With the past few decades bringing exponential growth in the fields of neuroscience and cell biology, we have begun to understand physiology in a way that our predecessors could only hypothesize. We have identified countless proteins, genes, receptors and channels, traced their interactions, manipulated their properties and even developed drugs that can interact to treat diseases. However, there are still many areas that lack understanding of the players involved in some of the most basic biological processes. Stephen Liberles, PhD, is an associate professor in Cell Biology at Harvard Medical School. His research involves internal and external sensory systems, mainly olfaction and pheromone signaling and sensory modalities of the vagus nerve. Here I will discuss the findings from a 2013 Nature paper regarding juvenile mouse pheromone signaling and inhibition of sexual behavior.

Pheromones come in many forms: urinary volatiles, steroid derivatives and proteins in urine, tears and saliva. However, the function of many of these pheromones is unknown. qPCR using cDNA from a range of ages in mice demonstrated stark age-dependent differences in some peptide expression levels in the extraorbital lacrimal glad (LG). Amongst these peptides was exocrine-gland secreting peptide 22 (ESP22) which was produced in juveniles, but not in adults (see attached figure). The expression levels were highest at 2-3 weeks, corresponding to the time just before puberty. The levels were similar in males and females, and was isolated to the LG. RNA in situ hybridization localized ESP22 to a set of acinar lacrimal cells, the cells responsible for releasing contents into tears. Western blot analysis supported that ESP22 was a component of juvenile tears, using mass spectrometry the Liberles lab was able to identify the primary structure of the peptide.

Once ESP22 had been identified in juvenile mice, the sensory perception pathway was examined. Electrophysiological recordings determined that neurons in the vomeronasal organ (VNO) respond to ESP22, and more specifically require the ion channel TRPC2. This was affirmed by juvenile tears activating VNO neurons, while adult tears produced no recordings. Immunohistochemical staining (cFos) was used to trace the neural circuitry. It was demonstrated that upon activation with ESP22 cFos expression was increased in the medial amygdala (MeA) which receives input from the VNO by the accessory olfactory bulb and sends projetions to the hypothalamic areas controlling reproductive responses. In order to further examine the signaling, TRPC2 knockout (KO) mice were used and demonstrated no activation of the amygdala.

Once the signaling pathway was determined, implicating the need for both ESP22 and TRPC2 receptor, behavioral studies were performed in mice lacking ESP22 (C3H strain) and TRPC2 KO mice. While wild type male mice have a mating preference for adult females, they tend to not mount juveniles. However, when ESP22 was not present, the adult mice had an increased time spent mounting juveniles, and decreased latency between mountings. Similarly, the TRPC2 KO mice also spent more time mounting juveniles. When exogenous ESP22 was added to the C3H juvenile mice, the males with TRPC2 stopped the mating behavior.

Taking together this information supports the findings that ESP22 is a pheromone produced by the acinar cells in the lacrimal gland of juvenile mice before puberty. It activates TRPC2 in adult male mice, which acts on the medial amygdala to inhibit mating behavior. Once the mice reach puberty, the production of ESP22 decreases from the females, thus removing the inhibition from the males, and allowing for regular mating behavior.

For more information and publications from the Liberles lab, please visit the website at https://liberles.med.harvard.edu/home

Amy Taylor is a third year MSTP student in the Neuroscience PhD program at University of California San Diego. Her research focus is on the identification and manipulation of EEG biomarkers in schizophrenia and their relation to functional outcomes.

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