Anatomical and functional characterization of clock gene expression in neuroendocrine dopaminergic neurons

Abstract

Oscillations of gene expression and physiological activity in suprachiasmatic nucleus (SCN) neurons result from autoregulatory feedback loops of circadian clock gene transcription factors. In the present experiment, we have determined the pattern of PERIOD1 (PER1), PERIOD2 (PER2), and CLOCK expression within neuroendocrine dopaminergic (DAergic) neurons (NDNs) of ovariectomized (OVX) rats. We have also determined the effects of per1, per2, and clock mRNA knockdown in the SCN with antisense deoxyoligonucleotides (AS-ODN) on DA release from NDNs. Diurnal rhythms of PER1 and PER2 expression in tuberoinfundibular DAergic (TIDA) and periventricular hypophyseal DAergic (PHDA) neurons, peaked at circadian time (CT)18 and CT12, respectively. Rhythms of PER1 expression in tuberhypophyseal neuroendocrine DAergic (THDA) neurons were undetectable. Rhythms of PER2 expression were found in all three populations of NDNs, with greater levels of PER2 expression between CT6 and CT12. AS-ODN injections differentially affected DA turnover in the axon terminals of the median eminence (ME), neural lobe (NL) and intermediate lobe (IL) of the pituitary gland, resulting in a significant decrease in DA release in the early subjective night in the ME (TIDA), a significant increase in DA release at the beginning of the day in the IL (PHDA), and no effect in the NL (THDA). AS-ODN-treatment induced a rhythm of DA concentration in the anterior lobe, with greater DA levels in the middle of the day. These data suggest that clock gene expression, particularly PER1 and PER2, within NDNs may act to modulate diurnal rhythms of DA release from NDNs in the OVX rat.prolactin; dopamine; suprachiasmatic nucleus; hypothalamus DOPAMINE (DA) of hypothalamic origin exerts tonic inhibitory control over prolactin (PRL) secretion (for review see Ref. 17). DA is released directly into hypothalamo-hypophyseal portal blood from three populations of neuroendocrine DAergic neurons (NDNs) (5, 17). These subpopulations include the tuberoinfundibular DAergic (TIDA) and tuberohypophyseal neuroendocrine DAergic (THDA,A12) neurons with cell bodies in the arcuate nucleus (ARN) and periventricular hypophyseal DAergic (PHDA,A14) neurons with cell bodies in the periventricular region (5, 17). THDA and PHDA axons traverse the pituitary stalk and terminate on fenestrated short portal vessels within the neural (NL) and intermediate (IL) lobes of the pituitary gland (21). TIDA axons terminate on fenestrated capillary beds within the external zone of the median eminence (ME) that drain into long portal vessels, transporting DA to the anterior lobe (AL) of the pituitary gland (8, 10). TIDA neurons are well established as the primary PRL inhibitory neurons, although growing importance has been assigned to both THDA and PHDA neurons in the regulation of PRL secretion (13, 21, 43, 52, 61).We have observed diurnal rhythms of DA turnover in the nerve terminals of TIDA, THDA, and PHDA neurons in the ovariectomized (OVX) rat (57, 58). Whereas both TIDA and PHDA neurons display circadian ...

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

“…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 ( Table 1). As demonstrated previously in our laboratory, this cocktail leads to a knockdown of PER1, PER2, and CLOCK expression in the SCN by Ͼ60% within 6 h of injection at a dose of 3 nmol (2.5 mg/ml, 800 nl injection) (59). In the work reported here, we further confirmed the efficiency of the AS-ODN treatment by demonstrating that the number of CLOCK-positive neurons in the SCN at ZT 2 (0800) and ZT 10 (1600) was lower than that produced by RS-ODN injection.…”

“…In a previous study (59), we have shown that the expression of clock genes in the SCN is reduced by 60% after AS-ODN cocktail injection, so the remaining clock protein could still be present in the SCN controlling the LC neuronal activity. Its presence might explain why the abolition of LC circadian rhythm has only been seen in DD, when both clock-gene circadian inputs from the SCN and the light cue were missing.…”

“…Consequently, these clock genes seem to be essential for driving of circadian rhythms in mammals by the SCN neurons. We have shown (59) that an injection of an antisense deoxyoligonucleotide (AS-ODN) cocktail for clock genes (Per1, Per2, and Clock) into the SCN decreases by 60% the expression of the clock genes in these neurons and in turn compromises the circadian release of corticosterone and drinking behavior in OVX rats. Here we describe a specific effect, revealed by this approach, of SCN neuronal circadian rhythm on the mechanism timing PRL surges.…”

Pregnancy-induced adaptations of the central circadian clock and maternal glucocorticoids

“…Similarly, while Crh expression showed a trend for overall time-of-day variation, this did not match the conventional cosinor pattern observed for the clock genes. These observations may appear surprising given that ablation studies (both anatomical and transgenic) have highlighted the importance of the SCN in the regulation of the diurnal secretion of corticosterone (Moore & Eichler 1972, Sellix et al 2006. Interestingly, while a previous study also observed arrhythmic hypothalamic Crh mRNA expression in the mouse, rhythmic expression of Crh heteronuclear (hn) RNA was detected, indicative of circadian variation in Crh at this pre-processing stage (Watts et al 2004).…”

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

“…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.…”