Neural thyroid hormone metabolism integrates seasonal changes in environmental temperature with the neuroendocrine reproductive axis.

We asked if environmental temperature alters thyroid hormone metabolism within the hypothalamus, thereby providing a neuroendocrine mechanism by which temperature could be integrated with photoperiod to regulate seasonal rhythms. We used immunohistochemistry to assess the effects of low-temperature winter dormancy at 4 °C or 12 °C on thyroid-stimulating hormone (TSH) within the infundibulum of the pituitary as well as deiodinase 2 (Dio2) and 3 (Dio3) within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis). Both the duration and, in males, magnitude of low-temperature dormancy altered deiodinase immunoreactivity within the hypothalamus, increasing the area of Dio2-immunoreactivity in males and females and decreasing the number of Dio3-immunoreactive cells in males after 8-16 weeks. Reciprocal changes in Dio2/3 favor the accumulation of triiodothyronine within the hypothalamus. Whether TSH mediates these effects requires further study, as significant changes in TSH-immunoreactive cell number were not observed. Temporal changes in deiodinase immunoreactivity coincided with an increase in the proportion of males exhibiting courtship behavior as well as changes in the temporal pattern of courtship behavior after emergence. Our findings mirror those of previous studies, in which males require low-temperature exposure for at least 8 weeks before significant changes in gonadotropin-releasing hormone immunoreactivity and sex steroid hormones are observed. Collectively, these data provide evidence that the neuroendocrine pathway regulating the reproductive axis via thyroid hormone metabolism is capable of transducing temperature information. Because all vertebrates can potentially use temperature as a supplementary cue, these results are broadly applicable to understanding how environment-organism interactions mediate seasonally adaptive responses.

Role of Melatonin in Temperature-Induced Activation of the Neuroendocrine Reproductive Axis in Garter Snakes.

An animal's ability to respond optimally to changing environmental conditions is paramount to successfully reproducing and thus maximizing fitness. Studies on photoperiod-induced changes in neural thyroid hormone metabolism have conclusively linked environmental cues to the neuroendocrine reproductive axis of birds and mammals. Whether this conserved mechanism also transduces changes in environmental temperature, however, has not been fully addressed. We investigated whether the hormone melatonin mediates the effects of low-temperature dormancy on thyroid hormone metabolism within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis parietalis). To address this question, we used immunohistochemistry to assess changes in thyroid-stimulating hormone (TSH) in the infundibulum of the pituitary and deiodinase 3 (Dio3) and gonadotropin-releasing hormone (GnRH) in the hypothalamus. We also asked if changes in TSH, Dio3, and/or GnRH immunoreactivity are associated with changes in male courtship behavior. In contrast to our predictions, 6 weeks of dormancy at 4°C significantly decreased the number of TSH-labeled cells in the infundibulum. It is possible that the observed decrease in TSH is related to the release of snakes from temperature refractoriness, but this idea needs further testing. Treatment of snakes with the melatonin precursor 5-hydroxytryptophan during dormancy at 4°C both reversed the temperature-induced change in TSH immunoreactivity and disrupted the temporal pattern of male courtship behavior. These results suggest that TSH cells within the infundibulum are both modulated by temperature and sensitive to changes in melatonin. As predicted, male snakes hibernated at an elevated temperature of 12°C for 6 weeks and treated with vehicle showed no change in TSH-, Dio3-, or GnRH-immunoreactive cell number. Treatment of snakes with the melatonin receptor antagonist luzindole was not sufficient in rescuing the effects of dormancy at 12°C on TSH immunoreactivity or courtship behavior. However, luzindole-treated snakes showed a significant increase in GnRH-immunoreactive cell number, suggesting that melatonin exerts an inhibitory effect on GnRH in garter snakes. In summary, our results provide critical insights into the mechanisms that mediate the effects of temperature on reproductive physiology and behavior.

