of labeled cells was also observed during the fall, but only within the nucleus sphericus and the combined preoptic area/hypothalamus. The immunoreactive cell number did not vary significantly with days post-BrdU treatment in either season or in any brain region. While it is possible that the higher rates of cell proliferation in the fall simply reflect increased growth of all body tissues, including the brain, our data show that seasonal changes in cell migration into the parenchyma are region specific. In red-sided garter snakes and other reptiles, the dorsal and medial cortex is important for spatial navigation and memory, whereas the nucleus sphericus, septal nucleus, and preoptic area/hypothalamus are central to reproductive regulation. Thus, our results provide support for the hypothesis that adult neurogenesis plays a role in mediating seasonal rhythms in migratory and reproductive behaviors. © 2014 S. Karger AG, Basel

Deborah I. Lutterschmidt Department of Biology, Portland State University 1719 SW 10th Ave Portland, OR 97201 (USA) E-Mail d.lutterschmidt @ pdx.edu

Abbreviations used in this paper

ac Acc aDVR AOB aot aSN BNST BrdU BrdU-ir C co d DM dRF Fun GC Ico III IIIN Ip l lfb LHN lSN m mfb mSN

anterior commissure nucleus accumbens anterior dorsal ventricular ridge accessory olfactory bulb accessory olfactory tract anterior septal nucleus bed nucleus of the stria terminalis 5-bromo-2′-deoxyuridine BrdU-immunoreactive cerebellum cortex dorsal cortex dorsomedial thalamic nucleus dorsal retrobulbar formation nucleus of the dorsal funiculus central gray intercollicular nucleus oculomotor nerve nucleus of the oculomotor nerve interpeduncular nucleus lateral cortex lateral forebrain bundle lateral posterior hypothalamic nucleus lateral septal nucleus medial cortex medial forebrain bundle medial septal nucleus

Introduction

Plasticity in the adult central nervous system has been described in all vertebrate classes as well as in some invertebrate groups, including insects and crustaceans [e.g. see review in Lindsey and Tropepe, 2006]. One form of neuroplasticity is postnatal neurogenesis, defined as the birth and maturation of new neurons that add to or replace neurons in the existing circuitry [Lindsey and Tropepe, 2006]. Neurogenesis generally involves 4 different phases: cell proliferation within the ependymal layer of a ventricle, cell migration into the parenchyma, cell differentiation and maturation, and cell survival. Numerous laboratory studies have focused on elucidating the molecular, neural, and endocrine mechanisms regulating neurogenesis [e.g. Berg et al., 2013] and the environmental factors that influence the rates of cell proliferation and cell survival [e.g. Nithianantharajah and Hannan, 2006; Simpson and Kelly, 2011]. However, the vast majority of these studies have been conducted in relatively few species, with an emphasis on laboratory mice and rats [Lindsey and Tropepe, 2006]. 182

Brain Behav Evol 2014;84:181–196 DOI: 10.1159/000364778

NS oc Ols optr OS ot OT PBS pDVR PH POA pvo ra Ris sn SN SVL Torc Torl va Vds Vedl Vmd VMH VTA XIIN

nucleus sphericus optic chiasm superior olive optic tract olfactostriatum olfactory tubercle optic tectum phosphate-buffered saline posterior dorsal ventricular ridge periventricular hypothalamic nucleus preoptic area paraventricular organ rostral amygdaloid nucleus isthmal reticular nucleus substantia nigra septal nucleus snout-vent length torus semicircularis centralis torus semicircularis laminaris ventral amygdaloid nucleus descending nucleus of the trigeminal nerve dorsolateral vestibular nucleus dorsal motor nucleus of the trigeminal nerve ventromedial hypothalamic nucleus ventral tegmental area hypoglossal nucleus

Among vertebrates, mammals exhibit relatively low levels of cell proliferation in the adult brain, and the regions of proliferative activity are limited to 2 areas: the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus within the hippocampus. In contrast, birds show ‘hot spots’ of cell proliferation within the periventricular zone of the lateral ventricles [Lindsey and Tropepe, 2006], while ectothermic vertebrates exhibit high levels of cell proliferation and migration throughout many areas of the telencephalon [reviewed in Kaslin et al., 2008]. As suggested by Lindsey and Tropepe [2006], the limited taxonomic diversity represented in the current neurogenesis literature limits our ability to assess the functional significance of adult neurogenesis for natural behaviors as well as the evolution of neurogenesis and its regulatory mechanisms. A more recent review by Bonfanti and Peretto [2011] further suggests that our current mammalian-based understanding of neurogenesis is also biased by a limited taxonomic representation within mammals. For example, in some mammalian species such as the New Zealand white HY/ CR rabbit (Orictolagus cuniculus), adult neurogenesis has Maine/Powers/Lutterschmidt

been described in regions outside of the well characterized and widely accepted neurogenic sites [e.g. Luzzati et al., 2006; Migaud et al., 2010]. Differences in neurogenesis have also been described between mammalian orders, species, and even strains of laboratory mice [reviewed by Lindsey and Tropepe, 2006; Gould, 2007; Bonfanti and Peretto, 2011; Brus et al., 2013]. Thus, the current evidence suggests that patterns of adult neurogenesis may be species specific, with variation in neurogenesis reflecting each species’ unique behavioral, social, and ecological niche [Bonfanti and Peretto, 2011]. Despite remaining questions about how and if patterns of neurogenesis vary phylogenetically, there is clear evidence in multiple vertebrate taxa that cell proliferation and cell migration in the brain are associated with learning, memory, and olfactory processes [Barker et al., 2011; reviewed in Shors et al., 2012]. For example, the survival of new brain cells is enhanced by spatial navigation learning during Morris water maze tests in rats and mice [reviewed in Shors, 2008; Shors et al., 2012]. Similarly, Hoshooley and Sherry [2007] showed that greater neuron recruitment in the hippocampus is associated with food caching in adult black-capped chickadees, i.e. Poecile atricapillus [also see review in Sherry and Hoshooley, 2010]. In reptiles, including turtles, lizards, and snakes, a larger hippocampal volume and/or increased cell proliferation in the brain is also associated with increased spatial memory demands [e.g. López et al., 2003; Roth et al., 2006; Delgado-González et al., 2008; Sampedro et al., 2008; Holding et al., 2012]. However, we still know very little about the role of neurogenesis in other brain regions or in the regulation of natural behaviors other than learning and spatial memory. Moreover, in contrast to the vast literature on laboratory rodents, relatively few studies have focused on evaluating whether seasonal changes in neurogenesis mediate seasonal rhythms in physiology and behavior in natural populations. There is increasing evidence that seasonal rhythms in postembryonic brain development play a role in orchestrating seasonal changes in physiology and behavior [Ebling and Barrett, 2008]. Many developmental processes in the brain vary seasonally, including cell proliferation, cell death, and neuron migration [Ebling and Barrett, 2008]. In fact, the first direct evidence that newly proliferated cells could be incorporated into the existing neural circuitry as functional neurons was provided by studies of the highly seasonal HVC song nucleus in adult male canaries [Paton and Nottebohm, 1984; reviewed in Nottebohm, 2004]. Further studies in this species demonstrated that seasonal changes in neurogenesis were temporally associated with seasonal changes in song learning Seasonal Neurogenesis in Garter Snakes

