Seasonal and Sex Differences in the Effects of Melatonin on Brain Arginine Vasotocin in Green Treefrogs (Hyla cinerea): Relationship to Melatonin Receptor 1a

Abstract

Critical life history events such as breeding, migration and hibernation must take place in the correct environmental context to minimize deleterious consequences on survival and reproductive fitness. Neuroendocrine mechanisms synchronizing internal physiological states with extrinsic environmental cues are vital to timing life history events appropriately. Secretion of the pineal hormone melatonin is sensitive to light and temperature cues, which provides a physiological indicator of time of day and time of year for organisms. Melatonin influences seasonal reproduction in a variety of vertebrates, likely by altering the synthesis and/or release of reproductive neuropeptides in the brain. The neuropeptides arginine vasotocin and its mammalian homologue, arginine vasopressin, are well-known modulators of reproductive and sociosexual behavior across vertebrate taxa, and are likely targets of melatonin in the context of seasonal reproduction. There is extensive evidence that vasotocin/vasopressin innervation in the brain is subject to seasonal variation, and that this variation is frequently sexually dimorphic. However, evidence that melatonin directly modulates this important neuropeptide system is lacking. Melatonin receptor 1a (MT1 in mammals) may be responsible for mediating melatonin's influence on brain vasotocin, as it is known to regulate seasonal reproduction in a variety of vertebrates. In the present study, I asked whether melatonin influences brain vasotocin in male green treefrogs (Hyla cinerea), and compared the distribution of melatonin receptor 1a in the brain of green treefrogs between sexes and seasons. Adult male and female green treefrogs were collected from field sites in Louisiana during the summer breeding season. Summer animals were acclimated to lab conditions for 3 weeks, then euthanized and their brains collected. Winter animals were maintained in the lab for four months under incrementally changing photo-, thermo-, and hygroperiod regimes that mimicked the transition to winter in their natural habitat, followed by euthanasia and brain collection. A subset of winter males (Experiment 1) were implanted with melatonin-filled or blank silastic capsules for a period of one month prior to euthanasia and brain collection. Brains of these males were processed for vasotocin immunohistochemistry. I quantified AVT-ir cell number in Experiment 1 males in the nucleus accumbens (NAcc), amygdala and caudal striatum (AMG), preoptic area (POA), suprachaismatic nucleus (SCN), and ventral hypothalamus (VH). Melatonin did not influence brain vasotocin-ir cell number in any brain region. Brains from untreated summer and winter males and females were collected and processed for MT1 immunohistochemistry. MT1-ir cells were quantified in the NAcc, striatum (STR), AMG, POA, SCN,