Exogenous leptin promotes reproductive behavior during aphagia in red-sided garter snakes (Thamnophis sirtalis parietalis).

Despite the established dichotomy between investment in either reproduction or self-maintenance, a hormonal mechanism that influences an organism's decision to prioritize these behaviors remains elusive. The protein hormone leptin is a likely candidate because it is secreted from adipocytes in proportion to the amount of stored fat in numerous species. Although the majority of studies suggest that leptin stimulates reproduction, the actions of leptin can be context-dependent. Leptin increases sexual behavior in fed individuals, but inhibits sexual behavior in food-restricted individuals. We investigated if exogenous leptin influences sexual behavior in red-sided garter snakes (Thamnophis sirtalis parietalis) experiencing a predictable bout of aphagia during the mating season. We tested two doses of recombinant murine leptin injected for three days. Males were subjected to three mating trials, one on each day of injections, while females were subjected to one mating trial on the last day of injections. Leptin affects male and female snakes similarly by increasing both appetitive (i.e., mating behavior score) and consummatory (i.e., number of copulations, proportion of individuals copulated) sex behavior. We found no evidence to suggest that leptin influenced latency to copulate or duration of copulation. Because leptin promotes reproductive behavior in non-feeding garter snakes, these findings do not align with research on food-restricted mammals. Further investigations into how leptin affects sexual behavior in snakes exposed to food-restriction manipulations would clarify if the role of leptin is evolutionarily divergent.

Physiological correlates of reproductive decisions: Relationships among body condition, reproductive status, and the hypothalamus-pituitary-adrenal axis in a reptile.

When opportunities to feed and reproduce are limited, females are often unable to recover sufficient energy stores to reproduce in consecutive years. Body condition has been used as a proxy for recent reproductive history in such species. We previously found that glucocorticoid responses to capture stress vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis), a species with limited seasonal breeding opportunities. Because variation in glucocorticoid receptor (GR) protein in the brain could explain these differences, we first assessed GR protein content in females in different body conditions. To investigate if body condition during the spring mating season accurately reflects recent reproductive history, we measured glucocorticoid responses to stress in females with different body conditions, assessed their mating behavior and brought mated females to our lab to determine which females would give birth during the summer (i.e., were parturient). Female red-sided garter snakes reproduce biennially, and therefore mated females that did not give birth were deemed non-parturient. In this study, glucocorticoid stress responses and mating behavior did not vary with body condition, nor was body condition related to brain GR or reproductive condition (parturient vs non-parturient). Only unreceptive females showed a significant stress-induced increase in glucocorticoids, suggesting that reduced stress responsiveness is associated with receptivity. Parturient females mated faster (were more proceptive) than non-parturient females. These data suggest that HPA axis activity modulates receptivity, while proceptivity is related primarily to reproductive condition.

Patterns of stress responses shift during seasonal life-history transitions: An analysis comparing baseline, maximal and integrated corticosterone in female red-sided garter snakes (Thamnophis sirtalis parietalis).

Glucocorticoids often rise and fall with a variety of external and internal cues and frequently vary among life-history stages. This suggests that changing glucocorticoids may coordinate life-history transitions. To explore this hypothesis, we asked if the time-course of stress-induced glucocorticoid levels differ between two life-history transitions (i.e., spring and fall migration) in female red-sided garter snakes (Thamnophis sirtalis parietalis). We collected non-migratory females from a communal den and migratory females from a road along the migration route and treated them with 4h of capture stress; plasma corticosterone was measured before, during and after capture stress. During the spring, den-collected females exhibited a stress-induced peak in corticosterone at an earlier sampling time than migrating, road-collected females. Because the pattern of corticosterone responses varied with migratory state, negative feedback on and/or sensitivity of the hypothalamus-pituitaryadrenal (HPA) axis may be linked to spring migration. During the fall, capture stress elicited an increase in corticosterone in den-collected females but not in migrating, road-collected females. Baseline corticosterone was higher and both maximal and integrated corticosterone responses were lower during the fall compared to spring, indicating that stress responses are smaller when baseline corticosterone is elevated, perhaps due to a "ceiling effect". These data suggest that HPA axis regulation changes during seasonal migration, possibly via altering negative feedback, HPA axis sensitivity, or some other mechanism. This study supports the hypothesis that glucocorticoids coordinate life-history events and suggests that examining a suite of stress response characteristics is most informative for understanding the function of HPA modulation.

