Prolactin-releasing Peptide mRNA Research Articles

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction.

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHβ and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

Colocalized neurotransmitters in the hindbrain cooperate in adaptation to chronic hypernatremia

Chronic hypernatremia activates the central osmoregulatory mechanisms and inhibits the function of the hypothalamic–pituitary–adrenal (HPA) axis. Noradrenaline (NE) release into the periventricular anteroventral third ventricle region (AV3V), the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN) from efferents of the caudal ventrolateral (cVLM) and dorsomedial (cDMM) medulla has been shown to be essential for the hypernatremia-evoked responses and for the HPA response to acute restraint. Notably, the medullary NE cell groups highly coexpress prolactin-releasing peptide (PrRP) and nesfatin-1/NUCB2 (nesfatin), therefore, we assumed they contributed to the reactions to chronic hypernatremia. To investigate this, we compared two models: homozygous Brattleboro rats with hereditary diabetes insipidus (DI) and Wistar rats subjected to chronic high salt solution (HS) intake. HS rats had higher plasma osmolality than DI rats. PrRP and nesfatin mRNA levels were higher in both models, in both medullary regions compared to controls. Elevated basal tyrosine hydroxylase (TH) expression and impaired restraint-induced TH, PrRP and nesfatin expression elevations in the cVLM were, however, detected only in HS, but not in DI rats. Simultaneously, only HS rats exhibited classical signs of chronic stress and severely blunted hormonal reactions to acute restraint. Data suggest that HPA axis responsiveness to restraint depends on the type of hypernatremia, and on NE capacity in the cVLM. Additionally, NE and PrRP signalization primarily of medullary origin is increased in the SON, PVN and AV3V in HS rats. This suggests a cooperative action in the adaptation responses and designates the AV3V as a new site for PrRP’s action in hypernatremia.

Fasting and refeeding induce differential changes in hypothalamic mRNA abundance of appetite-associated factors in 7 day-old Japanese quail, Coturnix japonica

There is little information regarding effects of fasting on feeding behavior and hypothalamic physiology in young Japanese quail. The aim was thus to measure food intake and hypothalamic mRNA in response to fasting and refeeding. Five d-old quail ate little during the dark cycle. Food intake was greatest during the first 2 h of the light cycle. Six day-old quail fasted for 6 h ate the most during the first 15 min of refeeding. In 7 d-old quail, 3 h of fasting up-regulated hypothalamic neuropeptide Y (NPY), NPY receptor sub-type 2 (NPYR2), agouti-related peptide (AgRP), orexin receptor 2 (ORXR2), melanocortin receptors 3 and 4 (MC3R and MC4R, respectively), and neuropeptide S (NPS) and decreased corticotropin-releasing factor receptor sub-type 1 (CRFR1) mRNA. Quail fasted for 3 h and refed for 1 h had greater NPY, AgRP, POMC, and MC3R but less CRFR1 mRNA than fed quail. Quail fasted for 6 h expressed more NPY, NPYR1, NPYR2, and MC3R and less ORXR2, prolactin releasing peptide (PrRP), cocaine- and amphetamine-regulated transcript (CART), and calcitonin (CAL) mRNA than fed quail. Quail fasted for 6 h and refed for 1 h expressed more NPY, NPYR1, NPYR2, AgRP, MC3R, MC4R, and NPS and less galanin, ORXR2, PrRP, CART, and CAL mRNA than fed birds. Hence, fasting induced changes in hypothalamic mRNA, with the largest changes occurring in genes associated with NPY and melanocortin signaling. Most genes remained elevated or downregulated after refeeding, suggesting that there was a time lag for transcription to respond to compensatory feeding.