and the recruitment of new song syllables [Kirn et al., 1994]. Similarly, Almli and Wilczynski [2009, 2012] demonstrated that seasonally breeding male and female green treefrogs (Hyla cinerea) respond to conspecific mating calls with an increase in cell proliferation in acoustically sensitive brain regions that regulate sexual behavior. These and other studies suggest that neurogenesis may play an integral role in mediating seasonal changes in the sensitivity to sensory stimuli, including social reproductive cues. Indeed, Migaud et al. [2011] showed that photoperiod-induced changes in neurogenesis are associated with the timing of reproductive activity in Île-de-France ewes, i.e. Ovis aries. To better understand the biological relevance of neurogenesis, studies addressing its functional significance for natural behaviors in a wide variety of species are needed [Lindsey and Tropepe, 2006]. This is underscored by the findings of Delgado-González et al. [2008], showing that the proliferative activity in Tenerife lizards (Gallotia galloti) is reduced in captivity, as well as the original studies of Kaplan [see review in Kaplan, 2001] and others describing increased neurogenesis in response to environmental enrichment in mammals [see reviews in Nithianantharajah and Hannan, 2006; Simpson and Kelly, 2011]. In the present study, we used a population of freeranging male red-sided garter snakes (Thamnophis sirtalis parietalis) in Manitoba, Canada, to test the hypothesis that seasonal shifts in physiology and behavior are associated with seasonal variation in cell proliferation in multiple brain regions. This population of snakes undergoes 8 months of winter dormancy in underground hibernacula followed by a brief, intense mating season from late April through May. Once snakes disperse from the den, they travel as far as 18 km to summer feeding grounds [Gregory and Stewart, 1975]. After approximately 3 months of summer foraging activity, snakes migrate back to the den site in preparation for winter dormancy. This highly seasonal model system provides an excellent opportunity to better understand whether seasonal neurogenesis is related to seasonal transitions between reproductive, migratory, and foraging behaviors. Specifically, we asked whether the rates of cell proliferation and cell migration in the brain vary between adult male snakes collected during spring and fall. Materials and Methods Animals and Experimental Design The experimental protocols were approved by the Portland State University Animal Care and Use Committee and were in

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compliance with the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This research was performed under the authority of Wildlife Scientific Permit WB12691, issued by the Manitoba Department of Conservation. We collected male red-sided garter snakes (T. sirtalis parietalis) from a single den site in the Interlake region of Manitoba, Canada, during the spring breeding season (April 27, 2009; n = 15) and the fall prehibernation period (September 14, 2009; n = 15), when snakes return to their den site in preparation for winter dormancy. The approximate photoperiod during the midbreeding season is 16:8 h LD; during the fall prehibernation period the photoperiod is approximately 11:13 h LD. Environmental temperatures can be extremely variable in the Interlake region during spring and fall. During these experiments, the mean high and low temperatures recorded at the weather station in nearby Fisher Branch, Manitoba, from April 27 to May 7, 2009, were 14.3 and –0.1 ° C, respectively. From September 14 to 24, 2009, the mean high and low temperatures were 25.2 and 10.3 ° C, respectively (Digital Archive of Canadian Climatological Data; Environment Canada). Animals were weighed and their snout-vent length (SVL) was measured. All animals used in this study were sexually mature adult males with an SVL greater than 39 cm (range 40.8–55.4), the minimum size generally indicative of adult status in T. sirtalis parietalis [Crews et al., 1985; Conant and Collins, 1998]. Similar to Almli and Wilczynski [2007], we treated each animal with 2 consecutive intraperitoneal injections of 100 mg/kg 5-bromo-2′-deoxyuridine (BrdU; item B5002; Sigma-Aldrich, St. Louis, Mo., USA) between 15.00 and 19.00 h on the day of capture. BrdU is a thymidine analog that is incorporated into the DNA of mitotic cells. BrdU was diluted to a concentration of 20 mg/ml in 25% DMSO in reptile Ringer’s solution; the total injection volume for each snake was 1% of the body mass. All animals were returned to the research station, where they were housed for up to 10 days in semi-natural outdoor arenas (1 × 1 × 1 m with an open top) containing a hide box and water bowl. Previous studies in red-sided garter snakes have demonstrated that these arenas do not induce significant increases in plasma glucocorticoid stress hormones, particularly during the spring mating season [e.g. Moore and Mason, 2001; Cease et al., 2007; Lutterschmidt and Mason, 2010]. Water was provided ad libitum, but the snakes were not fed during this study because males do not eat during the spring mating season or the fall prehibernation period. All spring- and fall-collected snakes were handled identically. To examine temporal variation in cell proliferation and/or cell migration within each season, we euthanized a subset of 5 snakes at 1, 5, and 10 days post-BrdU treatment with a lethal overdose of 1% sodium pentobarbital (0.2 ml/snake). Brains were immersionfixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 18 h at 4 ° C. Tissues were then transferred to 0.1 M phosphate buffer and stored at 4 ° C until sectioning. Brains were cryoprotected in 30% sucrose in 0.1 M phosphate buffer and cut on a cryostat (Leica 3050S) into 4 alternate series of 25-μm coronal sections. Tissues were thaw-mounted onto subbed slides (Fisherbrand Superfrost Plus) and stored at –20 ° C until immunohistochemical processing.  