Seasonal and sex differences in responsiveness to adrenocorticotropic hormone contribute to stress response plasticity in red-sided garter snakes (Thamnophis sirtalis parietalis).

As in many vertebrates, hormonal responses to stress vary seasonally in red-sided garter snakes (Thamnophis sirtalis parietalis). For example, males generally exhibit reduced glucocorticoid responses to a standard stressor during the spring mating season. We asked whether variation in adrenal sensitivity to adrenocorticotropic hormone (ACTH) explains why glucocorticoid responses to capture stress vary with sex, season and body condition in red-sided garter snakes. We measured glucocorticoids at 0, 1 and 4 h after injection with ACTH (0.1 IU g(-1)body mass) or vehicle in males and females during the spring mating season and autumn pre-hibernation period. Because elevated glucocorticoids can influence sex steroids, we also examined androgen and estradiol responses to ACTH. ACTH treatment increased glucocorticoids in both sexes and seasons. Spring-collected males had a smaller integrated glucocorticoid response to ACTH than autumn-collected males. The integrated glucocorticoid response to ACTH differed with sex during the spring, with males having a smaller glucocorticoid response than females. Although integrated glucocorticoid responses to ACTH did not vary with body condition, we observed an interaction among season, sex and body condition. In males, ACTH treatment did not alter androgen levels in either season, but androgen levels decreased during the sampling period. Similar to previous studies, plasma estradiol was low or undetectable during the spring and autumn, and therefore any effect of ACTH treatment on estradiol could not be determined. These data provide support for a mechanism that partly explains how the hypothalamus-pituitary-adrenal (HPA) axis integrates information about season, sex and body condition: namely, variation in adrenal responsiveness to ACTH.

Sex or candy? Neuroendocrine regulation of the seasonal transition from courtship to feeding behavior in male red-sided garter snakes (Thamnophis sirtalis parietalis).

This article is part of a Special Issue "Energy Balance". Seasonal modulation of glucocorticoids plays an important role in supporting critical life-history events, and probably facilitates transitions between different life-history stages. In a well-studied population of red-sided garter snakes (Thamnophis sirtalis parietalis), glucocorticoids are elevated during the mating season, but males dispersing to summer feeding grounds have significantly lower baseline glucocorticoids than courting males at the den. We tested the hypothesis that decreased plasma glucocorticoids mediate the behavioral switch between reproduction and foraging in this species. Using a two-choice Y-maze paradigm, we demonstrate that males treated with the glucocorticoid synthesis inhibitor metyrapone (1 and 3mg implants) prefer feeding cues (worm trail) over reproductive cues (female pheromone trail) significantly earlier than control-treated snakes. The metyrapone-induced changes in appetitive feeding behavior were independent of changes in plasma androgens and body mass loss. Metyrapone-treated males continued to court females at levels similar to those of control-treated snakes, suggesting that appetitive reproductive and ingestive behaviors are not mutually exclusive during this life-history transition. Consistent with this hypothesis, metyrapone treatment did not alter the number of arginine vasotocin-immunoreactive cells in any brain region, while it significantly increased neuropeptide Y-immunoreactive cell number in both the cortex and nucleus sphericus (homologues of the mammalian hippocampus and amygdala, respectively). Our results suggest that male red-sided garter snakes have the potential to maximize reproductive opportunities by continuing to court females they encounter even as they disperse from the den in search of food. Taken together, these data have important implications for understanding the neuroecology of seasonal life-history transitions.

