Kisspeptin Regulation of Oxytocin Neuron Activity in Late Pregnancy

Abstract

Oxytocin is secreted from the posterior pituitary gland by oxytocin neurons to trigger uterine contractions during birth. We have recently shown that the oxytocin neuron firing rate increases in response to intracerebroventricular (ICV) kisspeptin only in late‐pregnant rats. Furthermore, the kisspeptin projection from the periventricular nucleus of the hypothalamus (PeN) to the perinuclear zone (PNZ) surrounding the supraoptic nucleus (SON) increases in late pregnancy. This study aimed to determine the site and mechanism of central kisspeptin activation of oxytocin neuron activity in late pregnant rats. First, in vivo extracellular single unit recordings were made from SON neurons in urethane‐anaesthetised non‐pregnant and late‐pregnant rats during microdialysis application of kisspeptin (100 μM in the dialysate) into the SON. Intra‐SON kisspeptin consistently increased the firing rate of oxytocin neurons in late‐pregnant rats but not in non‐pregnant rats. Two‐way repeated measures (RM) ANOVA revealed a significant interaction between REPRODUCTIVE STATUS and TIME (F(6,88) = 10.31, p < 0.001); post hoc all pairwise Holm‐Sidak tests revealed a significant increase in firing rate in late‐pregnant rats at 30 (p < 0.05), 40, 50 and 60 minutes (all p < 0.001 versus pre‐kisspeptin), and a significantly higher firing rate than in time‐matched recordings from non‐pregnant rats at 50 and 60 minutes (p < 0.05).Then, to examine whether excitatory effects of central kisspeptin emerges directly within the SON and paraventricular nucleus (PVN) containing oxytocin neurons, we performed single label diaminobenzidine (DAB) immunostaining against phosphorylated extracellular regulated kinase (pERK), a second messenger activated by kisspeptin. ICV cannulae were implanted on day 13 or 14‐pregnant and non‐pregnant rats and one week later, day 21 pregnant and non‐pregnant rats were perfused 15 min after ICV administration of kisspeptin (2 μg/2 μl) or vehicle under pentobarbital anaesthesia. ICV kispeptin injection did not alter pERK expression in the SON or PVN. Two‐way RM ANOVA revealed a significant effect of REPRODUCTIVE STATUS (F(1,28) = 4.78, p = 0.03) but no significant effect of TREATMENT (kisspeptin/vehicle) (F(1,28) = 0.04, p = 0.82) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,28) = 0.81, p = 0.37 ) in the SON. There was no significant effect of REPRODUCTIVE STATUS (F(1,27) = 0.93, p = 0.34, two‐way RM ANOVA) or TREATMENT (F(1,27) = 0.93, p = 0.34) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,27) = 0.29, p = 0.59) in the PVN. Similarly, there was no significant effect of REPRODUCTIVE STATUS (F(1,27) = 2.28, p = 0.14, two‐way RM ANOVA) or TREATMENT (F(1,27) = 0.28, p = 0.59) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,27) = 0.07, p = 0.78) in the magnocellular division of PVN. Hence, it appears that kisspeptin excites oxytocin neurons at the end of pregnancy by a presynaptic action at local nerve terminals to stimulate peripheral oxytocin release and so might contribute to oxytocin neuron activation for parturition.