 

 

 

 

 

 

 

 

 

BrdU Immunohistochemistry Seasonal variation in cell proliferation was examined using immunohistochemistry for BrdU-positive nuclei on one series of tis-

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Brain Behav Evol 2014;84:181–196 DOI: 10.1159/000364778

sue. All slides were processed during one assay. Slides were allowed to air-dry and the tissues were outlined with a hydrophobic barrier (Liquid Blocker – Super Pap Pen; Electron Microscopy Sciences, Hatfield, Pa., USA). Tissues were further fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4) for 5 min to ensure the tissue adhered to the slides. The slides were washed 3 times for 5 min with 0.1 M PBS followed by fixative neutralization with 0.1% sodium borohydride (pH 8.5) for 20 min. The slides were again washed in PBS (3 × 5 min). Antigen retrieval was performed by incubating the slides in 2 N HCl in PBS at 37 ° C for 30 min to denature the DNA. The slides were neutralized via washing with 0.1 M boric acid buffer (pH 8.5; 2 × 5 min) followed by PBS (3 × 5 min). Endogenous peroxidase activity was quenched with 1% hydrogen peroxide in 0.1 M PBS for 30 min. The slides were washed in PBS (2 × 5 min) followed by PBS with 0.3% Triton X (PBS-T; 1 × 5 min) and then incubated for 60 min in PBS-T containing 10% normal goat serum and 10% avidin (Vector Labs, Inc., Burlingame, Calif., USA) to reduce nonspecific binding. BrdU immunoreactivity was examined using a rat anti-BrdU antiserum (item OBT0030; Accurate Chemical, Westbury, N.Y., USA) at a dilution of 1: 5,000 in PBS-T containing 10% normal goat serum and 10% biotin (Vector Labs). Sections were incubated with the primary antibody for 48 h at 4 ° C in a humid chamber. The slides were then washed in PBS (2 × 5 min) followed by PBS-T (1 × 5 min) and the primary antibody signal amplified by incubation for 60 min with biotinylated goat anti-rat secondary antibody (item BA-9400; Vector Labs) diluted 1: 400 in 0.1 M PBS-T. The slides were washed in PBS (2 × 5 min) followed by PBS-T (1 × 5 min). Tissues were incubated for 60 min in avidin conjugated to horseradish peroxidase (Elite ABC peroxidase kit; Vector Labs) and rinsed in PBS (2 × 5 min) followed by PBS-T (1 × 5 min). Primary antibody binding was visualized using 0.25 mg/ml diaminobenzidine (item 0430-5G; BioExpress, Kaysville, Utah, USA) in 0.2% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.2). The reaction was terminated by immersion in nanopure H2O (3 × 5 min). Tissues were dehydrated in a graded ethanol series, cleared with Citrisolv (Fisher Scientific, Pittsburgh, Pa., USA), and covered with Permount and coverslips. Control tests included omission of the primary antibody and the antigen retrieval step.  

 

 

 

Immunoreactive Cell Counting Stained tissue was examined using an Olympus BX40 microscope with a QIClick digital camera and QImaging software (QImaging; Surrey, B.C., Canada). The locations of BrdU-immunoreactive (BrdU-ir) cells were mapped onto standard anatomical brain sections adapted from Martinez-Marcos et al. [2001, 2005] and Krohmer et al. [2010]. Immunoreactive cells were counted manually in both hemispheres of the brain; animals were coded so that the observer was blind to the treatment group of individuals. Within each brain region, the total number of BrdU-ir nuclei was quantified in every tissue section throughout the region of interest under ×200 magnification and again under ×400 magnification. These counts were usually identical but in some cases differed by 1–3 cells. In these instances, the section was recounted under ×400 magnification to verify the cell count. Labeled nuclei were further categorized by their location relative to the ventricle. As in the study of Almli and Wilczynski [2007], a BrdU-ir cell was considered to be located within the ventricular zone if it was within 50 μm of a ventricle (fig. 1). If the BrdU-labeled cell was located more than 50 μm from the ependymal layer of the ventricle, it was cat-

Maine/Powers/Lutterschmidt

Color version available online

Fig. 1. Example photomicrograph showing BrdU-labeled cells in the dorsal cortex of male red-sided garter snakes (T. sirtalis parietalis). Arrows indicate BrdU-ir nuclei; scale bar = 50 μm. The dashed line delineates a distance of 50 μm from the ependymal layer of the lateral ventricle, which was used to further categorize labeled cells as being located within the ventricular zone (i.e. within 50 μm of the ventricle) or the parenchymal region (i.e. more than 50 μm from the ependymal layer of the ventricle).

Table 1. Final sample sizes for investigating seasonal variation in BrdU-ir cell number within the cortex, SN, combined NS and aDVR, and combined POA and hypothalamus of male red-sided garter snakes (T. sirtalis parietalis)

Brain region

Cortex SN NS and aDVR POA and hypothalamus

Sample sizes spring

fall

4, 3, 5 4, 4, 5 4, 4, 4 4, 5, 5

5, 5, 5 3, 4, 3 4, 4, 3 5, 5, 5

Values are for 1, 5, 10 days post-BrdU treatment.

al. [2008] to account for missing and/or damaged sections. Within each region, the number of BrdU-ir cells for an individual was calculated as the total number of labeled cells divided by the total number of usable brain sections multiplied by the average number of brain sections for a given region across all animals. These counting methods allowed us to retain more animals in the statistical analyses, in contrast to eliminating an animal if 2 or more consecutive sections were missing or damaged [e.g. Lutterschmidt and Wilczynski, 2012]. The mean number of missing or damaged sections across all animals and regions was 1.42 (±0.03 SE). On average, any animal missing more than 30% of its sections within a region of interest was excluded from statistical analyses. The final sample sizes for each region of interest are listed in table 1. Because tissue was divided into 4 different series of 25-μm sections, approximately 100 μm separated each section within a series. Thus, there is little possibility that double counting of nuclei split between 2 sections inflated our cell quantifications. In addition, a BrdU-labeled cell was counted only if the cell and nucleus were of the same general size and shape as the other BrdU-ir cells in the region of interest, thereby excluding partial nuclei from our quantifications. Statistics We compared the number of BrdU-ir cells between the left and right hemispheres in each bilateral brain region using paired t tests. All spring- and fall-collected animals at each sampling time were used for these analyses. To determine whether the BrdU-ir cell number varies between seasons or over time, we performed a twoway analysis of variance (ANOVA) for each region of interest, with season and days post-BrdU treatment as the between-subjects factors. Data were natural log-transformed where necessary to meet the assumptions required for parametric analysis. When data transformation could not correct for nonnormality and/or unequal variance, we used the nonparametric Scheirer-Ray-Hare extension of the Kruskal-Wallis analysis, as described by Lutterschmidt and Mason [2008, 2009].