Seasonal variation in cell proliferation and cell migration in the brain of adult red-sided garter snakes (Thamnophis sirtalis parietalis).

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 free-ranging 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 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.

Responses to capture stress and exogenous corticosterone vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis).

This study examined whether hormonal and behavioral responses to capture stress and exogenous corticosterone (CORT) vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis). Female snakes were collected during the spring mating season and treated with 4 h of capture stress. We measured plasma CORT and estradiol before, during and after capture stress treatment followed by latency to copulate, a measure of female receptivity. Body condition was determined as the residual from a regression of body mass on snout-vent-length. Baseline CORT did not differ between females in positive and negative body condition, but females in negative body condition showed a significantly larger increase in plasma CORT in response to capture stress. Estradiol, which is generally low during the mating season in this population, did not change in response to capture stress. Body condition, but not capture stress, influenced latency to copulate, suggesting that females are resistant to the behavioral effects of capture stress during the spring mating season. In a second experiment, only females in negative body condition increased latency to copulate in response to injection of a physiological (15 µg) dose of exogenous CORT, while all females responded to a pharmacological (60 µg) dose. These results indicate that behavioral responses to exogenous CORT vary with female body condition during the short mating season. Taken together, our data suggest that variation in body condition may be associated with differences in HPA axis sensitivity and/or glucocorticoid receptor density in the brain.

Pheromonal mediation of intraseasonal declines in the attractivity of female red-sided garter snakes, Thamnophis sirtalis parietalis.

During the breeding season, female red-sided garter snakes (Thamnophis sirtalis parietalis) produce and express a sexual attractiveness pheromone that elicits male courtship behavior. Composed of a homologous series of saturated and monounsaturated methyl ketones, this pheromone is expressed in female skin lipids. Recent studies have shown that the sexual attractivity of unmated female garter snakes declines as the breeding season progresses. Here, we investigated whether temporal changes in the quantity and/or quality of the female sexual attractiveness pheromone are responsible for the observed loss of attractivity. Female red-sided garter snakes were collected immediately following spring emergence and held under natural conditions for the duration of the breeding season. Behavioral experiments confirmed that unmated females become significantly less attractive to males within two weeks of emergence from hibernation. Additionally, these females had lower estradiol concentrations at two weeks post-emergence. Subsequent chemical analyses revealed qualitative variation between the pheromone profiles of newly emerged females and those of females at two weeks post-emergence. Together, these results support the hypothesis that changes in the female sexual attractiveness pheromone are responsible for declining post-emergence female attractivity in garter snakes.

Sexually dimorphic effects of melatonin on brain arginine vasotocin immunoreactivity in green treefrogs (Hyla cinerea).

Arginine vasotocin (AVT) and its mammalian homologue, arginine vasopressin (AVP), regulate a variety of social and reproductive behaviors, often with complex species-, sex- and context-dependent effects. Despite extensive evidence documenting seasonal variation in brain AVT/AVP, relatively few studies have investigated the environmental and/or hormonal factors mediating these seasonal changes. In the present study, we investigated whether the pineal hormone melatonin alters brain AVT immunoreactivity in green treefrogs (Hyla cinerea). Reproductively active male and female frogs were collected during the summer breeding season and a melatonin-filled or blank silastic capsule was surgically implanted subcutaneously. The duration of hormone treatment was 4 weeks, at which time frogs were eutha-nized and the brains and blood collected and processed for AVT immunohistochemistry and steroid hormone assay. We quantified AVT-immunoreactive (AVT-ir) cell bodies in the nucleus accumbens (NAcc), caudal striatum and amygda- la (AMG), anterior preoptic area, suprachiasmatic nucleus (SCN) and infundibular region of the ventral hypothalamus. Sex differences in AVT-ir cell number were observed in all brain regions except in the anterior preoptic area and ventral hypothalamus, with males having more AVT-ir cells than females in the NAcc, amygdala and SCN. Brain AVT was sensitive to melatonin signaling during the breeding season, and the effects of melatonin varied significantly with both region and sex. Treatment with melatonin decreased AVT immunoreactivity in both the NAcc and SCN in male H. cinerea. In contrast, brain AVT was relatively insensitive to melatonin signaling in females, indicating that the regulation of the AVT/AVP neuropeptide system by melatonin may be sexually dimorphic. Finally, melatonin did not significantly influence testosterone or estradiol concentrations of male or female frogs, respectively, suggesting that the effects of melatonin on AVT immunoreactivity are independent of changes in gonadal sex steroid hormones. Collectively, our results indicate that the AVT/AVP neuronal system may be an important target for melatonin in facilitating seasonal changes in reproductive physiology and social behavior.

