Circadian Pattern of Melatonin MT1 and MT2 Receptor Localization in the Rat Suprachiasmatic Nucleus

Abstract

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker. The pineal hormone melatonin is involved in the regulation of circadian phase. As a part of the circadian system, its synthesis and secretion is under SCN control. On the other hand, melatonin feeds back on the SCN to regulate its function. Melatonin has two specific windows of time at which it regulates SCN function, namely dusk and dawn. It has been suggested that melatonin exerts its effect on the SCN during that specific window of time via one or both of its specific receptors, MT1 or MT2. The hypothesis that the density of these receptors varies across the circadian cycle was tested. Using immunohistochemistry with receptor-specific antibodies, the localization and distribution of melatonin receptors MT1 and MT2 was studied in the SCN at different Zeitgeber times (ZT): ZT 11-13 (dusk), 23-01 (dawn), 5-7 (mid-day), and 17-19 (midnight). Our results show that MT1 receptor density significantly increased at dusk relative to dawn and midnight (p<0.01 and p<0.001 respectively). Although MT1 receptors were widespread in the SCN and parts of the optic chiasm at dusk, they were restricted to the SCN during the mid-day period. MT2 receptors were not detected in the SCN. Thus, we find that melatonin receptor MT1 density and distribution varies with circadian time. This creates a time window during which melatonin can affect the operation of the SCN. We also find that melatonin regulates SCN function via MT1 receptors with a minimal role for MT2.

Keywords: MT1; MT2; SCN; circadian rhythm; immunohistochemistry; melatonin; melatonin receptors.

Fig. 1

Immunoblotting of rat hypothalamus (HT) with MT1 and MT2 receptors antibodies. A) Immunoblot of rat hypothalamic tissue with anti-MT1 antibody. B) Same as in A but with anti-MT2 antibody.

Fig. 2

MT1 receptors localization in the cortex and the hippocampus. A) low and C) high magnification pictures showing label of hippocampal neurons with anti-MT1 antibody. Scale bars are 80 and 20 µm respectively. B) and D) show labeling with anti-MT1 antibody in cortical neurons in low and high magnification respectively. Scale bars are 80 and 20 µm respectively.

Fig. 3

MT1 receptors localization in rat SCN across the circadian cycle. Labeling with MT1 antibody in the SCN is shown at A) midday ZT 5–7, B) dusk ZT 11–13, C) midnight ZT 23–01, and D) dawn ZT 17–19, respectively. 3V indicates the third ventricle, OC is the optic chiasm. Scale bar in each picture is 80 µm.

Fig. 4

Circadian rhythm of MT1 receptors density in the SCN. Normalized pixel density is plotted at different CTs. * and ** indicates that p value is less than 0.5 and less than 0.01 respectively, ns statistically insignificant.

Fig. 5

MAP2 staining in the SCN and optic chiasm. A) Labeling of the dendrites within the SCN is shown with red color. The arrow points to a neuron within the optic chiasm. B) An overlay of both DAPI labeling the nuclei (blue) and MAP2 labeling the dendrites (Red). C) Same as in B but in a different section; notice a more distant neuron within the SCN (arrows). 3V indicates the third ventricle. OC is the optic chiasm. Scale bar is 20 µm.

Fig. 6

MT2 receptors immunostaining in the SCN. 3V indicates the third ventricle, OC is the optic chiasm. Scale bar is 80 µm.

Fig. 7

MT2 receptor immunolabeling in the paraventricular nucleus. 3V is the third ventricle. Scale bar is 80 µm.

Fig. 8

The possible feedback of melatonin on the SCN. RHT is the retinohypothalamic tract, GHT is the geniculohypothalamic tract, PVN is the paraventricular nucleus, and SCG is the superior cervical ganglia. Plus sign (+) indicates stimulation while negative sign (-) indicates inhibition.