Results

Distribution of BrdU-Labeled Cells As expected, omission of both the primary antibody and the antigen retrieval step (incubation in 2 N HCl at 37 ° C for 30 min) eliminated all immunoreactive staining. Newly proliferated BrdU-ir cells were regionally distributed in the adult male red-sided garter snake brain as shown in figure 2. BrdU-ir cells occurred mainly in the ventricular zone (i.e. within 50 μm of the ependymal cell layer of the ventricle), especially in forebrain regions. Immunoreactive cells often appeared in clusters or pairs of labeled cells. A large number of BrdU-ir cells was observed in the telencephalon, primarily around the lateral ventricles from the accessory olfactory bulbs rostrally to the caudal portions of the cortex. From the rostral to the caudal telencephalon, BrdU-ir cells were distributed around the dorsolateral region of the lateral ventricles in  

egorized as being located within the parenchymal region [Almli and Wilczynski, 2007]. The cell category was also confirmed by the nucleus shape, as cells located close to the ventricle within the ventricular zone have nuclei that are circular in shape, while cells migrating away from the ventricle (i.e. within the parenchymal region) have nuclei that are elongated and more oval in shape (fig. 1) [Almli and Wilczynski, 2007, 2009; Delgado-González et al., 2011]. For each region of interest, we therefore report the total number of BrdU-labeled nuclei, the number of BrdU-ir cells in the ventricular zone, and the number of BrdU-ir cells in the parenchymal region. The number of BrdU-ir cells in each brain region was quantified in one tissue series and totaled for each individual. We followed the counting methods and criteria described by Laberge et

Seasonal Neurogenesis in Garter Snakes

 

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E

I

F

J

A

B

C

G

Pineal gland

K

D

H

L

Fig. 2. Composite distribution of BrdU-ir cells in the brain of adult male red-sided garter snakes (T. sirtalis parietalis) across all individuals and seasons. A Schematic showing the dorsal view of the snake brain; the rostral portion of the brain is oriented to the left. The labeled vertical lines (B–L) indicate the level of sections through BrdU-ir cell populations from the rostral telencephalon

(B) through the caudal hindbrain (L). We quantified BrdU-labeled nuclei in the lateral, dorsal, and medial cortex (B–H), the SN (D–F), the combined NS and anterior dorsal ventricular ridge (C–H), and the combined anterior POA and hypothalamus (E–H). Solid dots indicate the locations of immunoreactive nuclei.

the lateral, dorsal, and medial cortex, and in the ventromedial region of the lateral ventricles in the nucleus sphericus (NS) and the septal nucleus (SN; anterior, lateral, and medial regions) (fig. 2). Many immunoreactive cells were also observed around the ventral and lateral

regions of the third ventricle in the preoptic area (POA) and hypothalamus. Immunoreactive cells were occasionally found in the bed nucleus of the stria terminalis, the dorsomedial thalamic nucleus, the interpeduncular nucleus, the posterior optic tectum, the substantia nigra, and

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the lateral, medial, and rostral ventral amygdaloid nucleus. The brainstem had few BrdU-ir cells. The regional distribution of the newly proliferated cells we observed within the brain of red-sided garter snakes is similar to that reported in other adult vertebrates, including the occasional cells localized within the bed nucleus of the stria terminalis, the thalamic nucleus, the interpeduncular nucleus, the optic tectum, and the substantia nigra [e.g. Shan et al., 2006; Akbari et al., 2007; Almli and Wilczynski, 2007, 2009]. Within the cortex, BrdU-ir cells were distributed similarly throughout the lateral, dorsal, and medial regions in both the ventricular zone and parenchymal regions (fig. 2). We observed relatively more BrdU-labeled cells in the parenchymal regions of the middle and caudal SN compared to the rostral SN, whereas BrdU-ir cells in the ventricular zone were uniformly distributed throughout the SN. Labeled cells were numerous in the rostral portion of the anterior dorsal ventricular ridge, where it is adjacent to the NS. Few labeled cells were observed within the posterior dorsal ventricular ridge. As shown in figure 2, labeled cells in the NS were uniformly distributed throughout the region in both the ventricular zone and the parenchymal region. Within the POA, labeled cells were located lateral to the third ventricle rather than close to or within the ependymal layer of the ventricle, similar to the pattern described by Font et al. [2001]. This pattern of cell labeling continued throughout the ventromedial hypothalamic nucleus, the lateral posterior hypothalamic nucleus, and the periventricular hypothalamic nucleus. No BrdU-ir cells were found within the periventricular organ. We quantified the BrdU-ir cell number in the lateral, dorsal, and medial regions of the cortex, the NS, the anterior dorsal ventricular ridge, the anterior, lateral, and medial SN, the POA, and the hypothalamus. Because distinct boundaries could not be consistently established between the BrdU-ir cell populations in the NS and the anterior dorsal ventricular ridge, labeled cells located in the anterior dorsal ventricular ridge were grouped into and analyzed with the NS. Similarly, distinct boundaries could not be identified between labeled nuclei in the POA and hypothalamus, and thus labeled cells within the hypothalamus were grouped into and analyzed with the POA. Seasonal Variation in BrdU-ir Cell Number There were no significant differences in the body size (SVL) of animals between seasons (F1, 29 = 0.616, p = 0.440) or among days post-BrdU treatment (F2, 29 = 0.377, p = 0.690; from a two-way ANOVA), indicating that we Seasonal Neurogenesis in Garter Snakes

