Prolactin has numerous biological actions in the brain, and transgenic mice are increasingly being used to investigate these actions. The present study aimed to provide a detailed mapping of the prolactin-responsive neurons in the female mouse forebrain by describing the distribution of prolactin receptor mRNA by in situ hybridization, and measuring prolactin-induced phosphorylation of signal transducer and activation of transcription 5 (pSTAT5) by immunohistochemistry. For in situ hybridization, a probe designed to detect both long and short receptor isoforms showed mRNA expression in a heterogeneous manner within the forebrain. Strong expression was observed in the rostral hypothalamus, particularly in periventricular regions, as well as in the arcuate and ventromedial nuclei of the mediobasal hypothalamus. There was also significant expression in some nonhypothalamic regions, notably high expression in the choroid plexus, and lower levels of expression in the medial amygdala, bed nucleus of the stria terminalis, and lateral septum. Prolactin-induced pSTAT5, detected by immunohistochemistry, provided a functional index of prolactin receptor activation in neurons. Prolactin-induced pSTAT5 was only observed in areas containing prolactin receptor mRNA, and was particularly prominent in the rostral and mediobasal hypothalamus. Most other areas that contained prolactin receptor mRNA also showed positive signal for prolactin-induced pSTAT5. The major exceptions were paraventricular nucleus and median preoptic nucleus, in which prolactin receptor mRNA was observed, but no induction of pSTAT5 by prolactin. The data provide key neuroanatomical information facilitating the use of the mouse model for furthering our understanding of prolactin actions in the brain.

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.

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.

Altered physiological states require neuronal adaptation. In late pregnancy and lactation, a sub-population of the mouse hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons alters their behavior to synthesize and release met-enkephalin rather than dopamine. These neurons normally release dopamine to inhibit prolactin secretion and are activated by prolactin in a short-loop feedback manner. In lactation, dopamine synthesis is suppressed in an opioid-dependent (naloxone-reversible) manner, meaning that prolactin secretion is disinhibited. Conditional deletion of the prolactin receptor in neurons reveals that this change in phenotype appears to be driven by prolactin itself, apparently through an alteration in intracellular signaling downstream of the prolactin receptor that favors enkephalin production instead of dopamine. Thus, prolactin effectively facilitates its own secretion, which is essential for lactation and maternal behavior. These studies provide evidence of a physiologically important, reversible alteration in the behavior of a specific population of hypothalamic neurons in the adult brain.

Kisspeptin has been shown to stimulate prolactin secretion. We investigated whether kisspeptin acts through the Kiss1 receptor (Kiss1r) to regulate dopamine and prolactin. Initially, we evaluated prolactin response in a Kiss1r-deficient mouse line, in which Kiss1r had been knocked into GnRH neurons (Kiss1r-/-R). Intracerebroventricular kisspeptin-10 (Kp-10) increased prolactin release in wild-type but not in Kiss1r-/-R female mice. In ovariectomized, estradiol-treated rats, the Kiss1r antagonist kisspeptin-234 abolished the Kp-10-induced increase in prolactin release but failed to prevent the concomitant reduction in the activity of tuberoinfundibular dopaminergic (TIDA) neurons, as determined by the 3,4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence. Using whole-cell patch clamp recordings in juvenile male rats, we found no direct effect of Kp-10 on the electrical activity of TIDA neurons. In addition, dual-label in situ hybridization in the hypothalamus of female rats showed that Kiss1r is expressed in the periventricular nucleus of the hypothalamus (Pe) and arcuate nucleus of the hypothalamus (ARC) but not in tyrosine hydroxylase (Th)-expressing neurons. Kisspeptin also has affinity for the neuropeptide FF receptor 1 (Npffr1), which was expressed in the majority of Pe dopaminergic neurons but only in a low proportion of TIDA neurons in the ARC. Our findings demonstrate that Kiss1r is necessary to the effect of kisspeptin on prolactin secretion, although TIDA neurons lack Kiss1r and are electrically unresponsive to kisspeptin. Thus, kisspeptin is likely to stimulate prolactin secretion via Kiss1r in nondopaminergic neurons, whereas the colocalization of Npffr1 and Th suggests that Pe dopaminergic neurons may play a role in the kisspeptin-induced inhibition of dopamine release.

Prolactin (PRL) is known to suppress LH secretion. Kisspeptin neurons regulate LH secretion and express PRL receptors. We investigated whether PRL acts on kisspeptin neurons to suppress LH secretion in lactating (Lac) and virgin rats. Lac rats displayed high PRL secretion and reduced plasma LH and kisspeptin immunoreactivity in the arcuate nucleus (ARC). Bromocriptine-induced PRL blockade significantly increased ARC kisspeptin and plasma LH levels in Lac rats but did not restore them to the levels of non-Lac rats. Bromocriptine effects were prevented by the coadministration of ovine PRL (oPRL). Virgin ovariectomized (OVX) rats treated with either systemic or intracerebroventricular oPRL displayed reduction of kisspeptin expression in the ARC and plasma LH levels, and these effects were comparable with those of estradiol treatment in OVX rats. Conversely, estradiol-treated OVX rats displayed increased kisspeptin immunoreactivity in the anteroventral periventricular nucleus, whereas oPRL had no effect in this brain area. The expression of phosphorylated signal transducer and activator of transcription 5 was used to determine whether kisspeptin neurons in the ARC were responsive to PRL. Accordingly, intracerebroventricular oPRL induced expression of phosphorylated signal transducer and activator of transcription 5 in the great majority of ARC kisspeptin neurons in virgin and Lac rats. We provide here evidence that PRL acts on ARC neurons to inhibit kisspeptin expression in female rats. During lactation, PRL contributes to the inhibition of ARC kisspeptin. In OVX rats, high PRL levels suppress kisspeptin expression and reduce LH release. These findings suggest a pathway through which hyperprolactinemia may inhibit LH secretion and thereby cause infertility.

Adaptive changes in glucose homeostasis during pregnancy require proliferation of insulin-secreting beta-cells in the pancreas, together with increased sensitivity for glucose-stimulated insulin secretion. Increased concentrations of maternal prolactin/placental lactogen contribute to these changes, but the site of action remains uncertain. Use of Cre-lox technology has generated pancreas-specific prolactin receptor (Prlr) knockouts that demonstrate the development of a gestational diabetic like state. However, many Cre-lines for the pancreas also express Cre in the hypothalamus and prolactin could act centrally to modulate glucose homeostasis. The aim of the current study was to examine the relative contribution of prolactin action in the pancreas and brain to these pregnancy-induced adaptations in glucose regulation. Deletion of prolactin receptor (Prlr) from the pancreas using Pdx-cre or Rip-cre led to impaired glucose tolerance and increased non-fasting blood glucose levels during pregnancy. Prlrlox/lox /Pdx-Cre mice also had impaired glucose-stimulated insulin secretion and attenuated pregnancy-induced increase in beta-cell fraction. Varying degrees of Prlr recombination in the hypothalamus with these Cre lines left open the possibility that central actions of prolactin could contribute to the pregnancy-induced changes in glucose homeostasis. Targeted deletion of Prlr specifically from the forebrain, including areas of expression induced by Pdx-Cre and Rip-cre, had no effect on pregnancy-induced adaptations in glucose homeostasis. These data emphasize the pancreas as the direct target of prolactin/placental lactogen action in driving adaptive changes in glucose homeostasis during pregnancy.