Abstract
Oxytocin triggers uterine contractions during parturition. My laboratory recently showed that the oxytocin neurons are excited by intracerebroventricular (ICV) administration of kisspeptin only in late-pregnant rats. Furthermore, a kisspeptin projection from the periventricular nucleus (PeN) of the hypothalamus to the perinuclear zone (PNZ) surrounding oxytocin neurons in the supraoptic nucleus (SON) emerges in late pregnancy. The research described in this thesis aimed to determine the site and mechanism of central kisspeptin activation of oxytocin neuron activity in late-pregnant rats.
To determine whether ICV kisspeptin directly activates oxytocin neurons, I performed immunohistochemistry for the kisspeptin-activated second messengers, phosphorylated extracellular regulated kinase 1/2 (pERK1/2) and phosphorylated-p38 (p-p38). I found that ICV kisspeptin (2 µg in 1 µg/µl) did not change pERK1/2 or p-p38 expression in oxytocin neurons in the SON and PVN, suggesting that exogenous kisspeptin does not directly activate oxytocin neurons.
I then investigated pERK1/2 and p-p38 expression in brain areas that project to oxytocin neurons to determine whether kisspeptin acts indirectly at cell bodies of afferent inputs to excite oxytocin neurons. ICV kisspeptin did not change pERK1/2 or p-p38 expression in the nucleus tractus solitarius (NTS), rostral or caudal ventrolateral medulla (VLM), or dorsal raphe nucleus (DRN) in the brainstem, or in the organum vasculosum lamina terminalis (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), or anteroventral periventricular nucleus (AVPe)/PeN in the forebrain. Hence, exogenous kisspeptin does not appear to activate afferent inputs to oxytocin neurons.
To determine whether kisspeptin acts locally within the SON, in vivo extracellular single-unit recordings were made from SON neurons during intra-SON microdialysis administration of kisspeptin (100 µM in the dialysate) in urethane-anaesthetised rats. I found that intra-SON kisspeptin consistently increased the firing rate of oxytocin neurons in urethane-anaesthetised late-pregnant rats but had no effect in non-pregnant rats. Hence, it appears that kisspeptin excites SON oxytocin neurons by a local action within the SON.
Noradrenergic projections to the SON from the NTS are prominently activated at parturition. Therefore, I next investigated whether kisspeptin enhances noradrenergic signalling to oxytocin neurons in late pregnancy. To determine whether there is an interaction between kisspeptin and TH-positive (a marker for noradrenaline neurons) fibres in the SON/PNZ, I performed immunohistochemistry for kisspeptin and TH in the SON/PNZ. Confocal image analysis showed similar low levels of colocalisation/appositions between kisspeptin and TH in the SON/PNZ of non-pregnant and late-pregnant rats, suggesting there is no increase in putative synapses between kisspeptin and TH fibres in late pregnancy. Next, I investigated whether kisspeptin pre-synaptically modulated excitatory noradrenergic inputs to oxytocin neurons in late pregnancy using in vivo electrophysiology to determine the functional interaction between kisspeptin and noradrenergic inputs to oxytocin neurons. Intravenous (IV) cholecystokinin-8S (CCK-8S) excites oxytocin neurons via noradrenergic inputs. Hence, the oxytocin neuron response to IV CCK-8S was determined before and during intra-SON administration of kisspeptin. Preliminary analysis showed no significant differences in oxytocin neuron responses to IV CCK-8S before or during kisspeptin administration in non-pregnant and late-pregnant rats, suggesting that kisspeptin excitation of oxytocin neurons might not be mediated by noradrenergic inputs.
Taken together, these results suggest that kisspeptin might excite oxytocin neurons at the end of pregnancy by a presynaptic action at their afferent inputs, but the specific input involved is yet to be identified.