measured rates of cell proliferation and cell migration in the same size/age class of snakes during both spring and fall. As expected, male snakes collected during the fall after the summer foraging period weighed more than males collected during the spring mating season (F1, 29 = 4.806, p = 0.038; from a two-way ANOVA). Body mass did not differ significantly among days post-BrdU treatment (F1, 29 = 0.475, p = 0.627). The interaction between season and days post-BrdU was statistically nonsignificant for both SVL (F1, 29 = 0.423, p = 0.660) and body mass (F1, 29 = 1.077, p = 0.357). We first compared the number of BrdU-ir nuclei between the left and right hemispheres of the brain in both spring- and fall-collected animals. There were no significant differences in the total number of labeled cells between hemispheres in the cortex (t = 0.141, d.f. = 18, p = 0.889), NS (t = –0.114, d.f. = 25, p = 0.910), or SN (t = 0.540, d.f. = 19, p = 0.596, all statistics from paired t tests). There were also no differences in the number of labeled cells located in the ventricular zone between hemispheres (cortex: t = –0.796, d.f. = 18, p = 0.436; NS: t = –0.070, d.f. = 25, p = 0.945, and SN: t = –0.056, d.f. = 19, p = 0.956, from paired t tests). Finally, there were no significant differences in the number of labeled cells in the parenchymal region between hemispheres (cortex: t = –0.337, d.f. = 18, p = 0.740; NS: t = –0.526, d.f. = 25, p = 0.603, and SN: t = 0.869, d.f. = 19, p = 0.396, from paired t tests). We therefore used cell counts from one hemisphere for all subsequent analyses. For each animal, the hemisphere with the lowest number of missing or damaged tissue sections was selected to maximize the sample sizes. Because BrdU-labeled cells in the POA and hypothalamus are located along the midline of the brain, we reported the number of cells in this region as one population. Cortex. The total number of BrdU-ir cells in the combined lateral, dorsal, and medial cortex was significantly higher during the fall compared to spring (fig. 3A; F1, 26 = 4.836, p = 0.039, from a two-way ANOVA). The days post-BrdU treatment did not significantly influence the total number of BrdU-labeled cells in the cortex (F2, 26 = 2.142, p = 0.142). The number of BrdU-ir cells located in the ventricular zone also varied significantly with season (fig. 3B; F1, 26 = 5.276, p = 0.032) but not with days postBrdU treatment (F2, 26 = 2.645, p = 0.095, from a two-way ANOVA). Neither season (F1, 26 = 2.764, p = 0.111) nor days post-BrdU treatment (F2, 26 = 2.031, p = 0.156, from a two-way ANOVA) significantly influenced the number of BrdU-ir cells in the parenchymal region of the cortex (fig. 3C). There were no significant interactions between season and days post-BrdU treatment in the number of Brain Behav Evol 2014;84:181–196 DOI: 10.1159/000364778

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total (F2, 26 = 0.450, p = 0.644), ventricular zone (F2, 26 = 0.424, p = 0.660), or parenchymal region (F2, 26 = 0.788, p = 0.468) immunoreactive cells. Septal Nucleus. A two-way ANOVA indicated that the total number of BrdU-ir cells in the SN did not vary significantly with season (fig. 4A; F1, 22 = 1.171, p = 0.294) or days post-BrdU treatment (F2, 22 = 1.627, p = 0.226). Within the ventricular zone of the SN, the number of BrdU-ir cells also did not vary significantly with days posttreatment (H2, 22 = 3.580, p = 0.167; from a ScheirerRay-Hare extension of the Kruskal-Wallis analysis). The number of ventricular zone BrdU-ir cells was higher during the fall compared to spring (fig. 4B; H1, 22 = 3.658, p = 0.056), but this effect did not reach the threshold for statistical significance at α = 0.05. However, collapsing days post-BrdU and using a parametric t test to analyze these data revealed that the number of BrdU-labeled cells within the ventricular zone was significantly higher during the fall compared to spring (t = –2.101, d.f. = 21, p = 0.048). The small discrepancy between these two analyses is likely a result of slight variation in the calculations underlying these tests, and we therefore treated the number of BrdUlabeled cells within the ventricular zone of the SN as exhibiting significant seasonal variation. Finally, neither season (H1, 22 = 0.002, p = 0.965) nor days post-BrdU treatment (H2, 22 = 0.585, p = 0.747, from a Scheirer-RayHare extension of the Kruskal-Wallis analysis) significantly influenced the number of BrdU-ir cells in the parenchymal region of the SN (fig. 4C). There were no significant interactions between season and days post-BrdU treatment in the number of total (F2, 22 = 2.136, p = 0.149), ventricular zone (H2, 22 = 3.155, p = 0.206), or parenchymal region (H2, 22 = 2.196, p = 0.334) immunoreactive cells. Nucleus Sphericus and Anterior Dorsal Ventricular Ridge. The total number of BrdU-ir cells in the combined NS and anterior dorsal ventricular ridge was significantly higher during the fall compared to spring (fig. 5A; F1, 22 = 15.880, p < 0.001, from a two-way ANOVA). Days post-BrdU treatment did not significantly influence the total number of BrdU-labeled cells in the NS (F2, 22 = 2.587, p = 0.104). The number of BrdU-ir cells located in the ventricular zone of the NS also varied significantly with season (fig. 5B; F1, 22 = 12.275, p = 0.003) but not with days post-BrdU treatment (F2, 22 = 2.038, p = 0.161, from a two-way ANOVA). Finally, the number of BrdUir cells located in the parenchymal region of the NS varied significantly with season (fig. 5C; F1, 22 = 13.464, p = 0.002) but not with days post-BrdU treatment (F2, 22 = 3.154, p = 0.068, from a two-way ANOVA). There were 188

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A

B

C

Fig. 3. Seasonal variation in the total number of BrdU-ir cells in the lateral, dorsal, and medial cortex of adult male red-sided garter snakes (T. sirtalis parietalis) (A). Labeled cells were further categorized as ventricular zone (B) or parenchymal region cells (C). Each bar is the mean (+1 SEM) number of BrdU-labeled cells in one brain hemisphere for all snakes within each season. Asterisks indicate significant main effects of season on the BrdU-labeled cell number (statistics from two-factor ANOVA). Days post-BrdU treatment did not affect cell number in any category and therefore data are shown collapsed across days posttreatment for clarity. Collapsed sample sizes are shown above the x-axis in C and are identical for all panels (i.e. total, ventricular zone, and parenchymal region cell number).

Maine/Powers/Lutterschmidt

A

A

B

B

C

Fig. 4. Seasonal variation in the total number of BrdU-ir cells in

the septal nucleus (SN) of adult male red-sided garter snakes (T. sirtalis parietalis) (A). Labeled cells were further categorized as ventricular zone (B) or parenchymal region cells (C). Each bar is the mean (+1 SEM) number of BrdU-labeled cells in one brain hemisphere for all snakes within each season. The asterisk indicates significant main effects of season on the BrdU-labeled cell number (statistics from two-factor ANOVA). Days post-BrdU treatment did not affect cell number in any category and therefore data are shown collapsed across days posttreatment for clarity. Collapsed sample sizes are shown above the x-axis in C and are identical for all panels.

