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

Abstract

Poletini MO, McKee DT, Kennett JE, Doster J, Freeman ME. Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats. Am J Physiol Endocrinol Metab 293: E1325-E1334, 2007. First published August 28, 2007; doi:10.1152/ajpendo.00341.2007.-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 estradiolinduced 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. norepinephrine; PERIOD1; PERIOD2 IN FEMALE RATS, PROLACTIN (PRL) secretion has been proposed to result from synchronization among a circadian neural signal from the suprachiasmatic nucleus (SCN), both inhibitory and stimulatory neurotransmitter actions on the pituitary gland, and modulating effects of ovarian steroids (19). As a consequence of this synchronization, female rats display cyclical increases of PRL secretion characterized by two surges: the preovulatory surge of proestrous afternoon (64) and the secondary surge of estrous afternoon (12,70).The preovulatory surge of PRL depends on the levels of plasma ovarian steroids, because the increase of estradiol titers induces (45) and the increase of progesterone amplifies (76) this surge. Estradiol treatment in ovariectomized (OVX) rats induces daily PRL surges that occur at roughly the same time of day as the proestrous surge (13). Shifting of the light phase results in a coincident shift of the proestrous surge (10), and the estradiol-induced PRL surges of OVX rats free run in constant light (52), but the m...

Central Clock Regulates the Cervically Stimulated Prolactin Surges by Modulation of Dopamine and Vasoactive Intestinal Polypeptide Release in Ovariectomized Rats

“…In the present study, an AS-ODN injection that decreased the expression of per1 , per2, and clock [14] within neurons of the SCN abolished both PRL surges in cervically stimulated OVX rats. We have previously demonstrated this same effect on the PRL secretory surge induced by estradiol in OVX and proestrus rats [13]. Taken together, these data link PRL secretion observed in different stages of the reproductive cycle to the molecular clock mechanism in the SCN.…”

“…Control animals were injected with RS-ODNs, which had the same nucleotide content (% AGCT) as the AS-ODN but were not complementary to clock gene mRNA sequences. As demonstrated previously in our laboratory, this treatment leads to a knockdown of PER1, PER2, and CLOCK expression in the SCN by more than 60% within 6 h of injection and to a decrease in the number of CLOCK-positive neurons in the SCN at 08.00 and 16.00 h [13,14]. We have also previously reported that this treatment disrupts the circadian drinking behavior of OVX rats [14].…”

“…At 18.00 h (Zeitgeber, ZT, 12) animals received bilateral injections into the SCN of an antisense deoxyoligonucleotide cocktail (AS-ODN) of clock , per1 and per2 genes or of random-sequence deoxyoligonucleotides (RS-ODN). We have previously shown that this treatment disrupts circadian drinking behavior for 72 h [13,14], suggesting that it compromises the feedback loop of clock gene expression, and consequently SCN rhythmic function for this period of time. On the following day, blood samples were taken at 13.00 h and every 2 h afterward until 05.00 h for measurement of plasma PRL by radioimmunoassay (RIA).…”

“…Disruption of the molecular clock mechanism leads to impairment of several circadian rhythms, such as locomotor behavior and the secretion of corticosterone [8,9,10,11,12]. We have shown that an injection of deoxyoligonucleotide antisense sequence of rPer 1, rPer 2, and rclock (which promotes an acute and transitory knockdown of these genes and their respective protein expression within the SCN) disrupts the preovulatory surge of PRL as well as the PRL surge induced by estradiol in ovariectomized (OVX) rats [13]. This treatment also disrupts the circadian rhythm of drinking behavior, the circadian release of corticosterone, and CLOCK expression [13,14].…”

“…We have shown that an injection of deoxyoligonucleotide antisense sequence of rPer 1, rPer 2, and rclock (which promotes an acute and transitory knockdown of these genes and their respective protein expression within the SCN) disrupts the preovulatory surge of PRL as well as the PRL surge induced by estradiol in ovariectomized (OVX) rats [13]. This treatment also disrupts the circadian rhythm of drinking behavior, the circadian release of corticosterone, and CLOCK expression [13,14]. A great deal of evidence indicates that the SCN controls PRL secretion during mating.…”