Courtship pheromone–induced c-Fos-like immunolabeling in the female salamander brain

Plethodontid salamanders display intricate courtship behaviors. Proteinaceous courtship pheromones were recently discovered in the submandibular (mental) gland of the male Plethodon shermani, the red-legged salamander. Behavioral studies showed that these male pheromones are delivered by direct contact to the female snout and modulate her receptivity during courtship. Previous reports demonstrated that experimental application of courtship pheromones activates vomeronasal sensory neurons in P. shermani. The present study investigated the CNS response to courtship pheromones in that species using immunocytochemical detection of the immediate-early gene product c-Fos. The results show that application of a male gland extract to females activated Fos-like immunolabeling in the extended vomeronasal amygdala of the accessory olfactory system, as well as in the preoptic area and ventromedial hypothalamus; regions of the brain known to mediate reproductive responses in vertebrates. The gland extract additionally activated Fos-like labeling in the raphe median, possibly indicating a serotonergic activation. Application of individual purified courtship pheromone proteins resulted in increases in Fos-like labeling in some of the regions activated by the complete submandibular gland extract, but the pattern of labeling was not as clear as that of the complete extract. Unlike other known vertebrate reproductive pheromones, courtship pheromones in P. shermani were effective only at a high concentration. This could result from the particular mode of pheromone transfer in that species, which involves sustained direct contact between male and female. It is concluded that salamander courtship pheromones exert their influence on behavior through the vomeronasal pathway and its direct projections to the preoptic and hypothalamic regions.