The Effects of Corticotropin-Releasing Hormone (CRH) on Gonadotropin-Releasing Hormone (GNRH) Neurons in Female Mice

Abstract

Reproductive function is regulated through the hypothalamic-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons form final commons pathway that integrates inputs and releases GnRH, which then stimulates downstream elements of the HPG axis. Several factors can alter GnRH neuron output and thus reproductive function. One such signal is stress. Stress activates several pathways including the hypothalamic-pituitary-adrenal (HPA) axis. Corticotropin-releasing hormone (CRH) is released in response to activation of HPA axis and has been shown in several studies to be involved in stress-induced alteration of reproductive function. In this dissertation, our central hypothesis is that CRH acts on the HPG axis to alter GnRH neuron activity. To determine if CRH modifies firing activity of GnRH neurons, we performed extracellular recordings of GFP-identified GnRH neurons before and during treatment. Several studies suggested that the interactions between HPA and HPG axes are modulated by estradiol. Therefore, GnRH neurons were recorded from ovariectomized mice either with an estradiol capsule implanted (OVX+E) or not treated further (OVX) to determine the influence of estradiol on the GnRH neuron response to CRH. In OVX mice, CRH did not affect GnRH neuron activity. In contrast, CRH displayed dose-dependent stimulatory and inhibitory effects on firing frequency of GnRH neurons in brain slices from OVX+E mice. This suggested that estradiol is required for CRH to alter GnRH neuron activity. The dose-dependent effects of CRH resulted from the activation of different CRH receptors. A CRH receptor (CRHR)-1 agonist increased firing activity in GnRH neurons, whereas activation of CRHR-2 led to decrease in firing frequency. We further analyzed if CRH alter short-term burst firing patterns, which has been associated with hormone secretion from neuroendocrine cells. We found that CRH affected burst frequency and other properties, including burst duration, spikes/burst, and/or intraburst interval. These results indicate that GnRH neuron activity is regulated by CRH. We further explored how CRH acts on GnRH neurons both through indirect and direct mechanisms. First, we tested if CRH has an acute effect on activity of arcuate kisspeptin neurons, which provide estradiol-sensitive activation of GnRH neurons. CRH did not acutely alter activity of arcuate kisspeptin neurons in either OVX or OVX+E (both daily surge and low E models) mice. Second, we tested if GABAergic inputs, which is a major excitatory fast synaptic transmission to GnRH neurons, are influenced by CRH. Activation of CRHR-1 increased GABAergic postsynaptic current frequency in GnRH neurons from OVX+E, whereas activation of CRHR-2 showed no effect. This increase in GABAergic inputs was not observed in GnRH neurons from OVX mice treated with CRHR-1 agonist. This implies that CRH stimulate GnRH neuron activity by increasing GABAergic inputs to GnRH neurons and this effect is also estradiol-dependent. Third, we tested the direct action of CRH on GnRH neuron voltage-gated potassium currents, which play roles in regulating resting membrane potential and excitability of GnRH neurons. The amplitude of fast transient and sustained potassium currents in GnRH neurons from OVX+E was not affected by either activation of CRHR-1 or CRHR-2. Fourth, the direct action of CRH on GnRH neuron excitability was tested. No differences were observed in action potential firing in response to current injection from GnRH neurons treated with CRH. Overall, these data suggest that CRH exerts both stimulatory and inhibitory on GnRH neuron firing activity and the CRH is likely act on GnRH neurons through indirect mechanisms.