Prolactin-releasing peptide is a potent mediator of the innate immune response in leukocytes from Salmo salar

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression or its effects on innate immunity are unclear even in mammals. To study the possible presence of PrRP in peripheral organs, PrRP expression patterns and their effect on innate immunity were characterised in SHK-1 cells and head kidney (HK) leukocytes purified from the salmonid, Salmo salar. We detected immunoreactive cells in leukocytes from blood and HK of S. salar and found that PrRP mRNA was abundantly expressed in these cells. We have recently reported that physiological concentrations of native PRL, downstream of neuropeptide PrRP were able to induce expression of pro-inflammatory cytokines and the production of reactive oxygen species (ROS) in HK leukocytes and macrophages from S. salar and Sparus aurata. It is of interest to note that in this work we have revealed that synthetic PrRP was able to induce expression of pro-inflammatory cytokines (interleukins) IL-1β, IL-6, IL-8, IL-12 and PRL. We also show here that PrRP increased both (ROS) production and phagocytosis. Taken together, our results demonstrate for the first time that PrRP may be a local modulator of innate immune responses in leukocytes from S. salar.

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6ppt) and hyper-osmotic (33ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6ppt-adapted fish did not change. Specific signals for Na+–K+-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na+–K+-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50ppt for 4weeks and in fish abruptly transferred from 33ppt to 6ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.

Prolactin-releasing peptide mRNA expression in mouse medulla remains relatively stable during pregnancy and lactation

We compared levels of prolactin-releasing peptide (PrRP) mRNA expression in mouse medulla at different stages of pregnancy and lactation. Mouse medulla samples were collected on days 6, 12 and 18 of pregnancy and lactation, respectively (six per group), for mRNA. Expression levels of PrRP mRNA in the medulla were measured by semi-quantitative RT-PCR, with glyceraldehyde 3-phosphate dehydrogenase as a control. PrRP mRNA was highly expressed in mouse medulla oblongata on day 6 of pregnancy (0.53), followed by 0.43 at lactation day 6, and 0.42 at lactation day 12. The expression level of PrRP mRNA on days 12 and 18 of pregnancy and day 18 of lactation shared the same value of 0.36. PrRP mRNA levels during lactation decreased slightly compared with that during pregnancy, but the differences between them were not significant. In summary, PrRP mRNA levels in the medulla oblongata remain relatively stable during pregnancy and lactation. This is evidence that medulla PrRP is not involved in the regulation of prolactin secretion.

Carnitine and carnitine orotate affect the expression of the prolactin-releasing peptide gene

Carnitine is involved in fatty acid metabolism in mammals and is widely used as a nutritional supplement; carnitine orotate is a more absorbable form of carnitine. We investigated the effects of carnitine and carnitine orotate on mouse prolactin-releasing peptide (PrRP) mRNA expression. Twenty-four female mice were randomly divided into four groups of six; control mice were orally drenched with physiological saline solution (250 mg/kg body weight) and treatment mice were orally drenched with carnitine (250 mg/kg) or carnitine orotate (250 or 750 mg/kg), once a day, for 20 days from parturition. The carnitine or carnitine orotate was dissolved in saline solution before administration. The hypothalamus, pituitary and ovary were sampled on day 21 after parturition, and PrRP mRNA levels in these tissues were measured by semi-quantitative PCR, with glyceraldehyde 3-phosphate dehydrogenase as a control. Expression of PrRP in mice treated with carnitine and carnitine orotate was significantly increased in the ovary and significantly reduced in the pituitary gland. Compared with the control, hypothalamus PrRP mRNA increased significantly in the carnitine and low-dose carnitine orotate groups and decreased significantly in the high-dose carnitine orotate group. We conclude that carnitine and carnitine orotate regulate expression of PrRP in the pituitary gland and ovaries.

Downregulation of prolactin-releasing peptide gene expression in the hypothalamus and brainstem of diabetic rats

We investigated the prolactin-releasing peptide (PrRP) mRNA levels in the hypothalamus and brainstem of streptozotocin (STZ)-induced diabetic rats and fa/ fa Zucker diabetic rats, using in situ hybridization histochemistry. PrRP mRNA levels in the hypothalamus and brainstem of STZ-induced diabetic rats were significantly reduced in comparison with those of control rats. PrRP mRNA levels in the diabetic rats were reversed by both insulin and leptin. PrRP mRNA levels in the fa/ fa diabetic rats were significantly reduced in comparison with those of Fa/ ? rats. PrRP mRNA levels in the fa/ fa diabetic rats were significantly increased by insulin-treatment, but did not reach control levels in the Fa/ ? rats. We also investigated the effect of restraint stress on PrRP mRNA levels in STZ-induced diabetic rats. The PrRP mRNA levels in the control and the STZ-induced diabetic rats increased significantly after restraint stress. The diabetic condition and insulin-treatment may affect the regulation of PrRP gene expression via leptin and other factors, such as plasma glucose level. The diabetic condition may not impair the role of PrRP as a stress mediator.

