Key Roles of Cyclooxygenase 2-Protein Kinase A-Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 Pathway in the Regulation of Oxytocin Neuronal Activity in Lactating Rats with Intermittent Pup-Deprivation

•Early postpartum is a critical time for the milk-ejection reflex and breastfeeding.•That can be disrupted by the separation of babies from the mothers.•The separation increases intrahypothalamic but not pituitary oxytocin secretion.•Oxytocin uncouples oxytocin receptor-associated signals Cox-2 and PKA with HCN3.•Intranasal application of oxytocin largely reverses these changes. Suckling-evoked pulsatile release of oxytocin (OT) from the posterior pituitary plays a key role in breastfeeding, which relies on burst-like discharges of OT neurons. To explore cellular mechanisms regulating OT neuronal activity, using lactating rats with pup-deprivation (PD) during postpartum day 1–5, we observed the involvement of prostaglandin, cyclic AMP/protein kinase A (PKA) and hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) signaling pathway in OT neuronal activity. PD gradually reduced lactation efficiency. Intermittent PD (IPD) was largely reversed by intranasally-applied OT (IAO) but not by hypodermically-applied OT. IPD caused involution-like histological changes in the mammary glands, increased hypothalamic OT release but did not influence plasma OT concentrations. In the supraoptic nucleus, IPD increased OT receptor (OTR) expressions in OT neurons as well as Gαq subunit, Gβ subunit and cyclooxygenase 2 (Cox-2). These effects except that on Gβ subunit were reversed by IAO. Notably, IPD increased the expression of catalytic subunit of PKA in the SON, specifically in vasopressin neurons but not in OT neurons. In addition, IPD increased the expression of HCN3. IAO partially reversed these changes in the SON. Lastly, blocking HCN3 blocked excitation and burst firing in OT neurons-evoked by prostaglandin E2, a key mediator of OT-evoked burst firing; blocking Cox-2 or PKA reduced the molecular association between OTR and HCN3. Thus, there is a prostaglandin-cAMP/PKA–HCN3 pathway in the regulation of OT neuronal activity. PD disrupts lactation performance through uncoupling OTR and PKA-HCN3 signaling. The reversal effect of IAO highlights its therapeutic potential in PD-evoked hypogalactia.