Chronobiology of reproduction in garter snakes: neuroendocrine mechanisms and geographic variation.

The majority of studies on reproductive neuroendocrinology in snakes have focused on one particular snake population in Manitoba, Canada, the red-sided garter snake (Thamnophis sirtalis parietalis). Although traditionally these studies have emphasized its unusual temporal dissociation between mating behavior and peak gonadal activity, current evidence suggests that reproductive regulation in this population may be more similar to the norm than previously thought. Like other ectotherms, temperature plays a critical role in activating reproductive behavior in red-sided garter snakes. Diel melatonin and corticosterone rhythms appear to be important in transducing temperature cues, and it is clear that both hormones regulate courtship behavior during spring. Current evidence also suggests that sex steroid hormones are in fact central to reproductive regulation in males, although the timing of their action occurs during winter dormancy. Whether this is also true for female T. sirtalis parietalis requires further study, but it should be noted that patterns of sex steroid hormones are sexually dimorphic during winter dormancy, as are melatonin rhythms during spring emergence. While continuing to advance our understanding of reproductive regulation in this extremely well-studied population is prudent, future comparative studies are critical for understanding if and how reproductive regulatory mechanisms differ across environments, populations, and phylogenies. For example, melatonin and corticosterone responses to environmental cues vary significantly among populations of T. sirtalis in a common garden, as do male courtship behavior and androgen concentrations. These data support the hypothesis that neuroendocrine-mediated responses to environmental cues underlie phenotypic plasticity in reproductive life history traits.

Trans-seasonal activation of the neuroendocrine reproductive axis: Low-temperature winter dormancy modulates gonadotropin-releasing hormone neurons in garter snakes.

All animals use external cues from the environment to accurately time life-history events. How the brain decodes environmental stimuli to effect changes in physiology and behavior, however, is poorly understood, particularly with regard to supplementary environmental cues such as temperature. We asked if low-temperature dormancy alters the synthesis and/or release of gonadotropin-releasing hormone (GnRH). We used the well-studied red-sided garter snake (Thamnophis sirtalis) for this study, as low-temperature exposure is both necessary and sufficient to induce reproduction in northern populations of this species. Snakes were collected from the field and hibernated at 4°C or 10°C in complete darkness for up to 16 weeks. In males, increasing duration of low-temperature dormancy significantly increased GnRH-immunoreactive cell number and GnRH soma size (a proxy for relative cell activity) in the forebrain. These changes mirrored those in male reproductive behavior (reported previously) and plasma androgen concentrations. The changes in GnRH cell area observed in males were specific to the neuroendocrine population of cells in the medial preoptic area; soma size in the rostral GnRH cells did not change. Finally, temperature-induced changes in GnRH were sexually dimorphic: neither hibernation temperature nor the duration of winter dormancy significantly modulated GnRH cell number or soma size in females, despite the fact that plasma estradiol and corticosterone increased significantly in response to both. These data demonstrate that the neuroendocrine GnRH system is sensitive to environmental temperature and suggest that GnRH neurons play a conserved but trans-seasonal role in mediating changes in reproductive physiology and behavior in dissociated breeders.

