Defining kisspeptin neuronal networks and function in the mouse hypothalamus (Resubmission).

Fertility in mammals is controlled by a small part of the brain called the hypothalamus. At puberty, a protein called kisspeptin initiates the release of a hormone called GnRH, which is required for normal growth of the genitalia and fertility. Kisspeptin is produced by nerves in two specific areas of the hypothalamus known as the AVPV and the ARC regions. This project will study the way in which kisspeptin producing neurons in these regions are connected to other parts of the brain. This will allow us to understand how the activity of kisspeptin neurons is controlled by signals from other regions of the brain. To do this, we will use a specialized virus that can only spread between neurons that are physically connected to each other. The virus will be activated in kisspeptin neurons and the movement of the virus tracked over time as it spreads to connected neurons. The identity of these neurons will be determined from their anatomical location and by looking at the chemical messengers (neurotransmitters) that they express. The functional significance of these connections will be determined by examining whether kisspeptin neurons can respond to these neurotransmitters. This project will provide us with fundamental knowledge about how fertility is regulated by hormonal changes and under different conditions such as day length, lactation and ageing.

Kisspeptin neuropeptides are key regulators of GnRH release and activation of the mammalian reproductive axis at puberty. Kiss1 neurons are located in two main regions of the hypothalamus; the AVPV region, which controls the pre-ovulatory LH surge and the ARC region, which controls basal GnRH pulsatility. One of the fundamental steps in understanding how Kiss1 neurons are regulated by upstream signals requires an accurate definition of their neuronal connections. To determine this, we will use a new transgenic mouse line in which Kiss1 neurons express the CRE protein and use conditional viral tracing to define afferent neuronal inputs to Kiss1 neurons. We will use a genetically modified pseudorabies virus (Ba2001) that requires a CRE-mediated recombination event to express the neuronal tracer tau-GFP and the thymidine kinase (TK) gene required for viral replication. The virus will be delivered by stereotaxic injection into either the AVPV or the ARC regions of the hypothalamus to infect sub-populations of Kiss1 neurons. Viral spread visualized by GFP fluorescence will be monitored at different time points after infection to establish the hierarchy of neuronal connections to the Kiss1 neurons. Differences in neural connectivity between males and females will be identified as well as changes that occur during the estrous cycle and in response to sex steroids. We will also test the hypothesis that changes in neuronal connectivity to Kiss1 neurons influence circadian responses, lactational ammenorhea and the decline in fertility with age prior to menopause. The anatomic localization of the labelled neurons will provide insight into their function and their identity will be confirmed by immunohistochemical analysis of their neurotransmitter expression profiles. The functional significance of these neuronal connections will be tested in vivo by central delivery of the relevant neurotransmitters and activation of Kiss1 neurons visualized C-FOS induction.

This project will provide impact by generating fundamental scientific knowledge about the neuronal networks in the brain that regulate mammalian reproduction and fertility. The immediate beneficiaries of this knowledge will be the scientific community working in the area of reproductive physiology but in a broader sense the project will also have an impact in other research areas. Fundamental knowledge gained from this study may be relevant to understanding the pathogenesis of several reproductive disorders including precocious puberty, idiopathic infertility, idiopathic amenorrhea and the prevention of spontaneous abortions. Thus, the project has the potential to have impact in the reproductive health of the nation.The project will also be of benefit to undergraduate students by providing suitable research projects for training and education. The project may also benefit the pharmaceutical industry by providing them with a new therapeutic target for drug development.


This project will provide important insights into how the Kiss1 neurons regulate mammalian fertility and the factors that control their physiology. By understanding the neuronal connectivities, it might be possible to develop novel compounds to regulate the reproductive axis. This may lead to the development of new contraceptives or substances that induce earlier puberty or re-entry into the breeding cycle in domestic animals. Alternatively, by appropriate stimulation of these connections, it might be possible to increase sperm production in infertile men due to a reduced sperm count. Various aspects of these regulatory networks will be examined including how they change with age which may provide a better understanding of why there is a decline in testosterone production in older males. Increasingly, women are delaying childbirth until after 30 years old but fertility drops greatly after this age, even though the ovary still has many eggs. This project may provide an insight into why fertility drops in females long before the menopause. As such, this research may benefit the reproductive health of the ageing population by maintaining optimal fertility for longer.