Expression of brain prolactin releasing peptide (PrRP) changes in the estrous cycle of female rats

Prolactin releasing peptide (PrRP) is a neuropeptide with 31 or 20 amino acid residues and regarded as a potent and specific stimulator of pituitary prolactin. PrRP immunoreactive (PrRP-ir) neurons and mRNA are found in medulla oblongata and hypothalamus and the fibers containing PrRP are widely distributed in rat brains. Therefore, it is postulated that PrRP might act as a neurohormone or a neurotransmitter as well as a neuromodulator in the brain. In the present study, we probed the expression of brain PrRP in the estrous cycle of female rats and the relationship between brain PrRP and GnRH. Female rats were divided into four groups: the diestrus, the proestrus, the estrus and the metaestrus, which were identified by the vaginal cytological examination. Immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescent double labeling histochemistry combining confocal laser scanning microscope (CLSM) were used. The results showed that PrRP immunoreactive neurons in nucleus of solitary tract (NTS) and ventrolateral reticular nucleus (VLRN) in the proestrus were less than those in the diestrus, the estrus and the metaestrus. Similarly, the relative optical density of PrRP-ir fibers of the bed nucleus of stria terminalis (BST) in the proestrus was decreased compared with those in other three groups. However, the brain PrRPmRNA level was higher in the proestrus and estrus than those in the metaestrus and diestrus. We also observed the co-localization of GPR10-immunoreactive (GPR10-ir) and GnRH-immunoreactive (GnRH-ir) neurons in hypothalamic medial preoptic area (MPO). The present results provide morphological evidences that PrRP in the female rat brains might participate in the regulation of the rat estrous cycle at least in a direct way.

Electroacupuncture stimulates the expression of prolactin-releasing peptide (PrRP) in the medulla oblongata of ovariectomized rats

Electroacupuncture (EA) in reproductive medicine has become established in Western medicine as a therapy over the last decade. EA performs a variety of neuromodulatory functions in the central nervous system (CNS). Prolactin-releasing peptide (PrRP) is a neuropeptide identified as an endogenous ligand for the orphan G protein-coupled receptor hGR3. PrRP can affect the function of hypothalamus-pituitary-ovary axis (HPOA) and hypothalamus-pituitary-adrenal axis (HPAA). The present study was undertaken to characterize the effect of EA on the expression of PrRP in the medulla oblongata in ovariectomized (OVX) rats by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). In addition, estrogen (E 2) levels were detected by radioimmunoassay (RIA). The results suggest that EA significantly increase the blood level of E 2 and the expression of PrRP in the medulla oblongata of OVX rats. The number of PrRP immunoreactive (ir) neurons was higher in the group ovariectomized with EA than that in the OVX group. The numbers of PrRP-ir neurons in intact (INT) and intact with EA (INT + EA) were not significantly different between the two groups. The expression of PrRP mRNA was increased in the OVX + EA group than that in the OVX group. These results suggest that the mechanism that EA improved reproductive disorders induced by ovariectomy in rats is related to the modulation of the blood E 2 level and the expression of PrRP in the medulla oblongata.

