Central clock regulates the cervically stimulated prolactin surges by modulation of dopamine and vasoactive intestinal polypeptide release in ovariectomized rats.

BACKGROUND/AIMS: Cervical stimulation induces a circadian rhythm of prolactin secretion and antiphase dopamine release. The suprachiasmatic nucleus (SCN) controls this rhythm, and we propose that it does so through clock gene expression within the SCN. METHODS: To test this hypothesis, serial blood samples were taken from animals injected with an antisense deoxyoligonucleotide cocktail for clock genes (generated against the 5' transcription start site and 3' cap site of per1, per2, and clock mRNA) or with a random-sequence deoxyoligonucleotide in the SCN. To determine whether disruption of clock genes in the SCN compromises the neural mechanism controlling prolactin secretion, we sacrificed another group of rats (under the same treatments) at 12.00 or 17.00 h. Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured using HPLC/electrochemical detection in the median eminence as well as the intermediate and the neural lobe of the pituitary gland, and the DOPAC:dopamine ratio was used as an index of dopamine activity. Vasoactive intestinal polypeptide (VIP) content was determined in tissue punches of the SCN and paraventricular nucleus (PVN), an SCN efferent. RESULTS: Treatment with clock gene antisense deoxyoligonucleotide cocktail abolished both the diurnal and nocturnal prolactin surges induced by cervical stimulation. This treatment abolished the antiphase relationship established by cervical stimulation between dopamine neuronal activity and prolactin secretion. Also, VIP content increased in the SCN and decreased in the PVN. CONCLUSION: These results suggest that the SCN clock determines the circadian rhythm of prolactin secretion in cervically stimulated rats by regulating dopamine neuronal activity and VIP inputs to the PVN.

Vasoactive intestinal polypeptide modulates the estradiol-induced prolactin surge by entraining oxytocin neuronal activity.

In female rats, estradiol is responsible for a circadian secretory prolactin (PRL) pattern which requires an intact suprachiasmatic nucleus (SCN). SCN outputs involved in this secretory profile remain elusive. Because oxytocin has been proposed to stimulate PRL secretion, we investigated whether the projections of vasoactive intestinal polypeptide (VIP) from the SCN to neurons producing oxytocin in the paraventricular and periventricular nuclei (PVN and PeVN, respectively) are responsible for timing PRL surges induced by estradiol (E(2)). E(2)-treated ovariectomized rats received an injection of antisense or random-sequence oligodeoxynucleotide for VIP in the SCN and blood samples were taken for PRL measurements by radioimmunoassay. Additionally, the percentage of oxytocin-positive neurons immunoreactive to FOS-related antigens was determined in the PVN and PeVN, as an index of neuronal activity. In the PVN, oxytocinergic neuronal activity increased in the early evening regardless of E(2) treatment, whereas E(2) induced an increase of activity in the PeVN. VIP antisense attenuated this increase observed in both neuronal populations. Additionally, in the PeVN, VIP antisense advanced this increase by 2 h (from 19:00 h to 17:00 h). This same effect was observed in the PRL surge that occurred at 17:00 h in the VIP antisense injected animals. Thus, the SCN influences the precise timing of the E(2)-induced PRL surge via VIP projections to oxytocinergic neurons of the PVN and PeVN.

Antagonism of oxytocin prevents suckling- and estradiol-induced, but not progesterone-induced, secretion of prolactin.

In female rats, estradiol (E(2)) and suckling induce prolactin (PRL) secretion. This involves inhibition of hypothalamic dopaminergic tone and stimulation by a PRL-releasing hormone, possibly oxytocin (OT). Infusing an OT antagonist (OTA) i.v., we evaluated the role of OT on suckling- and E(2)-induced PRL secretion. Three days after parturition at 0900 h, lactating dams were fitted with 24-h osmotic minipumps filled with saline or OTA. On d 5 of lactation, pups were separated from their dams for 6 h. Immediately or 20 min after the resumption of suckling, dam trunk blood was collected. Also, ovariectomized (OVX) rats were treated with E(2) (OVE) and OTA at 1000 h on d 1. Blood samples were obtained from 1300 to 2100 h on d 2 for PRL measurements. Additionally, OVX rats were evaluated on d 2 after receiving progesterone (P(4)). OTA blocked suckling and E(2)-induced release of PRL but not that induced by E(2)+P(4). Pups from treated dams failed to gain weight when allowed to nurse for 20 min on d 5 but gained more than 7 g when nursed on d 7 of lactation, indicating that the OTA was active 48 h later. Western blot analysis showed that E(2) treatment increased OT receptors in the anterior pituitary when compared with OVX animals. No further increase was observed in response to the P(4), suggesting that the enhancing effect of P(4) on E(2)-induced PRL release may act through mechanisms independent of OT. These data demonstrate the role of OT in the control of suckling and steroid-induced PRL secretion.

Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats.

The nature of the circadian signal from the suprachiasmatic nucleus (SCN) required for prolactin (PRL) surges is unknown. Because the SCN neuronal circadian rhythm is determined by a feedback loop of Period (Per) 1, Per2, and circadian locomotor output cycles kaput (Clock) gene expressions, we investigated the effect of SCN rhythmicity on PRL surges by disrupting this loop. Because lesion of the locus coeruleus (LC) abolishes PRL surges and these neurons receive SCN projections, we investigated the role of SCN rhythmicity in the LC neuronal circadian rhythm as a possible component of the circadian mechanism regulating PRL surges. Cycling rats on proestrous day and estradiol-treated ovariectomized rats received injections of antisense or random-sequence deoxyoligonucleotide cocktails for clock genes (Per1, Per2, and Clock) in the SCN, and blood samples were taken for PRL measurements. The percentage of tyrosine hydroxylase-positive neurons immunoreactive to Fos-related antigen (FRA) was determined in ovariectomized rats submitted to the cocktail injections and in a 12:12-h light:dark (LD) or constant dark (DD) environment. The antisense cocktail abolished both the proestrous and the estradiol-induced PRL surges observed in the afternoon and the increase of FRA expression in the LC neurons at Zeitgeber time 14 in LD and at circadian time 14 in DD. Because SCN afferents and efferents were probably preserved, the SCN rhythmicity is essential for the magnitude of daily PRL surges in female rats as well as for LC neuronal circadian rhythm. SCN neurons therefore determine PRL secretory surges, possibly by modulating LC circadian neuronal activity.