Period Determination

The circadian period imposed by the SCN mas­ter pacemaker is determined at the level of a single cell, as revealed by studies of mutant SCN neurons. Behavioral circadian rhythms of tau mutant hamsters have short pe­riods, and SCN neurons from those hamsters also have short periods. Behavioral circa­dian rhythms of Cry2^-/- mice, on the other hand, have long periods, and SCN neurons from those mice also have long periods. Similar results apply for mice and SCN neu­rons heterozygous for the Clock d19 mutation, which exhibit long periods. Thus, the cir­cadian period measured at the whole-animal level reflects the intrinsic properties of circadian oscillators within individual SCN neurons; in this sense, circadian period is a cell-autonomous property.

Per2::luc Imaging

One limitation of multielectrode studies is that only a small minority of cells within a local area can be recorded, leaving open the pos­sibility that small clusters of SCN neurons may form local coupled networks, and that these networks may be necessary for rhythmicity. This possibility was addressed by bio­luminescence imaging of PER2 expression in dispersed SCN neurons. Cells were cul­tured from PER2::LUC knockin mice, express­ing a reporter protein containing PER2 fused to firefly luciferase, to reflect both transcrip­tional and posttranscriptional regulation. In this study, phases of cells were randomly dis­tributed by 6-7 days after a medium change (Figure 3).

Even among cells less than 500 |j.m apart, cells that were closer together did not tend to have more similar phases or periods (17; D.K. Welsh & S.A. Kay, unpublished results). This observation rules out any measurable local coupling, and confirms that SCN neurons are indeed autonomous oscillators, in the sense that no net rhythmic input is necessary for them to oscillate.

Figure 3. A movie of circadian rhythms of PER2::LUC bioluminescence recorded from mouse SCN neurons in dispersed culture over a period of two weeks, showing that the cells oscillate independently. Bioluminescence intensity of one cell highlighted near the center of the image is plotted below.

Isolated Cells

Cells were not completely isolated in these ex­periments, however, so it was still possible that SCN neuron rhythms might require tonic input from other cells. This possibility was addressed in a recent study from Erik Herzog’s group, in which SCN neurons were cultured at various densities. In multielectrode array exper­iments, reducing cell density to ~500 cells/sq mm reduced the proportion of rhythmic cells from 61% to 33%. In addition, SCN neurons were cultured from PER2::LUC mice at very low density (100 cells/sq mm). In these cul­tures, only 27% of cells expressed PER2 at de­tectable levels for 3 days, but of these cells 65% (252/386) were rhythmic. In a few cases, a sin­gle rhythmic neuron was selected, and all other neurons in the same dish were mechanically ab­lated; 3 out of 5 of these isolated neurons re­mained rhythmic. Thus, tonic input from other cells is not absolutely required for SCN neurons to oscillate, but it does appear to increase lev­els of PER2 expression and the proportion of rhythmic cells.