Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlrlox/lox mice). The Prlrlox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlrlox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are responsive to prolactin and may play a role in the orexigenic effects of prolactin, whereas prolactin does not directly affect Agrp neurones.

Effects of Prolactin and Lactation on A15 Dopamine Neurones in the Rostral Preoptic Area of Female Mice.

There are several distinct populations of dopamine neurones in the hypothalamus. Some of these, such as the A12 tuberoinfundibular dopamine neurones and the A14 periventricular dopamine neurones, are known to be regulated by the anterior pituitary hormone prolactin, whereas others, such as the A13 zona incerta dopaminergic neurones, are not. The present study aimed to investigate the role of prolactin in the regulation of a fourth population of hypothalamic dopamine neurones: the A15 dopamine population in the rostral hypothalamus. These neurones may play a role in the regulation of gonadotrophin-releasing hormone (GnRH) secretion, and we hypothesised that they might contribute to the suppression of GnRH release and infertility caused by hyperprolactinaemia. Under basal (low prolactin) conditions, only 8% of A15 dopamine neurones in the anteroventral periventricular nucleus (AVPV) of vehicle-treated dioestrous mice expressed phosphorylated signal transducer and activator of transcription 5 (pSTAT5), as labelled by immunohistochemistry. We have previously shown that this transcription factor can be used as an index of prolactin-receptor activation. Following acute prolactin administration, 35% of AVPV dopamine neurones co-expressed pSTAT5, whereas, during lactation, when endogenous prolactin levels are chronically elevated, 55% of AVPV dopamine neurones expressed pSTAT5. There was also a significant increase in dopamine turnover in the rostral hypothalamus, both in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis and in the rostral preoptic area during lactation, with the 3,4-dihydroxyphenylacetic acid/dopamine ratio increasing from 0.28 ± 0.04 and 0.14 ± 0.01 in dioestrous mice to 0.82 ± 0.06 and 0.38 ± 0.03, respectively, in day 7 lactating mice. It is not yet known whether this change is driven by the hyperprolactinaemia of lactation, or another lactation-specific signal. These data demonstrate that the A15 dopaminergic neurones of the rostral hypothalamus are responsive to exogenous prolactin and may be regulated by endogenous prolactin during lactation.

Prolactin regulation of kisspeptin neurones in the mouse brain and its role in the lactation-induced suppression of kisspeptin expression.

Hyperprolactinaemia is a major cause of infertility in both males and females, although the mechanism by which prolactin inhibits the reproductive axis is not clear. The aim of the present study was to test the hypothesis that elevated prolactin causes suppression of kisspeptin expression in the hypothalamus, resulting in reduced release of gonadotrophin-releasing hormone (GnRH) and consequent infertility. In oestrogen-treated ovariectomised mice, chronic prolactin-treatment prevented the rise in luteinising hormone (LH) seen in vehicle-treated mice. Kiss1 mRNA was significantly suppressed in both the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus after prolactin treatment. Exogenous prolactin treatment induced phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in kisspeptin neurones, and suppression of endogenous prolactin using bromocriptine reduced levels of pSTAT5 in kisspeptin neurones, suggesting that prolactin acts directly on kisspeptin neurones. By contrast, fewer than 1% of GnRH neurones expressed pSTAT5 in either dioestrous or lactating mice. As reported previously, there was significant suppression of kisspeptin mRNA and protein in the RP3V on day 7 of lactation, although not in the arcuate nucleus. Bromocriptine treatment significantly increased Kiss1 mRNA expression in the RP3V, although not to dioestrous levels. Unilateral thelectomy, aiming to eliminate sensory inputs from nipples on one side of the body, failed to alter the reduction in the number of kisspeptin neurones observed in the RP3V. These data demonstrate that chronic prolactin administration suppressed serum LH, and reduced Kiss1 mRNA levels in both the RP3V and arcuate nucleus, consistent with the hypothesis that prolactin-induced suppression of kisspeptin secretion might mediate the inhibitory effects of prolactin on GnRH secretion. During lactation, however, the suppression of Kiss1 mRNA in the RP3V was only partially reversed by the administration of bromocriptine to block elevated levels of prolactin, suggesting that, although elevated prolactin contributes to lactational anovulation, additional non-neural factors must also contribute to the lactation-induced suppression of kisspeptin neurones.

Lactational anovulation in mice results from a selective loss of kisspeptin input to GnRH neurons.