Seasonal aromatase activity in the brain of the male red-sided garter snake.

We investigated regional and seasonal variations in neural aromatase activity (AA), the enzyme that converts androgens into estrogens, to examine a possible indirect role of testosterone (T) in mediating spring reproductive behavior of red-sided garter snakes, a species exhibiting a dissociated reproductive pattern. Neural AA in male snakes varied significantly among brain regions. Additionally, there were significant interactions between brain region and season. In the spring, actively courting males had greater AA in the olfactory region (O) compared to the septum/anterior-hypothalamus preoptic area (S/AHPOA), nucleus sphericus (NS) and midbrain (Mb). Fall animals collected as they returned to the den prior to winter dormancy had significantly greater AA in the S/AHPOA compared to all other regions. These findings were consistent using either regional (gross) dissection or punch microdissection, which allowed us to separate the S and AHPOA. There were no significant differences in AA production between the S and AHPOA. This study provides the first documentation of seasonal and regional variations in AA in a snake brain and suggests that aromatization of androgens may play a role in regulating reproduction in red-sided garter snakes. During spring mating, elevated AA in the O may activate pathways essential for detection of courtship pheromones, while increased AA in the S and AHPOA of fall animals suggests that circulating androgens play an indirect role in programming critical neural pathways involved in reproduction. Thus, as in many other vertebrates, estrogenic metabolites of testosterone may be a critical hormonal component regulating reproductive behavior in this dissociated breeder.

Temporally distinct effects of stress and corticosterone on diel melatonin rhythms of red-sided garter snakes (Thamnophis sirtalis).

Circadian and circannual rhythms in physiology and behavior are temporally organized via hormonal signals that reflect changing environmental cues. Interactions between endocrine signals are in turn important for integrating multiple physiological and behavioral rhythms. In the present study, we examined interactions between melatonin, the hypothalamus-pituitary-adrenal (HPA) axis, and corticosterone in a well-studied population of red-sided garter snakes (Thamnophis sirtalis parietalis). We demonstrate that 4h of capture stress significantly increased photophasic melatonin and decreased scotophasic melatonin concentrations of male snakes. Treatment with exogenous corticosterone (15 and 60 µg) did not mimic the effects of stress on diel melatonin rhythms. To determine if capture stress decreases scotophasic melatonin by depleting the precursors necessary for melatonin synthesis, we used a paradigm in which snakes were treated with the melatonin precursor 5-hydroxytryptophan (0.6 and 1.2mg) to elevate melatonin concentrations. Pretreatment of snakes with both capture stress and exogenous corticosterone blocked the effect of 5-hydroxytryptophan on scotophasic melatonin. Thus, although corticosterone itself does not influence melatonin rhythms of snakes, corticosterone can inhibit the synthesis of melatonin from 5-hydroxytryptophan. These experiments suggest that the initial versus later phases of an acute physiological stress response have temporally distinct effects on melatonin synthesis: activation of the sympathoadrenal system increases melatonin, while increased glucocorticoids can inhibit melatonin synthesis. Collectively, we demonstrate that a physiological coupling between melatonin, glucocorticoids, and the sympathoadrenal system is conserved in this ectothermic model and propose that such interactions may mediate stress-induced changes in physiology and behavior.

Energy Metrics of Red-Sided Garter Snakes (Thamnophis sirtalis parietalis) Vary with Sex but Not Life-History Stage.

