Prolactin regulation of the HPA axis is not mediated by a direct action upon CRH neurons: evidence from the rat and mouse.

Centrally acting prolactin has been shown to have anti-stress effects by modulating the activity of the hypothalamic-pituitary-adrenal axis. We tested the hypothesis that prolactin directly targets hypothalamic corticotropin-releasing hormone (CRH) neurons. In situ hybridisation confirmed expression of mRNA encoding the long, but not the short, isoform of the prolactin receptor (PRLR) within the paraventricular nucleus (PVN) of the virgin rat; however, only 6% of CRH neurons expressed long-form Prlr mRNA. Examination of the functional response of CRH neurons to intracerebroventricular prolactin (500 ng) showed that these neurons did not respond with activation of phosphorylated signal transducer and activator of transcription 5 (pSTAT5), a marker of long-form PRLR activation. However, as only a subset of neurons expressing Crh mRNA could be detected using immunohistochemistry, we utilised a transgenic mouse model to label CRH neurons with a fluorescent reporter (CRH-Cre-tdTomato). In lactating animals, chronically elevated prolactin levels resulted in significantly increased pSTAT5 expression in the PVN. Overall, few tdTomato-labelled CRH neurons were double-labelled, although a small subset of CRH neurons in the caudal PVN were pSTAT5 positive (approximately 10% of tdTomato neurons at this level, compared to 1% in the rostral PVN). These data suggest that most CRH neurons do not respond directly to prolactin. To confirm that prolactin was not activating another signalling pathway, we used a transgenic mouse line to label PRLR-expressing neurons with Cre-dependent green fluorescent protein (GFP) expression (CRH-Cre-Prlr lox/lox ). No GFP-expressing cells were evident in the PVN, indicating that in the mouse, as in the rat, the CRH neurons do not express either PRLR isoform. Together these data showed that the anti-stress effects of prolactin are not the result of prolactin directly regulating CRH neurons.

Prolactin regulation of oxytocin neurone activity in pregnancy and lactation.

KEY POINTS: During lactation, prolactin promotes milk synthesis and oxytocin stimulates milk ejection. In virgin rats, prolactin inhibits the activity of oxytocin-secreting neurones. We found that prolactin inhibition of oxytocin neurone activity is lost in lactation, and that some oxytocin neurones were excited by prolactin in lactating rats. The change in prolactin regulation of oxytocin neurone activity was not associated with a change in activation of intracellular signalling pathways known to couple to prolactin receptors. The change in prolactin regulation of oxytocin neurone activity in lactation might allow coordinated activation of both populations of neurones when required for successful lactation. ABSTRACT: Secretion of prolactin for milk synthesis and oxytocin for milk secretion is required for successful lactation. In virgin rats, prolactin inhibits oxytocin neurones but this effect would be counterproductive during lactation when secretion of both hormones is required for synthesis and delivery of milk to the newborn. Hence, we determined the effects of intracerebroventricular (i.c.v.) prolactin on oxytocin neurones in urethane-anaesthetised virgin, pregnant and lactating rats. Prolactin (2 µg) consistently inhibited oxytocin neurones in virgin and pregnant rats (by 1.9 ± 0.4 and 1.8 ± 0.5 spikes s-1 , respectively), but not in lactating rats; indeed, prolactin excited six of 27 oxytocin neurones by >1 spike s-1 in lactating rats but excited none in virgin or pregnant rats (χ22  = 7.2, P = 0.03). Vasopressin neurones were unaffected by prolactin (2 µg) in virgin rats but were inhibited by 1.1 ± 0.2 spikes s-1 in lactating rats. Immunohistochemistry showed that i.c.v. prolactin increased oxytocin expression in virgin and lactating rats and increased signal transducer and activator of transcription 5 phosphorylation to a similar extent in oxytocin neurones of virgin and lactating rats. Western blotting showed that i.c.v. prolactin did not affect phosphorylation of extracellular regulated kinase 1 or 2, or of Akt in the supraoptic or paraventricular nuclei of virgin or lactating rats. Hence, prolactin inhibition of oxytocin neurones is lost in lactation, which might allow concurrent elevation of prolactin secretion from the pituitary gland and activation of oxytocin neurones for synthesis and delivery of milk to the newborn.

Differential sensitivity of specific neuronal populations of the rat hypothalamus to prolactin action.

Prolactin stimulates dopamine release from neuroendocrine dopaminergic (NEDA) neurons in the hypothalamic arcuate nucleus (ARC) to maintain low levels of serum prolactin. Elevated prolactin levels during pregnancy and lactation may mediate actions in other hypothalamic regions such as the paraventricular nucleus (PVN) and rostral preoptic area (rPOA). We predicted that NEDA neurons would be more sensitive prolactin targets than neurons in other regions because they are required to regulate basal prolactin secretion. Moreover, differences in the accessibility of the ARC to prolactin in blood may influence the responsiveness of this population. Therefore, we compared prolactin-induced signaling in different hypothalamic neuronal populations following either systemic or intracerebroventricular (icv) prolactin administration. Phosphorylation of the signal transduction factor, STAT5 (pSTAT5), was used to identify prolactin-responsive neurons. In response to systemic prolactin, pSTAT5-labeled cells were widely observed in the ARC but absent from the rPOA and PVN. Many of these responsive cells in the ARC were identified as NEDA neurons. The lowest icv prolactin dose (10 ng) induced pSTAT5 in the ARC, but with higher doses (>500 ng) pSTAT5 was detected in numerous regions, including the rPOA and PVN. NEDA neurons were maximally labeled with nuclear pSTAT5 in response to 500 ng prolactin and appeared to be more sensitive than dopaminergic neurons in the rPOA. Subpopulations of oxytocin neurons in the hypothalamus were also found to be differentially sensitive to prolactin. These data suggest that differences in the accessibility of the arcuate nucleus to prolactin, together with intrinsic differences in the NEDA neurons, may facilitate homeostatic feedback regulation of prolactin release.