Regulation of Oxytocin Magnocellular Neurosecretory Cells by Kisspeptin in Late Pregnancy

Abstract

Oxytocin magnocellular neurosecretory cells (MNCs) are located in the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus. MNC activity during parturition results in secretion of oxytocin into the bloodstream to facilitate uterine contraction, which aids delivery of the offspring. The regulation of oxytocin MNCs during late pregnancy and parturition is not completely understood. Kisspeptin is a peptide that has become widely recognised as a positive regulator of gonadotropin releasing hormone neurons and is essential for normal fertility. Previous work in the Brown lab has shown that kisspeptin also excites oxytocin MNCs from late-pregnant rats in vivo, but does not affect oxytocin MNCs from virgin rats. Furthermore, the expression of kisspeptin in fibres of the perinuclear zone (PNZ) of the SON is higher in late-pregnant rats compared to virgin rats. These results suggest that kisspeptin might be involved in excitation of oxytocin MNCs in late pregnancy to release oxytocin during parturition. The aim of this thesis is to further elucidate the mechanisms regarding the activation of oxytocin MNCs in late-pregnant rats.

Firstly, I confirmed earlier results by showing that kisspeptin fibre density in the PNZ was significantly higher in late-pregnant rats compared to virgin rats. Furthermore, I showed that kisspeptin expression in cell bodies was significantly higher in the rostral periventricular region of the third ventricle but was lower in the arcuate nucleus. To identify the origin of PNZ kisspeptin fibres, I conducted retrograde tracing and showed that only kisspeptin neurons from the periventricular nucleus co-expressed retrograde tracer, indicating that this is the region from which the neurons project.

Next, I performed patch clamp electrophysiology to determine whether kisspeptin excitation of oxytocin MNCs in vivo is evident in brain slices and whether the membrane properties of oxytocin MNCs are affected by kisspeptin. Using cell-attached recordings, I showed that kisspeptin did not affect action current frequency of MNCs in slices from virgin or late-pregnant rats. Because kisspeptin might affect membrane currents that are not strong enough to increase activity in the slice, I used whole-cell recordings of SON MNCs to measure postsynaptic currents. Kisspeptin did not affect excitatory or inhibitory postsynaptic current frequency or amplitude, suggesting that kisspeptin does not affect MNCs indirectly through modulation of glutamatergic or GABAergic inputs. Furthermore, kisspeptin did not affect MNC holding currents, indicating that kisspeptin does not act directly on MNCs. To determine whether central kisspeptin increased oxytocin MNC activation, I conducted immunohistochemistry for the marker of neuronal activation, Fos protein. Kisspeptin did not increase Fos expression in oxytocin MNCs in virgin or pregnant rats. Furthermore, central injection of antagonists to the kisspeptin receptor (Kiss1R) or neuropeptide FF receptor (NPFFR), receptors through which kisspeptin has been shown to mediate its effects, did not affect Fos expression.

Finally, to determine whether kisspeptin neurons are activated at the time of parturition, I conducted immunohistochemistry for Fos and found that there were no significant changes in Fos expression in kisspeptin neurons from any known population during parturition. These results suggest that kisspeptin neurons are not activated at parturition. However, Fos expression is not always associated with increased neuronal activity.

Here, I have shown that kisspeptin expression is increased in the periventricular nucleus in late pregnancy, and that kisspeptin is transported to the PNZ, where it might be involved in regulation of oxytocin MNCs. The mechanism by which kisspeptin excites oxytocin MNCs in late pregnancy was not established, but it does not involve direct activation, or indirect activation via local glutamatergic or GABAergic inputs. The results presented here do not provide evidence of activation of kisspeptin neurons in parturition, but further studies will be needed to confirm this.