Because reproduction is energetically expensive, an organism's energy stores are likely involved in mediating transitions between reproductive and self-maintenance activities. We investigated whether body condition index, adipocyte follicle size, and liver glycogen differ with the life-history transition from reproduction to migration and foraging in red-sided garter snakes (Thamnophis sirtalis parietalis). Females primarily investing in mating behavior located at the den had a significantly higher body condition index than females migrating to summer feeding grounds. The body condition index of male snakes did not differ between snakes located at the den and those migrating to summer feeding grounds. Neither adipocyte follicle area nor liver glycogen stores differed significantly between snakes performing mating activities at the den and those migrating to summer feeding grounds. We did find a sexual dimorphism in that female red-sided garter snakes had significantly larger adipocyte follicles and higher liver glycogen compared with males. Our findings support the across-species phenomenon of females and males displaying a sexual dimorphism in stored energy substrates. Conversely, we did not find evidence to suggest that red-sided garter snakes primarily utilize fatty acids to fuel the initiation of migration, a finding that is not consistent with other long-distance migrators, such as birds. Because we did not find evidence to suggest that stored energy metrics influence the decision to migrate, a physiological mechanism that induces migration in red-sided garter snakes remains elusive.

Endocrine mechanisms mediating temperature-induced reproductive behavior in red-sided garter snakes (Thamnophis sirtalis parietalis).

We investigated the mechanisms by which temperature induces seasonal reproductive behavior in red-sided garter snakes (Thamnophis sirtalis parietalis). Specifically, we addressed whether elevated temperatures during winter dormancy influence (1) diel melatonin and corticosterone rhythms; (2) sex steroid hormone and corticosterone profiles; and (3) the expression of reproductive behavior following emergence. Elevated hibernation temperatures (i.e. 10 degrees C versus 5 degrees C) significantly increased overall melatonin and decreased corticosterone concentrations of snakes. The temperature-induced differences in melatonin rhythms between the 5 degrees C and 10 degrees C treatment groups persisted even after both groups were again acclimated to 10 degrees C, indicating that cold temperature exposure has a lasting influence on melatonin rhythms. Elevated hibernation temperatures also significantly altered androgen and corticosterone profiles of snakes, providing a potential mechanism to explain reported annual variation in steroid hormones. Although previous studies indicate that male red-sided garter snakes exhibit a dissociated reproductive strategy, we demonstrate the presence of intersexual variation in sex steroid hormone profiles, as estradiol concentrations of female snakes increased significantly prior to spring mating activity. Importantly, the percentage change in body mass did not differ significantly between snakes in the hibernation treatments, indicating that the observed changes in hormone profiles are indeed temperature induced and not simply an indirect result of significant changes in the energy balance of snakes. Finally, in males maintained at 10 degrees C during winter dormancy the onset of courtship behavior following emergence was delayed. Our results suggest that environmental temperatures induce reproductive behavior, in part, via changes in diel melatonin and/or corticosterone rhythms in this seasonally breeding reptile.

Kisspeptin-like immunoreactive neuron distribution in the green anole (Anolis carolinensis).

Kisspeptins are a recently identified class of neuropeptides belonging to the RFamide peptide family. Despite growing evidence supporting kisspeptin as a key regulator of reproduction, data addressing whether kisspeptin is a conserved reproductive signal are lacking. We investigated the distribution of kisspeptin in adult green anole lizards (Anolis carolinensis) via immunohistochemistry. Additionally, we examined the possibility of a sexual dimorphism in kisspeptin expression. Kisspeptin immunoreactivity was observed rostrally in the preoptic area and caudally in an area lateral to the dorsal hypothalamic nucleus in both male and female anoles. These kisspeptin immunoreactive cells are associated with vesiculated fibers traveling through the paraventricular zone of the hypothalamus and preoptic area and extending into the rostral telencephalon. Preabsorption of the antiserum with gonadotropin inhibitory hormone (GnIH), a second RFamide peptide known to cross-react with the kisspeptin antiserum, eliminated staining of the caudal population of cells but retained staining in the rostral population, suggesting that kisspeptin is present in this area. Preabsorption with kisspeptin eliminated all immunoreactivity. These preabsorption results suggest that kisspeptin is restricted to a single population in the preoptic area in anoles. No sex differences were found in kisspeptin immunoreactive cell number. The presence of kisspeptin RFamide peptide in the green anole suggests that this reproductive regulatory signal is indeed evolutionarily conserved. Whether this reproductive signal functions similarly in regulating the reproductive axis of ectotherms requires further study.