Prolactin-releasing peptide, food intake, and hydromineral balance in goldfish

A potential role for prolactin-releasing peptide (PrRP) in appetite regulation and hydromineral balance in goldfish was examined. PrRP was found to be expressed in discrete regions of the goldfish brain, in particular, the hypothalamus. Intraperitoneal (IP) or intracerebroventricular administration of PrRP had dose-dependent effects to suppress food intake in goldfish. Hypothalamic PrRP mRNA expression significantly increased after feeding, as well as after 7 days of food deprivation. Refeeding fish after 7 days food deprivation did not result in a postprandial increase in PrRP mRNA expression. These data suggest an anorexigenic role for PrRP in the short term around a scheduled meal time, but not over the longer term. IP injection of PrRP significantly increased pituitary prolactin (PRL) mRNA levels, suggesting involvement in the regulation of lactotroph activity. Acclimating goldfish to an ion-poor environment decreased serum osmolality and increased PrRP and PRL mRNA levels, providing evidence for PrRP involvement in hydromineral balance through its actions on lactotrophs. Acclimation to ion-poor water diminished the anorexigenic properties of PrRP in goldfish, indicating that a role for PrRP in goldfish satiation is counterbalanced by alternate systemic needs (i.e., osmoregulatory). This was further supported by an ability to reinstate the anorexigenic actions of PrRP in fish acclimated to ion-poor water by feeding a salt-rich diet. These studies provide evidence that PrRP is involved in regulating appetite and hydromineral balance in fish, and that the degree of involvement in either process varies according to overall systemic needs in response to environmental conditions.

Prolactin-releasing peptide is a potent mediator of stress responses in the brain through the hypothalamic paraventricular nucleus

The effects of i.c.v. administration of prolactin-releasing peptide on neurons in the paraventricular nucleus of rats and plasma corticosterone levels were examined by measuring changes in Fos-like immunoreactivity, c- fos mRNA using in situ hybridization histochemistry, and plasma corticosterone using a specific radioimmunoassay. Approximately 80% of corticotropin-releasing hormone immunoreactive cells exhibited Fos-like immunoreactivity in the parvocellular division of the paraventricular nucleus 90 min after i.c.v. administration of prolactin-releasing peptide. The greatest induction of the c-fos mRNA expression in the paraventricular nucleus was observed 30 min after administration of prolactin-releasing peptide, and occurred in a dose-related manner. Plasma corticosterone levels were also significantly increased 30 min after administration of prolactin-releasing peptide. Next, the effects of restraint stress, nociceptive stimulus and acute inflammatory stress on the expression of the prolactin-releasing peptide mRNA in the dorsomedial hypothalamic nucleus, nucleus of the solitary tract and ventrolateral medulla were examined using in situ hybridization histochemistry for prolactin-releasing peptide mRNA. Restraint stress and acute inflammatory stress upregulated the prolactin-releasing peptide mRNA expression in the nucleus of the solitary tract and ventrolateral medulla. Nociceptive stimulus upregulated the prolactin-releasing peptide mRNA expression in the ventrolateral medulla. Finally, we observed that pretreatment (i.c.v. administration) with an anti-prolactin-releasing peptide antibody significantly attenuated nociceptive stimulus-induced c- fos mRNA expression in the paraventricular nucleus. These results suggest that prolactin-releasing peptide is a potent and important mediator of the stress response in the brain through the hypothalamic paraventricular nucleus.

Expression of prolactin-releasing peptide and prolactin in the euryhaline mudskippers (Periophthalmus modestus): prolactin-releasing peptide as a primary regulator of prolactin

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression is unclear even in mammals. To study the possible presence of PrRP-PRL axes not only in the brain-pituitary but also in peripheral organs, the expression patterns of PrRP, PRL and growth hormone (GH) were characterized in amphibious euryhaline mudskippers (Periophthalmus modestus). PrRP mRNA is abundantly expressed not only in the brain but also in the liver, gut and ovary, while less abundant expression was also detected in the skin and kidney. Corresponding to the distribution of PrRP mRNA, PRL mRNA was also detectable in these organs. During adaptation to different environments, the changes in mRNA levels of PrRP paralleled those in PRL in the brain-pituitary, liver and gut in an organ-specific manner. Brain PrRP mRNA and the pituitary PRL mRNA increased under freshwater and terrestrial conditions (P < 0.05); expression of PrRP and PRL in the gut of freshwater fish was higher (P < 0.05) than those in sea-water fish although there were no changes in fish kept out of water; no significant change was seen in the liver. Expressions of GH were not correlated with PrRP. In the gut, PrRP and PRL appear to be co-localized in the mucosal layer, especially in the mucous cells. Thus, PrRP may also be a local modulator of extrapituitary PRL expression and the PrRP-PRL axes in various organs may play an organ-specific role during environmental adaptation.