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C

Fig. 5. Seasonal variation in the total number of BrdU-ir cells in the nucleus sphericus (NS) of adult male red-sided garter snakes (T. sirtalis parietalis) (A). Cells quantified within the anterior dorsal ventricular ridge were grouped with the NS for analysis. Labeled cells were further categorized as ventricular zone (B) or parenchymal region cells (C). Each bar is the mean (+1 SEM) number of BrdU-labeled cells in one brain hemisphere for all snakes within each season. Asterisks indicate significant main effects of season on BrdU-labeled cell number (statistics from two-factor ANOVA). Days post-BrdU treatment did not affect cell number in any category and therefore data are shown collapsed across days posttreatment for clarity. Collapsed sample sizes are shown above the xaxis in C and are identical for all panels.

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no significant interactions between season and days post-BrdU treatment in the number of total (F2, 22 = 0.283, p = 0.757), ventricular zone (F2, 22 = 0.718, p = 0.502), or parenchymal region (F2, 22 = 0.004, p = 0.996) BrdU-ir cells. Preoptic Area and Hypothalamus. The total number of BrdU-ir cells located in the combined POA and hypothalamus was significantly higher during the fall compared to spring (fig. 6A; F1, 28 = 6.791, p = 0.016, from a two-way ANOVA). Days post-BrdU treatment did not significantly influence the total number of BrdU-labeled cells in the POA (F2, 28 = 1.201, p = 0.319). The number of BrdU-ir cells located in the ventricular zone of the POA also varied significantly with season (fig. 6B; F1, 28 = 7.770, p = 0.010) but not with days post-BrdU treatment (F2, 28 = 1.375, p = 0.273, from a two-way ANOVA). Finally, the number of BrdU-ir cells located in the parenchymal region of the POA varied significantly with season (fig. 6C; F1, 28 = 6.530, p = 0.018) but not with days post-BrdU treatment (F2, 28 = 1.007, p = 0.381, from a two-way ANOVA). There were no significant interactions between season and days post-BrdU treatment in the number of total (F2, 28 = 1.681, p = 0.208), ventricular zone (F2, 28 = 1.846, p = 0.180), or parenchymal region (F2, 28 = 1.828, p = 0.183) immunoreactive cells.

A

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C

Discussion

Fig. 6. Seasonal variation in the total number of BrdU-ir cells in

the combined preoptic area (POA) and hypothalamus of adult male red-sided garter snakes (T. sirtalis parietalis) (A). Labeled cells were further categorized as ventricular zone (B) or parenchymal region cells (C). Each bar is the mean (+1 SEM) number of BrdU-labeled cells for all snakes within each season. Asterisks indicate significant main effects of season on BrdU-labeled cell number (statistics from two-factor ANOVA). Days post-BrdU treatment did not affect cell number in any category and therefore data are shown collapsed across days posttreatment for clarity. Collapsed sample sizes are shown above the x-axis in C and are identical for all panels.

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Brain Behav Evol 2014;84:181–196 DOI: 10.1159/000364778

This is the first study to report seasonal variation in cell proliferation in the brain of a snake. We demonstrated that proliferative activity occurs regionally in the brain of adult male red-sided garter snakes (T. sirtalis parietalis), with BrdU-labeled cells localized mainly within the telencephalon. In addition, we demonstrated that proliferative rates differ significantly between snakes collected during spring and fall. In all regions of interest, the number of BrdU-labeled cells located within the ventricular zone was higher in the fall compared to spring (table 2). In the parenchymal region, a significantly higher number of labeled cells was also observed during fall, but only within the NS and POA/hypothalamus. The immunoreactive cell number did not vary significantly with days postBrdU treatment in either season or in any brain region. Our results suggest that seasonal differences in cell proliferation and cell migration in the adult garter snake brain are regulated differentially in different brain regions. We discuss these results in the context of the role of these brain regions in seasonal behavior, particularly reproduction and migration. Maine/Powers/Lutterschmidt

Table 2. Summary of seasonal differences in BrdU-labeled cell number in adult male red-sided garter snakes (T. sirtalis parietalis) with-

in the cortex, SN, combined NS and anterior dorsal ventricular ridge, and combined POA and hypothalamus Brain region

Cortex SN NS and aDVR POA and hypothalamus

Total BrdU-labeled cells

Ventricular zone cells

Parenchymal region cells

seasonal difference

p

seasonal difference

p

seasonal difference

p

Fall > spring Nonsignificant Fall > spring Fall > spring

0.039 0.294 0.001 0.016

Fall > spring Fall > spring Fall > spring Fall > spring

0.032 0.056 0.003 0.010

Nonsignificant Nonsignificant Fall > spring Fall > spring

0.111 0.965 0.002 0.018

All statistics represent the main effects of the factor season from a multifactor ANOVA within each brain region. Within the ventricular zone of the SN, note that when days post-BrdU is collapsed and the data are reanalyzed using a t test to increase power, the seasonal difference in BrdU-labeled nuclei is statistically significant at p = 0.048.

Cell Proliferation and Cell Migration In the adult brain, new cells are born from dividing ventricular zone cells [López-García et al., 1988; PérezCañellas et al., 1997]. As in other vertebrates, cell proliferation during embryonic development and in the adult reptilian brain exhibits similar patterns, with new cells migrating along radial glial cells to their final destination, usually into the parenchyma where they can become incorporated into the existing neural circuitry [e.g. Paton and Nottebohm, 1984]. Similar to previous studies [Radmilovich et al., 2003; Delgado-González et al., 2011], we observed labeling at the lateral ventricles within 24 h following injection of BrdU. We also observed very few cells within the ventricular zone of the third ventricle, a pattern that has been described in other reptiles [reviewed in Font et al., 2001]. We found no significant variation in BrdU-ir cell number with days post-BrdU treatment in any brain region. Our results suggest that neither significant transient amplification (i.e. the continued cell division of newly proliferated cells during migration into the parenchyma) nor significant differences in cell survival occurred during this 10-day study. A longer time course with larger sample sizes may be needed to determine rates of cell migration and/or survival. In a preliminary study using identical BrdU injection procedures in the same population of red-sided garter snakes, the number of BrdU-labeled cells did not vary significantly over a 28day period during the spring breeding season [Lutterschmidt, unpubl. data]. However, a similar time course during the fall, when overall levels of cell proliferation are higher, would be helpful in determining patterns of cell migration into the parenchyma as well as rates of cell survival.

