Increased expression of both short and long forms of prolactin receptor mRNA in hypothalamic nuclei of lactating rats.

This study investigated expression of prolactin receptor (PRL-R) mRNA in selected hypothalamic nuclei of lactating rats (days 7-10 post partum) compared with dioestrous rats. Rat brains were frozen with liquid nitrogen and cut into coronal sections of 300 microm. From these sections, tissues were micropunched from the parietal cortex (CTX), choroid plexus (ChP), and five hypothalamic regions: supraoptic (SO), paraventricular (Pa), arcuate (Arc) and ventromedial hypothalamic (VMH) nuclei, and median eminence (ME). Expression of both short and long forms of PRL-R mRNA were evaluated by reverse transcription-PCR and Southern hybridisation. The results showed that the relative amount of short form mRNA in the ChP of lactating rats was significantly higher than in dioestrous rats. The short form of PRL-R mRNA was undetectable in the SO, Pa, VMH of dioestrous rats but was expressed at a significant level in lactating rats. Levels of long form mRNA in the ChP, SO, Pa and VMH in lactating rats were significantly increased compared with dioestrous rats. Moreover, the long form mRNA was induced in the CTX of lactating rats. In the Arc, levels of both forms of PRL-R mRNA tended to increase in lactating rats compared with dioestrous rats but changes were not statistically significant. Neither form of PRL-R mRNA was detectable in the ME in the two animal models. Increased expression of PRL-R mRNA in specific brain regions during lactation is consistent with the variety of PRL effects on the brain, and may help to explain profound physiological changes in the lactating mother.

Increased prolactin receptor immunoreactivity in the hypothalamus of lactating rats.

Prolactin (PRL) exerts numerous effects in the brain including induction of maternal behaviour, increased food intake, and inhibition of GnRH secretion. Knowledge about the distribution of PRL receptors (PRL-R) in the brain will be critical for investigating mechanisms of PRL-brain interactions during lactation. The present study aimed to investigate the distribution of PRL-R in specific hypothalamic nuclei of lactating rats by immunohistochemistry and to compare this distribution with that in dioestrous rats. Rats were perfused with 2% paraformaldehyde and brains were cut into coronal sections (18 microm) for immunostaining. Immunoreactivity was detected by the avidin biotin complex method using mouse monoclonal antibody U5. In dioestrous rats, PRL-R immunoreactivity was observed in the choroid plexus, three hypothalamic nuclei: medial preoptic, periventricular and arcuate, and in the median eminence. The number of labelled profiles per section in the medial preoptic and arcuate nuclei increased significantly (P<0.05) in lactating rats (days 7-10 to post partum) when compared with dioestrous rats. Furthermore, in lactating rats, PRL-R immunoreactive neurons were identified in the cerebral cortex, substantia nigra and numerous additional hypothalamic nuclei including the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic, paraventricular hypothalamic, supraoptic, suprachiasmatic, and ventromedial hypothalamic nuclei. These observations assist our understanding of the multiple sites of PRL effects on brain function during lactation.

Differential expression of the two forms of prolactin receptor mRNA within microdissected hypothalamic nuclei of the rat.

The prolactin receptor (PRL-R) has recently been identified in various hypothalamic nuclei of female rats. In this study, expression of both the short- and long-forms of PRL-R mRNA was investigated in 11 microdissected hypothalamic nuclei of ovariectomized, estrogen-treated rats. Specific nuclei were micropunched from 300-micrometer thick frozen coronal sections with autoclaved stainless steel needles of 300 or 500 micrometer diameter. Total RNA was extracted from the punched tissue, and the two forms of PRL-R mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers. The RT-PCR product was verified by Southern hybridization with a digoxigenin-labelled oligonucleotide probe common to both forms. The results showed that both forms of PRL-R mRNA were expressed to varying degrees in the choroid plexus, cerebral cortex and various hypothalamic nuclei, including: ventromedial preoptic nucleus, ventrolateral preoptic nucleus, medial preoptic nucleus, suprachiasmatic nucleus, supraoptic nucleus, paraventricular hypothalamic nucleus, periventricular hypothalamic nucleus, arcuate nucleus, ventromedial hypothalamic nucleus, and median eminence. Of these brain regions, the choroid plexus expressed the highest level while the suprachiasmatic nucleus contained the lowest level of mRNA. There was no expression detected in the dorsomedial hypothalamic nucleus. The choroid plexus, supraoptic nucleus and paraventricular hypothalamic nucleus had higher levels of the short-form of the PRL-R mRNA than the long-form, whilst other hypothalamic nuclei preferentially expressed the long-form of the PRL-R mRNA. The differential expression of PRL-R gene suggests that the two forms may be differentially regulated in specific brain regions and may mediate different functions of PRL.

Distribution of prolactin receptor immunoreactivity in the brain of estrogen-treated, ovariectomized rats.

Although there is extensive evidence for effects of prolactin (PRL) on the brain, knowledge about the PRL receptor (PRL-R) in the brain is limited. By using monoclonal antibodies raised against purified rat liver PRL-R, the distribution of PRL-R was investigated by immunohistochemistry in brains of the estrogen-treated ovariectomized (OVX+E) rat and the adult male rat. Immunohistochemistry was performed by using the avidin biotinylated horse radish peroxidase macromolecular complex method. In both male and OVX+E rats, strong immunostaining was detected in the choroid plexus of all cerebral ventricles. This immunostaining was localized predominately on epithelial cell membranes. In the OVX+E female rat, scattered immunoreactive perikarya were observed in the arcuate nucleus, periventricular hypothalamic nucleus, preoptic area, suprachiasmatic nucleus, and supraoptic nucleus of the hypothalamus. Immunostaining in hypothalamic nuclei was localized on neuronal cell bodies as well as on neuronal processes. In addition, there was extensive PRL-R immunoreactivity throughout the globus pallidus and ventral pallidum. Immunostaining in these striatal regions was not associated with neuronal cell bodies but appeared to be localized on processes or glial cells. In the male rat, less immunostaining was observed in the hypothalamus, and there was no immunostaining in the corpus striatum. No significant staining was observed in the cerebral cortex, thalamus, or hindbrain of either male or OVX+E rats. The implication of PRL-R existence in these brain regions remains to be investigated.