Identification of the prolactin-releasing peptide-producing cell in the rat adrenal gland

Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo–pituitary–adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland.

Neuropeptide ff, but not prolactin-releasing peptide, mRNA is differentially regulated in the hypothalamic and medullary neurons after salt loading

Hypothalamic paraventricular and supraoptic nuclei are involved in the body fluid homeostasis. Especially vasopressin peptide and mRNA levels are regulated by hypo- and hyperosmolar stimuli. Other neuropeptides such as dynorphin, galanin and neuropeptide FF are coregulated with vasopressin. In this study neuropeptide FF and another RFamide peptide, the prolactin-releasing peptide mRNA levels were studied by quantitative in situ hybridization after chronic salt loading, a laboratory model of chronic dehydration. The neuropeptide FF mRNA expressing cells virtually disappeared from the hypothalamic supraoptic and paraventricular nuclei after salt loading, suggesting that hyperosmolar stress downregulated the NPFF gene transcription. The neuropeptide FF mRNA signal levels were returned to control levels after the rehydration period of 7 days. No changes were observed in those medullary nuclei that express neuropeptide FF mRNA. No significant changes were observed in the hypothalamic or medullary prolactin-releasing peptide mRNA levels. Neuropeptide FF mRNA is drastically downregulated in the hypothalamic magnocellular neurons after salt loading. Other neuropeptides studied in this model are concomitantly coregulated with vasopressin: i.e. their peptide levels are downregulated and mRNA levels are upregulated which is in contrast to neuropeptide FF regulation. It can thus be concluded that neuropeptide FF is not regulated through the vasopressin regulatory system but via an independent pathway. The detailed mechanisms underlying the downregulation of neuropeptide FF mRNA in neurons remain to be clarified.

Prolactin-releasing peptide affects pain, allodynia and autonomic reflexes through medullary mechanisms

Prolactin-releasing peptide (PrRP) and neuropeptide FF (NPFF) are RF-amide peptides expressed in brain areas involved in pain modulation. NPFF displays multiple effects on acute, inflammatory and neuropathic pain. The potential role of PrRP in pain was addressed by intrathecal and intracerebral injections of PrRP on pain-related responses in both neuropathic and normal rats. Particularly in the dorsal medulla, PrRP produced significant antinociception in normal rats and an antiallodynic effect in neuropathic rats. To understand the basis of PrRP-induced pain modulation, distributions of PrRP, PrRP receptor, and NPFF were compared in the rat central nervous system. PrRP and NPFF mRNA were expressed in different parts of the nucleus of the solitary tract. In the medulla, PrRP receptor mRNA expression was abundant only in area postrema. Of the peptides studied, only NPFF mRNA was found in the dorsal horn of the spinal cord and spinal nucleus of the trigeminal nerve. PrRP-immunoreactivity corresponded to the mRNA distribution. Even if the neuronal groups producing NPFF and PrRP were distinct, the fiber networks immunoreactive for PrRP and NPFF overlapped. The results show that PrRP modulates nociception due to supraspinal rather than spinal action, and that its antinociceptive mechanism differs from that previously characterized for NPFF.

