Control of rat hypothalamic pro-opiomelanocortin neurons by a circadian clock that is entrained by the daily light-off signal.

Previous studies have clearly demonstrated that the immediate-early gene, c-fos can regulate, through its protein product Fos, the expression of the pro-opiomelanocortin gene. In the present study, immunohistochemistry for Fos and beta-endorphin was used to assess the basal activity of hypothalamic pro-opiomelanocortin-producing neurons throughout a 12 h light/12 h dark cycle. Here, we showed that Fos is undetectable in most beta-endorphin neurons from late morning until 30 min after light offset in the evening, whereas Fos is spontaneously expressed in these neurons after 1 h following dark onset. The number of beta-endorphin neurons expressing Fos increases continuously during the first half of the dark phase, is maximal at the middle of this phase and decreases through late night and early morning, reaching a nadir 2-3 h after light onset. Acute shifts of lighting parameters allowed us to demonstrate that the light-off signal per se is neither sufficient nor necessary for Fos expression in beta-endorphin neurons. However, when recurrent, this signal is able to entrain Fos expression after a period of adaptation to the new light/dark schedule. Moreover, an expression of Fos in beta-endorphin neurons persists during subjective night in rat exposed to constant light or constant dark for two to three days. Thus, the occurrence of the daily rhythmic increase in the expression of Fos protein in hypothalamic pro-opiomelanocortin neurons exclusively at (subjective) night suggests that these neurons are, most likely, controlled by a (circadian) nocturnal oscillator. Our data also reveal an interesting property of this oscillator: its entrainment by the daily light-to-dark transition signal.

The daily pattern of Fos synthesis by hypothalamic pro-opiomelanocortin neurons is unaffected by adrenalectomy in the rat.

At the onset of dark, a large population of rat mediobasal hypothalamic (MBH) pro-opiomelanocortin (POMC) neurons starts spontaneously expressing Fos-immunoreactivity (Fos-IR). Here we studied the effect of adrenalectomy upon this expression since circulating corticosteroids, which increase in the rat with the onset of behavioural wakening, are thought to modulate the basal expression of MBH POMC mRNA. Hence, groups of intact, adrenalectomised and sham-operated rats were sacrificed at times when Fos synthesis by POMC neurons is known to show either nadir (at light-offset) or peak (6 h after light-offset) values. Brains were processed for Fos- and/or POMC immunohistochemistry. This allowed us to show that, in all experimental groups, Fos-IR is hardly expressed in MBH POMC neurons at the onset of dark, whereas it is strongly induced 6 h later. We concluded that such an induction is not triggered through the known evening rise of plasma corticosteroid levels.

Arachidonyl ethanolamide (anandamide) activates the parvocellular part of hypothalamic paraventricular nucleus.

Arachidonyl ethanolamide, anandamide (ANA) was administered to male rats via a single i.p. injection at a dose of 0.02 mg/kg. In an parallel experiment ANA injection was preceded by the injection of SR 141716 (1.0 mg/kg), a selective and potent cannabinoid receptor antagonist. We observed using FOS protein immunocytochemistry that the parvocellular part of hypothalamic paraventricular nucleus (PVN) was activated as soon as 45 min. after ANA injection, i.e. the PVN showed an increased FOS immunoreactivity (FOSir). The peak level of FOSir was observed 90 min, after treatment. Meanwhile serum ACTH and corticosterone levels, as measured by radioimmunoassay, also significantly increased. 180 min. following drug injection both FOSir and serum hormone levels and returned to normal. SR 141716 did not antagonize these effects of ANA. We postulate that the locus of action of ANA the activation of the hypothalamo-pituitary-adrenal (HPA) axis is the parvocellular part of PVN. This activation may occur via a possible central cannabinoid receptor for which SR 141716 is not an effective antagonist. The rapid central response and activation of the HPA axis further support the view that ANA may be a central neurotransmitter or neuromodulator.

Adrenalectomy does not affect the nocturnal peak of Fos expression within hypothalamic pro-opiomelanocortin neurons.

Circulating glucocorticoid (GC) levels are thought to modulate the basal activity of pro-opiomelanocortin (POMC) neurons within the mediobasal hypothalamus (MBH) of the male rat. In a recent study we demonstrated that Fos-immunoreactivity (Fos-IR) was spontaneously induced throughout the dark phase of the light/dark cycle within a large population of these MBH neurons. Here, we have investigated the effect of adrenalectomy on the nocturnal expression of Fos protein within POMC neurons. To this aim, groups of intact (IN), adrenalectomized (ADX) and sham-operated (sham) rats were killed 7 days after surgery (or no surgery) at times when Fos-IR is known to show either nadir (at light offset) or peak (6 h after light offset) values within MBH POMC neurons. Brains were processed for Fos- and/or POMC-immunohistochemistry. The results showed that, at both times studied, 7-day adrenalectomy did not affect the number of POMC/Fos double-stained neurons within the MBH. The rostro-caudal pattern of distribution of such labeled neurons throughout the MBH of ADX rats was also similar to that of IN or sham rats. The present data demonstrate that the nocturnal induction of Fos within MBH POMC neurons is not controlled via the nychtemeral rhythm of secretion of the adrenal gland. Furthermore, this study shows that basal levels of circulating GC do not alter the nocturnal peak of Fos synthesis within POMC neurons.