Graduation date: 2006 / Most vertebrates exhibit seasonality in many life history traits. Such seasonal rhythms are temporally organized via the transduction of environmental cues (e.g., photoperiod, temperature) into appropriate endocrine signals. However, among ectothermic vertebrates that undergo continuous winter dormancy, temperature is the only environmental cue available for synchronizing seasonal rhythms. Most intriguing is that in species where reproduction occurs immediately following spring emergence, the associated changes in neurophysiology and behavior that accompany reproduction likely occur during winter dormancy. The purpose of this dissertation research was to explore the mechanisms by which temperature cues affect the chronobiology and seasonal reproduction of red-sided garter snakes (Thamnophis sirtalis parietalis). Because of their roles in circadian organization and energy balance, melatonin and corticosterone are likely hormonal components of these time-keeping systems. I first characterized the interactions between melatonin and corticosterone to better understand the hormonal mechanisms facilitating temperature-induced reproduction. Melatonin and corticosterone additively inhibit reproductive behavior during the spring mating season. Experimental manipulations with a serotonin receptor antagonist suggest the mechanism underlying these effects involves a serotonin-regulated system. Although melatonin does not influence corticosterone responses to capture stress, capture stress significantly increases melatonin concentrations. To investigate the functional significance of these interactions in regulating temperature-induced reproduction, I measured body temperatures of snakes as well as circadian melatonin and corticosterone cycles during winter dormancy and spring emergence using a combination of field and laboratory experiments. Surprisingly, an increase in body temperature is not necessary for emergence from winter dormancy. Rather, critically low temperatures may serve as a zeitgeber entraining an endogenous circannual cycle that regulates emergence. Decreased environmental temperatures, in the absence of changing photoperiod cues, modulate circadian melatonin and corticosterone rhythms during hibernation. Such temperature-induced changes in hormone rhythms may facilitate seasonal reproductive behavior following spring emergence. Furthermore, a phase-shift in corticosterone rhythms during the mating season may regulate the seasonal transition between reproductive and non-reproductive states in red-sided garter snakes. Such studies investigating the environmental and hormonal mechanisms underlying time-keeping systems may provide valuable insight into the potential impact of environmental perturbations (e.g., climate change) on seasonal rhythms in physiology and behavior.

Environmental cues such as day length and temperature contribute to timing of biological rhythms in seasonal breeders. Life-history transitions such as spring emergence from hibernation, migration, or mating must be coordinated with environmental conditions or survival is compromised. Therefore, there must be chemical signaling pathways in the brain that transduce seasonally-changing sensory inputs into signals that initiate a hormonal cascade, culminating in reproductive behavior. The relative importance of environmental cues to reproductive timing varies with species, time of year, and sex, and the mechanisms driving these differences remain unknown. The role of photoperiod in regulating reproductive behavior has been explored in birds and mammals, but much less is known about the role of so-called supplementary cues such as temperature, which is crucial in the timing of ectotherm reproduction. This is a critical gap in our knowledge, because shifts in seasonal temperatures due to climate change could create a mismatch between peak reproductive behavior and resources necessary for gestation and offspring survival. Deiodinase 2 (DIO2) enzyme is a critical component of the pathway that mediates reproduction in photoperiod-activated seasonal breeders, but whether deiodinase 2 is sensitive to seasonal changes in environmental temperature is unknown. In this study, we used an ectothermic vertebrate known to be a temperature-activated seasonal breeder, the red-sided garter snake (Thamnophis sirtalis parietalis), to investigate changes in hypothalamic DIO2 in response to hibernation at 4°C and 12°C. We captured male and female snakes in Manitoba, Canada as they returned to their winter den site from summer feeding grounds. Snakes were hibernated in complete darkness at either 4°C or 12°C for up to 16 weeks. A subset of each sex and temperature group were euthanized at intervals, and the brains collected and processed for DIO2 immunohistochemistry. DIO2-specific staining was found in the anterior hypothalamus, in the periventricular hypothalamic nucleus and ventral pre-optic area, along the longitudinally central region of the olfactory tract, in the bed nucleus of the stria terminalis, caudally in the cortex and optic tectum, and in the lateral septal nucleus. DIO2-stained area in the anterior hypothalamus was quantified. Male T. sirtalis in both the 4°C and 12°C groups were found to have an increase in DIO2-specific staining in the anterior hypothalamus after 8 weeks in hibernation. Female T. sirtalis were found to have an increase in DIO2-specific staining in the anterior hypothalamus after 8 weeks in the 12°C group only. These findings shed light on the neuroendocrine pathway through which environmental cues other than photoperiod influence the timing of seasonal reproduction, and support the hypothesis that at least some components of this pathway are conserved across seasonal breeders.