Sexually Dimorphic Patterns of Cell Proliferation in the Brain Are Linked to Seasonal Life-History Transitions in Red-Sided Garter Snakes.

Seasonal rhythms in physiology and behavior are widespread across diverse taxonomic groups and may be mediated by seasonal changes in neurogenesis, including cell proliferation, migration, and differentiation. We examined if cell proliferation in the brain is associated with the seasonal life-history transition from spring breeding to migration and summer foraging in a free-ranging population of red-sided garter snakes (Thamnophis sirtalis) in Manitoba, Canada. We used the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to label newly proliferated cells within the brain of adult snakes collected from the den during the mating season or from a road located along their migratory route. To assess rates of cell migration, we further categorized BrdU-labeled cells according to their location within the ventricular zone or parenchymal region of the nucleus sphericus (homolog of the amygdala), preoptic area/hypothalamus, septal nucleus, and cortex (homolog of the hippocampus). We found that cell proliferation and cell migration varied significantly with sex, the migratory status of snakes, and reproductive behavior in males. In most regions of interest, patterns of cell proliferation were sexually dimorphic, with males having significantly more BrdU-labeled cells than females prior to migration. However, during the initial stages of migration, females exhibited a significant increase in cell proliferation within the nucleus sphericus, hypothalamus, and septal nucleus, but not in any subregion of the cortex. In contrast, migrating males exhibited a significant increase in cell proliferation within the medial cortex but no other brain region. Because it is unlikely that the medial cortex plays a sexually dimorphic role in spatial memory during spring migration, we speculate that cell proliferation within the male medial cortex is associated with regulation of the hypothalamus-pituitary-adrenal axis. Finally, the only brain region where cell migration into the parenchymal region varied significantly with sex or migratory status was the hypothalamus. These results suggest that the migration of newly proliferated cells and/or the continued division of undifferentiated cells are activated earlier or to a greater extent in the hypothalamus. Our data suggest that sexually dimorphic changes in cell proliferation and cell migration in the adult brain may mediate sex differences in the timing of seasonal life-history transitions.

Arginine Vasotocin and Neuropeptide Y Vary with Seasonal Life-History Transitions in Garter Snakes.

Transitions between life-history stages are often accompanied by dramatic behavioral switches that result from a shift in motivation to pursue one resource over another. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they modulate reproductive and feeding behavior, respectively. We asked if seasonal changes in AVT and NPY are concomitant with the seasonal migration to and from the feeding grounds in red-sided garter snakes (Thamnophis sirtalis parietalis). Male and female snakes were collected in different migratory states during both the spring and fall. The total number of AVT- and NPY-immunoreactive (ir) cells was then quantified in each brain region of interest. To correct for potential variation in region volume related to sexually dimorphic body size in this species, we first determined that snout-vent length is an accurate proxy for regional brain volume. We then corrected each individual's ir cell number by its SVL to directly compare seasonal changes in AVT and NPY between males and females. Within the supraoptic nucleus, both males and females had more AVT-ir cells during the fall compared with spring. As predicted, males had significantly more AVT-ir cells during the spring mating season in the hypothalamus (HYP) and bed nucleus of the stria terminalis, brain regions important in regulating reproductive behavior. Females also had significantly more AVT-ir cells in the HYP during the spring, as well as a significantly higher number of hypothalamic AVT cells than males. During the fall, males had significantly more NPY-ir cells in the cortex and posterior HYP compared with spring, possibly reflecting increased feeding behavior during summer foraging. Females did not exhibit significant main effects of season on NPY-ir cell number in any region. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, but we did observe significant changes related to seasonal transitions in reproductive state. Our results indicate that changes in brain AVT and NPY are associated with seasonal transitions in reproductive and foraging behaviors, and may be involved in mediating sex differences in the timing of life-history events.