Electrophysiological Characteristics of Kisspeptin Neurons in the Hypothalamus of the Mouse

Abstract

The gonadotropin-releasing hormone (GnRH) neurons are the principal neurons in a network within the hypothalamus that controls fertility in all mammals. GnRH neuron output is influenced by a wide range of neurotransmitters. One recently-discovered and essential neurotransmitter in this network is known as kisspeptin. Kisspeptin neurons exist in two distinct populations; in the rostral periventricular nucleus of the third ventricle (RP3V) and in the arcuate nucleus (ARN). Based upon anatomical evidence, the RP3V kisspeptin neurons are hypothesized to be important for the positive feedback mechanism of estrogen that drives the preovulatory GnRH surge. Also, anatomical evidence has led to the suggestion that the ARN kisspeptin neuronal population is involved in the negative feedback mechanism that homeostatically suppresses GnRH release and also is important to drive pulsatile GnRH secretion.

In my PhD, I have performed experiments to gain functional insight into the roles of these two kisspeptin neuronal populations. To identify kisspeptin neurons, I have used a knock-in kisspeptin-GFP mouse line, in which only kisspeptin neurons should appear fluorescent. I used immunohistochemistry to show that kisspeptin neurons can be identified in vitro using GFP fluorescence. I have then used patch-clamp electrophysiology techniques to describe the spontaneous firing rate of kisspeptin neurons and described this across the estrous cycle. I demonstrate that the spontaneous electrical activity is not strongly subject to fluctuating gonadal steroid levels. Unexpectantly, I have described a male-dominant sex difference in spontaneous firing rate of ARN kisspeptin neurons. Additionally, I show that RP3V kisspeptin neurons are not strongly activated at the time of the GnRH surge when measured in an acute brain slice. ARN kisspeptin neurons are thought to form an interconnected network, and utilize coexpressed neuropeptides to generate a pulsatile output that is important for generating the pulsatile pattern of GnRH release from the hypothalamus. I have used cell-attached and whole-cell electrophysiological recording techniques to show that neurokinin B and dynorphin potently modulate ARN kisspeptin neuronal output in an opposing manner, and described the receptors that mediate these effects. Following this, I have used whole-cell electrophysiological recording techniques to test for functional connections between ARN kisspeptin neurons. Whereas no evidence was found for monosynaptic glutamatergic synaptic connections, some evidence was found for neuropeptidergic neurotransmission between ARN kisspeptin neurons. These results provide electrophysiological data to inform and update theories of kisspeptin neuronal function which are built largely on anatomical data.