The Mammalian Biological Clock: The Heterogeneous Nature of the Suprachiasmatic Nucleus

In organisms ranging from cyanobacteria to humans, physiological and behavioral rhythms usually synchronize to periodically recurring environmental events (e.g. the light-dark cycle). These daily rhythms are called circadian rhythms. When all environmental time cues are held constant, many biological rhythms persist with near 24-hour periods, driven by an endogenous time keeping system in charge of the entrainment, regulation, and output of rhythms.

In mammals, the dominant circadian pacemaker is the suprachiasmatic nucleus (SCN) of the hypothalamus. The two nuclei of the SCN are located on each side of the third ventricle, just dorsal to the optic chiasm. When isolated in vitro, the SCN is capable of maintaining its oscillations for a long time. The oscillations of the SCN have been monitored using a real time bioluminescent reporter of gene activity. These bioluminescence studies have shown that different compartments of the SCN peak at different times of the day, suggesting that the SCN is not a homogenous structure but instead a heterogeneous clock composed of different types of cells with varying biological functions.

Two distinct sections of the SCN - the ventrolateral (VL) and the dorsomedial (DM) -peak at different times. Cells containing the neuropeptide arginine vasopressin (AVP) are found predominantly in the DM piece, and those with vasoactive intestinal polypeptide (VIP) are in the VL piece. Due to this division, we hypothesized that AVP and VIP perform distinct tasks within the circadian timing system. To study the individual functions of these neuropeptides, it is necessary to isolate them.

To separate the dorsomedial and ventrolateral sections, we sacrificed one to two months old Per1-luc rats (transgenic rats carrying the mPer1 - promoter-driving luciferase expression), cut their brains into 150 micron sections, and collected four sections that contained the SCN. These sections were divided by a V-cut to separate the DM and VL pieces, yielding a total of 8 SCN explants per brain, and cultured at 37 degrees Celsius under a photomultiplier tube five to seven days. The cultures were then treated with Colchicine overnight to reduce the movement of neuropeptides into the axon and then double stained for AVP and VIP using immunocytochemistry. The AVP and VIP cells were counted to determine the effectiveness of the cuts and the trends presented by the neuropeptides.

The dorsal-ventral distribution of AVP and VIP cells in vivo was maintained in vitro, with AVP concentrated in the DM and VIP concentrated in the VL. Rostral cuts held the majority of the SCN in the ventrolateral piece and caudal cuts isolated the majority of the SCN in the dorsomedial section. Cuts in the medial sections of the SCN most effectively separated DM from VL. AVP abundance varies little from rostral to caudal, while VIP is most abundant in the medial section and tapers at the rostral and caudal ends. Overall, the SCN contains about twice as many AVP cells as VIP cells.

Thus, it is possible to crudely divide the SCN into two neuropeptidergic parts. We will next correlate the proportions of AVP and VIP cells with the period and amplitude of Per1-luc oscillations to better understand the distinct circadian functions of these sub compartments in the SCN.