Ilona C. Kokay

Prolactin: A Pleiotropic Neuroendocrine Hormone

The neuroendocrine control of prolactin secretion is unlike that of any other pituitary hormone. It is predominantly inhibited by the hypothalamus and, in the absence of a regulatory feedback hormone, it acts directly in the brain to suppress its own secretion. In addition to this short‐loop feedback action in the brain, prolactin has been reported to influence a wide range of other brain functions. There have been few attempts to rationalise why a single hormone might exert such a range of distinct and seemingly unrelated neuroendocrine functions. In this review, we highlight some of the original studies that first characterised the unusual features of prolactin neuroendocrinology, and then attempt to identify areas of new progress and/or controversy. Finally, we discuss a hypothesis that provides a unifying explanation for the pleiotrophic actions of prolactin in the brain.

Pregnancy‐Induced Adaptation in the Neuroendocrine Control of Prolactin Secretion

During pregnancy, neuroendocrine control of prolactin secretion is markedly altered to allow a state of hyperprolactinaemia to develop. Prolactin secretion is normally tightly regulated by a short‐loop negative‐feedback mechanism, whereby prolactin stimulates activity of tuberoinfundibular dopamine (TIDA) neurones to increase dopamine secretion into the pituitary portal blood. Dopamine inhibits prolactin secretion, thus reducing prolactin concentrations in the circulation back to the normal low level. Activation of this feedback secretion by placental lactogen during pregnancy maintains relatively low levels of prolactin secretion during early and mid‐pregnancy. Despite the continued presence of placental lactogen, however, dopamine secretion from TIDA neurones is reduced during late pregnancy. Moreover, the neurones become completely unresponsive to endogenous or exogenous prolactin at this time, allowing a large nocturnal surge of prolactin to occur from the maternal pituitary gland during the night before parturition. In this review, we describe the changing patterns of prolactin secretion during pregnancy in the rat, and discuss the neuroendocrine mechanisms controlling these changes. The loss of response to prolactin is an important maternal adaptation to pregnancy, allowing the prolonged period of hyperprolactinaemia required for mammary gland development and function and for maternal behaviour immediately after parturition, and possibly also contributing to a range of other adaptive responses in the mother.

Male pheromones initiate prolactin-induced neurogenesis and advance maternal behavior in female mice

Prolactin is required for rapid onset of maternal behavior after parturition, inducing adaptive changes in the maternal brain including enhanced neurogenesis in the subventricular zone during pregnancy. The resultant increase in olfactory interneurons may be required for altered processing of olfactory cues during the establishment of maternal behavior. Pheromones act through olfactory pathways to exert powerful effects on behavior in rodents and also affect prolactin secretion. Hence, this study aimed to investigate the effect of male pheromones on neurogenesis and maternal behavior in female mice. Virgin female mice were housed individually or in split-cages where they had pheromonal but not physical contact with a male. Maternal behavior was assessed in a foster pup retrieval paradigm. Some mice were injected with bromodeoxyuridine, and the labeled cells visualized using immunohistochemistry. The data show that exposure to male pheromones, for a duration equivalent to a murine pregnancy, advanced maternal behavior in both virgin and postpartum female mice. The pheromone action was dependent on prolactin and ovarian steroids, and was associated with increased cell proliferation in the subventricular zone and subsequent increases in new neurons in the olfactory bulb. Moreover, the effect of pheromones on both cell proliferation and maternal behavior could be induced solely through administration of exogenous prolactin to mimic the pheromone-induced changes in prolactin secretion. The data suggest that male pheromones induce a prolactin-mediated increase in neurogenesis in female mice, resulting in advanced maternal behavior.

Distribution of prolactin-responsive neurons in the mouse forebrain

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. J. Comp. Neurol. 518:92–102, 2010.

Prolactin receptors in the brain during pregnancy and lactation: implications for behavior.

Numerous studies have documented prolactin regulation of a variety of brain functions, including maternal behavior, regulation of oxytocin neurons, regulation of feeding and appetite, suppression of ACTH secretion in response to stress, and suppression of fertility. We have observed marked changes in expression of prolactin receptors in specific hypothalamic nuclei during pregnancy and lactation. This has important implications for neuronal functions regulated by prolactin. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these functions may be enhanced or exaggerated in the maternal brain. The adaptations of the maternal brain allow the female to exhibit the appropriate behavior to feed and nurture her offspring, to adjust to the nutritional and metabolic demands of milk production, and to maintain appropriate hormone secretion to allow milk synthesis, secretion, and ejection. This review aims to summarize the evidence that prolactin plays a key role in regulating hypothalamic function during lactation and to discuss the hypothesis that the overall role of prolactin is to organize and coordinate this wide range of behavioral and neuroendocrine adaptations during pregnancy and lactation.