Brainstem prolactin-releasing peptide neurons are sensitive to stress and lactation

Prolactin-releasing peptide (PrRP) was originally thought to participate in the control of adenohypophyseal prolactin secretion, but its predominant expression in a subset of medullary noradrenergic neurons is more in line with roles in interoceptive and/or somatosensory information processing. To better define functional contexts for this peptide system, immuno- and hybridization histochemical methods were used to monitor the capacity of PrRP neurons to display activational responses to lactation, suckling, acute footshock or hypotensive hemorrhage. PrRP mRNA signal was reduced in the medulla of lactating dams, relative to both male and diestrus female controls, with cell counts revealing 42% and 43% reductions in the number of positively hybridized cells in the nucleus of the solitary tract (NTS) and ventrolateral medulla, respectively. Lactating mothers killed after a 90 min suckling episode (following 4 h pup removal) failed to show induced Fos expression in identified medullary PrRP neurons, despite the fact that responsive neurons were detected in other aspects of the caudal NTS. By contrast, acute exposure to hypotensive (25%) hemorrhage or footshock each activated substantial complements of medullary neurons expressing PrRP mRNA. A substantially greater fraction of the total medullary PrRP population exhibited sensitivity to footshock than hemorrhage (71 versus 39%, respectively). These results suggest that medullary PrRP neurons are negatively regulated by (presumably hormonal) changes in lactation, and are not recruited to activation by suckling stimuli. These populations exhibit differential sensitivity to distinct acute stressors, and may participate in the modulation of adaptive neuroendocrine and autonomic responses to each.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman™ RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly ( P<0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly ( P<0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the codon reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin secretion.

Prolactin-releasing peptide in the ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo.

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P<0.01) increased prolactin concentrations in the culture medium but the response was not observed in every experiment and was only seen when pituitary glands were dispersed with collagenase rather than trypsin. PrRP was much less potent than TRH which caused a significant (P<0.01) two- to threefold increase in prolactin concentrations in every experiment. Intravenous (10 and 50 nmol) or intracerebroventricular (10 and 50 nmol) injection of PrRP had no significant effect on either plasma prolactin concentration or pulsatile LH secretion whereas intravenous injection of TRH (10 nmol) produced a highly significant (P<0.01) and more than sevenfold stimulation of plasma prolactin concentrations. In conclusion, these results suggest that PrRP is unlikely to be an important prolactin-releasing factor in this species.

Developmental expression of prolactin releasing peptide in the rat brain: localization of messenger ribonucleic acid and immunoreactive neurons

Prolactin releasing peptide (PrRP) was recently identified as the stimulator of prolactin release from the anterior pituitary. PrRP mRNA is expressed in the medulla oblongata and the hypothalamus in the rat brain. The fibers containing PrRP are widely distributed in the brain, therefore, it was postulated that PrRP may act as a neurotransmitter or neuromodulator as well as an endocrine substance. To clarify the developmental changes in the expression of PrRP during brain development, we examined PrRP in rat fetuses and neonates using in situ hybridization and immunohistochemistry. The PrRP mRNA was expressed in the nucleus of the solitary tract (NTS) at embryonic day 18 (E18) and in the ventral and lateral reticular nucleus (VLRN) of the caudal medulla oblongata at E20. The PrRP mRNA in the hypothalamus was first expressed at postnatal day 13 (P13). Reverse transcription–polymerase chain reaction analysis (RT–PCR) for PrRP revealed that PCR product, a 268 bp band, was detected from either E18 in the medulla or P13 in the hypothalamus. Immunodetection with monoclonal antibody against prepro-PrRP revealed intensive staining of cells in the NTS at E18, in the VLRN at E20 or in the dorsomedial hypothalamus at P13. Immunohistochemistry using monoclonal antibody against mature PrRP at P6 showed PrRP fibers to be distributed in the paraventricular hypothalamic nucleus, periventricular hypothalamic nucleus, medial preoptic area, basolateral amygdaloid nucleus, dorsomedial hypothalamus, ventromedial hypothalamus, periventricular nucleus of the thalamus and bed nucleus of the stria terminalis as previously shown in the adult rat. PrRP fibers were also found in the optic chiasm, dorsal endopiriform nucleus, cingulum, intermediate reticular nucleus, and caudal ventrolateral reticular nucleus at P6 and P9. However, PrRP fibers were never found in the above regions in the adult animal. These findings suggest that PrRP fibers originating in the medulla oblongata have been widely distributed in the rat brain during the early postnatal day and PrRP may play various roles in the brain development.