Genetic Identification of GnRH Receptor Neurons: A New Model for Studying Neural Circuits Underlying Reproductive Physiology in the Mouse Brain

Abstract

GnRH signaling regulates reproductive physiology in vertebrates via the hypothalamic-pituitary-gonadal axis. In addition, GnRH signaling has been postulated to act on the brain. However, elucidating its functional role in the central nervous system has been hampered because of the difficulty in identifying direct GnRH signaling targets in live brain tissue. Here we used a binary genetic strategy to visualize GnRH receptor (GnRHR) neurons in the mouse brain and started to characterize these cells. First, we expressed different fluorescent proteins in GnRHR neurons and mapped their precise distribution throughout the brain. Remarkably, neuronal GnRHR expression was only initiated after postnatal day 16, suggesting peri- and postpubertal functions of GnRH signaling in this organ. GnRHR neurons were found in different brain areas. Many GnRHR neurons were identified in areas influencing sexual behaviors. Furthermore, GnRHR neurons were detected in brain areas that process olfactory and pheromonal cues, revealing one efferent pathway by which the neuroendocrine hypothalamus may influence the sensitivity towards chemosensory cues. Using confocal Ca(2+) imaging in brain slices, we show that GnRHR neurons respond reproducibly to extracellular application of GnRH or its analog [D-TRP(6)]-LH-RH, indicating that these neurons express functional GnRHR. Interestingly, the duration and shape of the Ca(2+) responses were similar within and different between brain areas, suggesting that GnRH signaling may differentially influence brain functions to affect reproductive success. Our new mouse model sets the stage to analyze the next level of GnRH signaling in reproductive physiology and behavior.

Prolactin action in the medial preoptic area is necessary for postpartum maternal nursing behavior

“…We inserted the IRES-Cre construct immediately downstream of exon 10 of the Prlr gene, resulting in Cre expression in cells that express the long form of the Prlr (Prlr-iCre). We then crossed the Prlr-iCre animals with mice expressing a Cre-dependent τGFP reporter (ROSA26-CAGS-τGFP; eR26-τGFP) (16) to fluorescently label prolactinresponsive neurons. Because τGFP associates with microtubules, it effectively labels neuronal processes of Prlr neurons as well as their cell bodies.…”

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

“…To start characterizing gonadotropin releasing hormone receptor (GnRHR) expressing neurons in the hypothalamus we made use of transgenic mice that express GFP after Cre-mediated excision in GnRHR-expressing neurons 4,11 . GFP fluorescent neurons were identified in various brain areas, including the hypothalamus.…”

“…Using our protocol we initially tested whether GnRHR neurons utilize similar Ca 2+ signals for stimulus detection in different areas of the hypothalamus in response to direct activation with GnRH ( Figure 4E). However, these signals differed in their waveform depending on stimulus strength and brain area 4 . To quantify the change in the dynamics of the Ca 2+ responses, the area-under-the-curve (AUC) can be calculated as a measure for the increase in intracellular Ca 2+ ( Figure 4E) 4 .…”

“…However, these signals differed in their waveform depending on stimulus strength and brain area 4 . To quantify the change in the dynamics of the Ca 2+ responses, the area-under-the-curve (AUC) can be calculated as a measure for the increase in intracellular Ca 2+ ( Figure 4E) 4 . Studies are currently underway to investigate the molecular basis underlying the Ca 2+ waves and oscillations, their dependence on sex and hormonal status of the animal, and whether they can be modulated by other natural stimuli.…”

“…We had previously good results using fura-red in combination with GFP-tagged olfactory neurons 3 . The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons 4 . Fura-red based Ca 2+ imaging was also successfully combined with GFP-tagged pancreatic β-cells and GFP-tagged receptors expressed in HEK cells 5,6 .…”