Identification of Prolactin-Sensitive GABA and Kisspeptin Neurons in Regions of the Rat Hypothalamus Involved in the Control of Fertility

High levels of circulating prolactin are known to cause infertility, but the precise mechanisms by which prolactin influences the neuroendocrine axis are yet to be determined. We used dual-label in situ hybridization to investigate whether prolactin-receptor (PRLR) mRNA is expressed in GnRH neurons. In addition, because γ-aminobutyric acidergic and kisspeptin neurons in the rostral hypothalamus are known to regulate GnRH neurons and, hence, might mediate the actions of prolactin, we investigated whether these neurons coexpress PRLR mRNA. (35)S-labeled RNA probes to detect PRLR mRNA were hybridized together with digoxigenin-labeled probes to detect either GnRH, Gad1/Gad2, or Kiss1 mRNA in the rostral hypothalamus of ovariectomized (OVX), estradiol-treated rats. Additional sets of serial sections were cut through the arcuate nucleus of OVX rats, without estradiol replacement, to examine coexpression of PRLR mRNA in the arcuate population of kisspeptin neurons. PRLR mRNA was highly expressed throughout the rostral preoptic area, particularly in periventricular regions surrounding the third ventricle, and there was a high degree of colocalization of PRLR mRNA in both Gad1/Gad2 and Kiss1 mRNA-containing cells (86 and 85.5%, respectively). In contrast, only a small number of GnRH neurons (<5%) was found to coexpress PRLR mRNA. In the arcuate nucleus of OVX rats, the majority of Kiss1 mRNA-containing cells also coexpressed PRLR mRNA. These data are consistent with the hypothesis that, in addition to a direct action on a small subpopulation of GnRH neurons, prolactin actions on GnRH neurons are predominantly mediated indirectly, through known afferent pathways.

Expression of mRNA for prolactin receptor (long form) in dopamine and pro-opiomelanocortin neurones in the arcuate nucleus of non-pregnant and lactating rats.

Under most conditions, prolactin secretion from the pituitary gland is subject to negative‐feedback regulation. Prolactin stimulates dopamine release from tuberoinfundibular (TIDA) neurones in the arcuate nucleus of the hypothalamus, which in turn suppresses the production of prolactin. However, during late pregnancy and continuing into lactation, this feedback mechanism becomes less responsive to prolactin and, as a result, a hyperprolactinaemic state develops. We investigated whether long‐form prolactin receptor (PRL‐RL) mRNA is present on TIDA neurones in nonpregnant and lactating rats. In addition, we examined whether PRL‐RL mRNA is colocalised on hypothalamic pro‐opiomelanocortin (POMC) neurones. Dual‐label in situ hybridisations using an 35S‐labelled cRNA probe specific for long‐form PRL‐R, together with a digoxigenin‐labelled RNA probe that encoded either tyrosine hydroxylase (TH) or POMC mRNA, were performed on brain sections. In both nonpregnant and lactating rats, the majority of TH mRNA‐positive cells (> 90%) were found to express long‐form PRL‐R mRNA. In sections from nonpregnant rats, few non‐TH positive cells expressed PRL‐RL mRNA. By contrast, during lactation, the proportion of PRL‐RL mRNA‐positive cells that were not TH mRNA‐positive increased to approximately 70%. Only a small number of neurones in this subpopulation of PRL‐RL mRNA‐positive neurones were found to be positive for POMC mRNA. These data show that the loss of responsiveness to prolactin occuring during lactation is not due to down regulation of long‐form PRL‐R gene expression on TIDA neurones. Moreover, the persistent expression of PRL‐RL in arcuate neuroendocrine circuits suggests that PRL‐R‐mediated signalling continues to be important in these neurones during lactation.

Loss of Hypothalamic Response to Leptin During Pregnancy Associated with Development of Melanocortin Resistance

Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro‐opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti‐related gene protein (AgRP) neurones are first‐order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double‐label immunohistochemistry was used to quantify leptin‐induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using α‐melanocyte‐stimulating hormone (MSH) as a marker. The percentage of α‐MSH neurones containing leptin‐induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with α‐MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of α‐MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first‐order leptin‐responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system.

Prolactin is involved in glial responses following a focal injury to the juvenile rat brain

A cerebral growth hormone axis is activated following brain injury in the rat and treatment with growth hormone is neuroprotective. We have now investigated whether the closely related prolactin axis has similar properties following injury to the developing rat brain. From one day following a unilateral hypoxic ischemic injury, prolactin immunoreactivity was increased in the affected cortex parallel to the development of the injury (P<0.001). Initial prolactin and prolactin receptor staining on penumbral neurons progressively decreased whereas astrocytes remained strongly immunopositive. Reactive microglia also became strongly prolactin immunoreactive. Unlike growth hormone, central treatment with prolactin failed to rescue neurons in this paradigm. This was confirmed in vitro; rat prolactin failed to protect neurons under conditions for which growth hormone was neuroprotective. However, prolactin had trophic and pro-proliferative effects on glia (P<0.001). We confirmed the expression of the prolactin receptor in vitro by reverse transcriptase polymerase chain reaction, and show its strong association with astrocytes as compared with neurons by immunocytochemistry. In summary, we show for the first time that hypoxia ischemia induces a robust activation of the prolactin axis in regions of the cerebral cortex affected by injury. The lack of neuroprotective properties in vivo and in vitro indicates that, unlike growth hormone, prolactin is not directly involved in neuronal rescue in the injured brain. Its strong relation to glial reactions and its gliatrophic effects suggest that the prolactin axis is primarily involved in a gliogenic response during recovery from cerebral injury.

Prolactin regulates kisspeptin neurons in the arcuate nucleus to suppress LH secretion in female rats.

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.