Most cells born within the ventricular zone are undifferentiated and can develop into either neurons or glial cells. Experiments carried out in Iberian wall lizards (Podarcis hispanica) and Moorish wall geckos (Tarentola mauritanica) using cell labeling with [3H]-thymidine and specific cell markers revealed that labeled cells found outside the ventricular zone differentiate into neurons rather than into glial cells [López-García et al., 1988; Pérez-Cañellas and García-Verdugo, 1996]. Further, López-García et al. [1990] reported that a minimum of 7 days is required for newly proliferated cells to differentiate into neurons in Iberian wall lizards (P. hispanica). In this study, we observed BrdU-labeled cells in the parenchyma in all brain regions in as little as 1 day posttreatment. As suggested by Delgado-González et al. [2011], these cells could be newly labeled blood vessel endothelial cells or neural progenitor cells dividing en route as they migrate away from the ventricular zone. Importantly, Delgado-González et al. [2011] showed that 90% of the labeled cells observed in the parenchyma of Tenerife lizards (G. galloti) also express doublecortin, a neuronal cell marker, suggesting that most labeled cells within the parenchymal region are developing neurons. Additional studies using double-labeling techniques for phenotype-specific cellular markers are needed to determine the fate of newly proliferated cells in the adult brain of red-sided garter snakes. For example, in many cases, whether newly proliferated cells differentiate into glial cells or neurons, and what neuronal phenotype these cells later express (e.g. dopaminergic, neuropeptide-producing, etc.), is unknown. Such studies would not only help determine if cell differentiation varies seasonally in the brain of this and other species, but they would also shed muchneeded light on the possible function of neurogenesis in facilitating seasonal changes in behavior.

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Seasonal Variation In all brain regions where cell proliferation was quantified (i.e. cortex, SN, NS, and combined POA/hypothalamus), we found that snakes collected during the fall had a significantly higher number of BrdU-labeled cells in the ventricular zone compared to snakes collected during spring. As reviewed in the paper by Kaslin et al. [2008], the higher level of cell proliferation observed in the fall may reflect an increased growth rate of snakes during summer foraging. For example, during spring, snakes emerge from winter dormancy and immediately enter an intense mating season lasting approximately 1 month. Snakes do not eat during winter dormancy or during the spring mating season [Crews et al., 1987], suggesting that growth rates during spring are minimal. Thus, it is possible that higher rates of neurogenesis in the fall, particularly within the ventricular zone, simply reflect increased growth of all body tissues, including the brain. Indeed, although there were no differences in SVL between spring- and fall-collected males, snakes collected during the fall after the summer foraging period weighed more than snakes collected during the spring mating season. Interestingly, the rate of cell migration into the parenchymal region was also significantly higher during the fall, but this was true only within the NS and combined POA/ hypothalamus. Thus, our results suggest that seasonal variation in cell migration is not simply a generalized result of increased body growth. Rather, our data suggest that seasonal changes in cell migration may be regulated differentially in different brain regions. Regardless of the underlying mechanism, seasonal differences in cell proliferation and cell migration may be regulated by environmental cues such as the photoperiod and temperature. Previous studies in golden hamsters (Mesocricetus auratus) and Ile de France ewes (O. aries) have indicated that the photoperiod plays a role in regulating neurogenesis, with short-day photoperiods resulting in more new cells in the hypothalamus and other brain regions compared to long-day photoperiods [Huang et al., 1998; Migaud et al., 2011]. In contrast, Tenerife lizards (G. galloti) exhibit higher rates of cell proliferation during spring compared to both autumn and winter [Delgado-González et al., 2008]. Moreover, long, summerlike photoperiods increase the number of newly proliferated cells in the brain of the spring-breeding Iberian wall lizard (P. hispanica), while cold, winter-like temperatures inhibit the migration of these newly proliferated cells [Ramirez et al., 1997]. In the Psammodromus lizard Psammodromus algirus [Peñafiel et al., 2001] and the blackbellied slider turtle Chrysemys d’orbigny [Radmilovich et 192

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al., 2003], the environmental temperature also modulates cell proliferation in the brain, with low temperatures decreasing neurogenesis in adults. Taken together, these data support the hypothesis that patterns of adult neurogenesis are species-specific, with differences in seasonal variation as well as responses to environmental cues reflecting each species’ unique behavioral, social, and ecological niche [e.g. Delgado-González et al., 2008; Bonfanti and Peretto, 2011]. Alternatively, the observed seasonal variation in cell proliferation and migration could reflect seasonal changes in hormone concentrations. For example, Lutterschmidt and Mason [2005, 2009] found that plasma androgen and glucocorticoid concentrations are elevated during the fall in male red-sided garter snakes, and both steroid hormones are capable of modulating varying aspects of vertebrate neurogenesis. Rasika et al. [1994] demonstrated that androgens affect cell recruitment and survival, but not cell proliferation, in Waterslager canaries. In contrast, social modulation of cell proliferation in the brain of green treefrogs (H. cinerea) is independent of sex steroid hormone concentrations in both males and females, and sex steroids do not directly influence cell proliferation alone [Almli and Wilczynski, 2012]. Similar findings that sex steroid hormones are not the primary factor regulating cell proliferation in the brain have been reported in canaries (Serinus canaria) [Brown et al., 1993] and weakly electric fish (Brachyhypopomus gauderio) [Dunlap et al. 2011]. Thus, we speculate that seasonal changes in neurogenesis in redsided garter snakes are probably not regulated by seasonal rhythms in sex steroid hormones, although a regulatory role of glucocorticoid ‘stress hormones’ is highly likely, particularly as it relates to seasonal changes in allostatic load [e.g. Schoenfeld and Gould, 2012]. In summary, the data presented here demonstrate significant seasonal variation in cell proliferation in all brain regions, as well as seasonal differences in cell migration into the parenchyma within specific brain regions. The environmental and/or hormonal factors responsible for producing such seasonal variation, however, are currently unknown. Future laboratory studies are needed to determine if and how photoperiod, environmental temperature, and hormonal factors influence cell proliferation and cell migration in the brain of red-sided garter snakes. Regional Variation The regions where large numbers of BrdU-labeled cells were observed are areas that are particularly important in the regulation of reproduction and spatial memory in this and other species. Our observations corroborate Maine/Powers/Lutterschmidt