In mammals, lactation is associated with a period of infertility characterized by the loss of pulsatile secretion of GnRH and cessation of ovulatory cycles. Despite the importance of lactational infertility in determining overall fecundity of a species, the mechanisms by which the suckling stimulus suppresses GnRH secretion remain unclear. Because kisspeptin neurons are critical for fertility, the aim of this study was to test the hypothesis that reduced kisspeptin expression might mediate the lactation-induced suppression of fertility, using mouse models. In the rostral periventricular area of the third ventricle (RP3V), a progressive decrease in RP3V Kiss1 mRNA levels was observed during pregnancy culminating in a 10-fold reduction during lactation compared with diestrous controls. This was associated with approximately 60% reduction in the numbers of kisspeptin-immunoreactive neurons in the RP3V detected during lactation. Similarly, in the arcuate nucleus there was also a significant decrease in Kiss1 mRNA levels during late pregnancy and midlactation, and a notable decrease in kisspeptin fiber density during lactation. The functional characteristics of the RP3V kisspeptin input to GnRH neurons were assessed using electrophysiological approaches in an acute brain slice preparation. Although endogenous RP3V kisspeptin neurons were found to activate GnRH neurons in diestrous mice, this was never observed during lactation. This did not result from an absence of kisspeptin receptors because GnRH neurons responded normally to 100 nM exogenous kisspeptin during lactation. The kisspeptin deficit in lactating mice was selective, because GnRH neurons responded normally to RP3V gamma aminobutryic acid inputs during lactation. These data demonstrate that a selective loss of RP3V kisspeptin inputs to GnRH neurons during lactation is the likely mechanism causing lactational anovulation in the mouse.

Differential actions of prolactin on electrical activity and intracellular signal transduction in hypothalamic neurons.

In many tissues, including brain, prolactin action is predominantly mediated by the Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway, leading to changes in gene transcription. However, prolactin can also exert rapid actions on electrical activity of hypothalamic neurons. Here, we investigate whether both responses occur in a single cell type, focusing on three specific populations known to be influenced by prolactin: GnRH neurons, tuberoinfundibular dopamine (TIDA) neurons, and neurons in the anteroventral-periventricular nucleus in female mice. We performed phosphorylated STAT5 (pSTAT5) immunohistochemistry to identify prolactin-responsive neurons after in vivo prolactin treatment. In addition, we carried out in vitro electrophysiology in slices from transgenic mice expressing green fluorescent protein driven by the GnRH or tyrosine hydroxylase promoters as well as from C57BL/6J mice to assess acute electrical responses to prolactin. Approximately 88% of TIDA neurons expressed pSTAT5 in diestrous mice, rising to 97% after prolactin treatment. All TIDA neurons also showed a rapid increase in firing rate after prolactin treatment. In contrast, very few GnRH neurons (11%) showed pSTAT5 in response to prolactin, and none showed a change in electrical activity. Finally, in the anteroventral-periventricular nucleus, most neurons (69%) responded to prolactin treatment with an increase in pSTAT5, but only 2/38 (∼5%) showed changes in electrical activity in response to prolactin. These observations show that prolactin recruits different combinations of electrical and transcriptional responses in neurons depending upon their anatomical location and phenotype. This may be critical in establishing appropriate responses to prolactin under different physiological conditions.

Differential changes in responses of hypothalamic and brainstem neuronal populations to prolactin during lactation in the mouse.

During lactation, there are numerous functional adaptations in the maternal brain. There is evidence that the high levels of circulating prolactin present during lactation might contribute to these adaptive changes. The present study aimed to investigate levels of functional prolactin-mediated signal transduction in the brain of lactating mice, using prolactin-induced phosphorylation of signal transducer and activator of transcription 5 (pSTAT5) as a marker, and compare these to the effect of exogenous prolactin during diestrus. On Day 7 of lactation, widespread induction of pSTAT5 was observed in numerous regions of the mouse forebrain and brainstem. In the medial preoptic nucleus, bed nuclei stria terminalis, paraventricular nucleus, and medial amygdala of the forebrain, and in the rostral periaqueductal gray, parabrachial nucleus, dorsal raphe, and the raphe obscurus nucleus of the brainstem, pSTAT5 expression was markedly increased during lactation compared with the response to exogenous prolactin during diestrus. In the anteroventral periventricular nucleus, arcuate nucleus, ventromedial nucleus, and dorsomedial nucleus, responses in lactation were comparable to diestrus. Conversely, in the area postrema of the brainstem, there was a reduction in response to prolactin, with a loss of pSTAT5 expression, during lactation. These differential responses following either acute or chronic elevations in prolactin were not accompanied by any changes in levels of prolactin receptor mRNA, when measured by in situ hybridization. These data are consistent with the hypothesis that prolactin might mediate widespread adaptive responses in the maternal brain.