Subtypes?

Not all SCN neurons are rhythmic, and rhyth­mic cells have different periods; this raises the question of whether there might be stable functional subclasses of SCN neurons, identifiable on the basis of neuropeptide expres­sion or other molecular markers. Early studies, however, found too high a proportion of rhyth­mic cells (50-60%) to be readily accounted for by any single neuropeptide population. Herzog's group recently addressed this issue by combining PER2::LUC imaging with im- munolabeling for neuropeptides. They found no association between circadian pheno­type (rhythmic/not, period, or amplitude) and neuropeptide expression (AVP or VIP). More­over, mutant SCN neurons sometimes exhibit rare, intermittent PER2 oscillations, suggesting that rhythmicity could be a stochastic phenomenon even in wild-type SCN neurons. Indeed, wild-type SCN neurons can switch from rhythmic to nonrhythmic (or vice versa), either spontaneously or upon adding forskolin. Furthermore, PER2::LUC rhythms per­sist in a minority of SCN neurons during appli­cation of TTX, but this is not a stable, repro­ducible subpopulation. Thus, there is no evidence that cell-autonomous circadian phe­notypes define stable, identifiable subtypes of SCN neurons.

Non-SCN Cells

Cell-autonomous circadian oscillation is not ex­clusive to SCN neurons. Circadian rhythms have long been observed in unicellular or­ganisms, including Gonyaulax and cyanobacteria. In the latter, imag­ing of luciferase reporters confirms cell- autonomous oscillations. In isolated basal retinal neurons of the sea snail Bulla gouldiana, circadian rhythms of membrane conduc­tance have been measured (by cross-sectional sampling). Zebrafish cells exhibit cell-autonomous rhythms of clock gene expres­sion, monitored by bioluminescence imaging. Chick pinealocytes in dispersed culture release melatonin in a circadian rhythm. In mammals, clock gene expression is rhythmic in many non-SCN tissues and cell popu­lations. Although non-SCN rhythms do tend to damp out gradually over time, low- amplitude rhythms persist for many days. PER2::LUC imaging of cultured fibroblasts re­veals that individual fibroblast oscillations per­sist without damping, and that the damping ob­served at the population level is due to gradual loss of synchrony among cells. Cells that are closer together do not have more similar phases, but rather oscillate independently. Similar re­sults were obtained in NIH3T3 cells using a fluorescent reporter of Rev-Erba. Thus, like SCN neurons, mammalian fibroblasts (and probably most other cell types) are autonomous circadian oscillators, leading to the suggestion that perhaps “the rat-1 fibroblast should replace the SCN as the in vitro model of choice”.

Limits of Autonomy

Although the unassuming fibroblast may be a good substitute for the SCN neuron from the point of view of a molecular biologist study­ing molecular mechanisms of the intracellu­lar clock, there is clearly much more to the SCN than its cell-autonomous circadian os­cillation. Unlike a population of fi­broblasts, the SCN is a neuronal network of coupled cellular oscillators, with a specific pat­tern of interconnections. As a tissue, the SCN shows specific modes of coupling mediated by specialized mechanisms, provides more robust and more precise time-keeping relative to sin­gle cells, responds to light input in complex and flexible ways, and produces variously phased output signals. In addition, unlike in other cells, the amplitude of the SCN intracellular clock is highly responsive to and dependent on inter­cellular signaling, which effectively reinforces the collective rhythmicity of the network and may silence errant cells. SCN neurons are not designed to function alone.