those of previous studies, including those in other nonmammalian vertebrates [e.g. Ramirez et al., 1997; Law et al., 2010; Delgado-González et al., 2011; Dunlap et al., 2011]. While the number of BrdU-labeled cells was significantly higher in the ventricular zone of the cortex, the SN, the NS, and the POA/hypothalamus during the fall, only the NS and the POA/hypothalamus had significantly higher numbers of labeled cells in the parenchymal region. These results indicate a higher rate of cell migration, survival, and/or subsequent division of newly formed, maturing brain cells in the NS and POA/hypothalamus during the fall. At this time, both the mechanisms and the functional significance of seasonal variation in cell proliferation and cell migration within these brain regions are unknown. We speculate that seasonal changes in neurogenesis within these regions of interest play a role in mediating seasonal rhythms in behavior. For example, as in many animals, the NS, SN, and POA/hypothalamus are important in the initiation and maintenance of sexual behavior in red-sided garter snakes. Both the NS and the POA/hypothalamus of T. sirtalis parietalis contain sex steroidconcentrating neurons [Halpern et al., 1982]. Further, lesions to both the NS and the SN prior to winter dormancy increase the courtship behavior of male red-sided garter snakes following emergence from winter dormancy, while lesions to the POA/hypothalamus lead to a delay in and a shorter duration of courtship behavior compared to controls [Krohmer and Crews, 1987a, b]. These results suggest that the NS and SN exert inhibitory control over sexual behavior, while the POA/hypothalamus positively regulates reproductive behavior. Finally, the NS and the anterior hypothalamus POA exhibit seasonal changes in aromatase activity, suggesting that the metabolism of androgens within these brain regions may regulate seasonal rhythms in reproductive physiology and behavior [Krohmer et al., 2010]. Whether (and how) changes in cell proliferation and/or cell migration within the NS, SN, and POA/hypothalamus influence seasonal reproductive behavior requires further study. Alternatively, it is possible that seasonal variation in neurogenesis is associated with emergence from (spring) or preparation for (fall) long-term winter dormancy. For example, Cerri et al. [2009] found higher rates of cell proliferation in the brains of hibernating common water frogs (Rana esculenta) and suggested that this may be a neuroprotective strategy in response to increased apoptosis during hibernation. Additional studies examining patterns of neurogenesis during winter dormancy are needed to evaluate this hypothesis.

In reptiles, the NS is a homologue of the mammalian amygdala [Ubeda-Bañon et al., 2011]. In addition to its role in regulating sexual behavior, the NS of garter snakes facilitates chemical communication between conspecifics. For instance, within T. sirtalis parietalis, the NS receives input directly from vomeronasal projections [Wang and Halpern, 1988; Lanuza and Halpern, 1998] and is therefore the major secondary vomeronasal structure [Lanuza and Halpern, 1997]. Because the courtship behavior of male red-sided garter snakes is facilitated by the detection of and response to the female sexual attractiveness pheromone [Joy and Crews, 1985; Mason et al., 1989], the vomeronasal-NS system plays a role in mediating responses to reproductive chemical cues. Seasonal variation in cell proliferation and cell migration within the NS may therefore be important for regulating seasonal changes in the sensitivity and responses to pheromone signals. Similarly, within the cortex, seasonal differences in cell proliferation may be related to seasonal changes in migratory behavior. In reptiles, the dorsal and medial cortex in particular is implicated as a structural and functional homologue of the mammalian and avian hippocampus [Rodriguez et al., 2002; Butler and Hodos, 2005]. Previous studies in cottonmouth snakes (Agkistrodon piscivorus) have shown that the volume of the medial cortex is correlated with territory size, suggesting that a large territory size requires a larger spatial memory map [Roth et al., 2006]. Using translocation studies, Holding et al. [2012] further demonstrated that the volume of the medial cortex of northern Pacific rattlesnakes (Crotalus o. oreganus) increased with experimentally increased spatial demands. In Moorish wall geckos (T. mauritanica), Pérez-Cañellas and Garcia-Verdugo [1996] reported that the highest rates of proliferation and neurogenesis occurred in the medial cortex, which is also associated with memory and spatial navigation in this species. During spring, red-sided garter snakes collected at the den site have recently emerged from winter dormancy and have not yet made the migration of up to 18 km to summer feeding grounds [Gregory and Stewart, 1975]. In contrast, snakes collected at the den site during the fall have completed their biannual migration, having returned from summer feeding grounds to the hibernaculum in preparation for winter dormancy. Thus, the higher rate of cell proliferation within the cortex during the fall may be related to differences in recent migratory history [e.g. Ladage et al., 2011]. Future studies comparing rates of cell proliferation and cell migration in both preand postmigratory snakes within each season would aid

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in understanding whether neuroplasticity within the cortex is involved in spatial memory formation. These additional studies would also aid in determining whether seasonal changes in cell proliferation are related to annual cycles of winter dormancy, reproduction, migration, or all of these behaviors.

Conclusions

We demonstrated that postembryonic cell proliferation and cell migration in the brain of male red-sided garter snakes vary both seasonally and by brain region. There are several areas of future research that are needed to better understand the functional significance of neurogenesis to seasonal physiology and behavior. For example, laboratory studies manipulating environmental cues and hormone signals are necessary to understand the mechanisms that regulate seasonal changes in adult cell proliferation. It is also unknown if the observed seasonal changes in cell proliferation are sexually dimorphic in T. sirtalis parietalis, as has been demonstrated in zebra finches (Taeniopygia guttata) [Katz et al., 2008]. Regarding the

fate of newly proliferated cells, future studies using double-labeling techniques are necessary to determine if BrdU-labeled cells survive and differentiate into neurons or glial cells. Such studies would aid in understanding whether transient amplification contributes to the relatively large number of labeled cells we observed within the parenchyma of all brain regions. Finally, longer-term studies examining the phenotype of mature, fully differentiated cells are needed to understand if and how neurogenesis alters the neural circuits that regulate seasonal rhythms. Collectively, these future research directions would aid in understanding the role of adult neurogenesis in organizing physiological and behavioral processes within a seasonally changing environment.

Acknowledgments We thank the Manitoba Department of Conservation and Dave Roberts for supporting these studies, and Leslie Dunham, Chris Friesen, Michael LeMaster, Rocky Parker, and Emily Uhrig for technical assistance. Partial funding for this work was provided by the Medical Research Foundation of Oregon and a Portland State University Faculty Enhancement Grant to D.I.L.

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