Suprachiasmatic Nucleus Interaction with the Arcuate Nucleus; Essential for Organizing Physiological Rhythms

Arrhythmic locomotor activity and temperature observed in DD following retrochiasmatic knife cuts (RC cut). A, Representative actograms of a SHAM-operated animal (left) and an RC-cut animal (right), in LD and DD conditions (shading indicates lights off). Following RC cuts, animals recovered in LD and showed normal diurnal rhythmicity, subsequent DD conditions rendered all RC-cut animals arrhythmic. B, χ² periodogram analysis of LD and DD periods of SHAM (left) and RC cut (right) animals. The slanted line in the periodogram indicates p = 0.01. C, For RC-cut animals temperature was arrhythmic in DD. Note the strong temperature decrease in RC-cut animals between ZT0 and ZT2 in reaction to light during LD. The graph is a double plot of temperature data in SHAM and RC-cut animals in LD (left) and DD (right). Each value represents seven animals (mean ± SEM). Only where a significant rhythm in expression was detected, data were fitted with a sine wave function (p < 0.05, F test).

In RC-cut animals corticosterone rhythm was lost while melatonin remained intact similar to SCN rhythmicity. Rhythm of ARC Per1 expression was attenuated, demonstrating SCN-ARC desynchronization. A, Corticosterone levels showed a significant peak at ZT12 in SHAM but not RC-cut or SCN_XARC^Cut animals, indicating loss of rhythmicity. Melatonin maintained its rhythm in LD and DD in both SHAM and experimental animals. Data represents N = 5-7, and N = 4-6 for the SCN_XARC^Cut group (mean ± SEM). *p < 0.05 (SHAM), +p < 0.05 (Experimental), ANOVA analysis. B, Representative photomicrographs of SCN Per1 mRNA expression (above) and ARC Per1-ir (below) in SHAM and RC-cut animals. Scale bar, 90 μm. 3V, third ventricle; ON, optic nerve; ME, median eminence. C, Per1 mRNA analysis of the SCN demonstrates significant circadian rhythmicity in RC-cut (p < 0.05) and in SHAM animals (p < 0.01). The ARC failed to show a significant rhythm in RC-cut animals (p > 0.05) as compared with SHAM. The higher point at CT6 suggests a phase advance; however, an F test failed to show a significant circadian rhythm. Data represents N = 3-4 with a double-plot of CT0 at CT24. Only where a significant rhythm in expression was detected, data were fitted with a sine wave function (p < 0.05, F test).

Peripheral liver clock genes in experimental animals show contrasting changes in rhythmicity as compared with SHAM animals. Per1 gene expression in experimental animals showed no significant changes in rhythmicity (p < 0.01) as compared with SHAM (p < 0.05), contrary to Bmal1 (p = 0.268) and Cry1 (p = 0.137) failing to demonstrate rhythmicity as compared with SHAM animals (p < 0.05). Each value represents three to four animals (mean ± SEM). CT24 is a double-plot of CT0. Solid squares indicate RC-cut animals and open circles SHAM animals. Only where a significant rhythm in expression was detected, data were fitted with a sine wave function (p < 0.05, F test).

c-Fos staining of the SCN and ARC showing significant changes in Fos expression following glucose intake in SHAM animals, whereas experimental animals show no significant alterations in Fos expression. A, Representative photomicrographs of SCN c-Fos expression in SHAM en RC-cut animals in ad libitum, fasting or fasting and glucose intake. B, ARC c-Fos expression in SHAM en RC-cut animals during above stated conditions. C, Bar charts of c-Fos quantification in the SCN and ARC of SHAM en RC-cut animals. All animals were killed at ZT4 following ad libitum, 48 h of fasting or fasting followed by 5-ml 3% glucose intake 2 h before killing. Each value in C represents three to four animals (mean ± SEM). Scale bar, 70 μm; *significant between group difference; +significant within group difference. */+p < 0.05, **/++p < 0.01, ***/+++p < 0.001 (ANOVA analysis).

RC cuts do not affect main SCN output other than the ARC. Clear VIP innervation of the DMH (A) and PVN (B) in RC-cut animals. C, Also VIP staining to the SPZ and retrochiasmatic area dorsal to the knife cut is still intact in RC-cut animals. D, Staining of VIP afferents in the ARC, clearly visible in SHAM animals, disappear following RC cut (E). F, Likewise, NPY SCN staining, shown to be predominantly IGL derived, is not damaged by RC cuts. Pictures A–C, E, F are from RC animals, only D shows staining in an intact animal. Scale bar, 130 μm (A and F), 175 μm (B), 250 μm (C), 70 μm (D and E).

ARC-specific AgRP staining shows intact innervation of (A) the PVN, (B) MnPO, (C) AVPV, and (D) DMH, not affected by the knife cuts. Scale bar, 175 μm (A–C) and 215 μm (D). All photomicrographs are from RC-cut animals. 3V, third ventricle; ON, optic nerve.