Intrinsic, nondeterministic circadian rhythm generation in identified mammalian neurons

Abstract

Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm(2) eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.

Fig. 1.

Intrinsic circadian rhythmicity is rare and found in diverse classes of SCN neurons. (A) A representative region of a recorded and subsequently immunolabeled very-low-density culture shows SCN neurons containing AVP (red), VIP (green), or neither neuropeptide (blue). (B) Of the neurons recorded over 6 days, 73% (n = 1,027 of 1,413 from 14 cultures) expressed PER2-mediated bioluminescence for <3 days (black), 18% were circadian (orange), and 9% were arrhythmic (pink). Most neurons with sustained PER2 expression (n = 252 of 386 in 14 cultures) were circadian (orange). (C) Representative bioluminescence traces from single identified SCN neurons show circadian or arrhythmic PER2 expression. (D) Neurons that did not express (Top), or showed circadian (Middle) or arrhythmic (Bottom) PER2 profiles were similar in their expression of AVP (red), VIP (green), or neither (blue). Thus, multiple cell types were intrinsically circadian, and neuropeptide expression did not distinguish between rhythmic and arrhythmic neurons.

Fig. 2.

SCN neurons grown at very low density are healthy and functionally isolated. (A) Representative whole-cell recording of spontaneous IPSCs at 0 mV from a neuron 7 days after plating at ≈10,000 cells/mm². Inset shows IPSCs at higher temporal resolution. (B) IPSC amplitudes and frequencies were high over 2 min in 8 neurons from 1 culture (data from each neuron in a different color) grown at high density. Bar indicates the time of 10 mM KCl exposure in A and B. Note the increase in evoked IPSCs. (C) Representative whole-cell recording of spontaneous IPSCs at 0 mV from a neuron 7 days after plating at ≈100 cells/mm². Insets show small current events that may reflect rare, spontaneous IPSCs. (D) Plots of IPSC amplitude in 12 neurons combined from 2 cultures grown at low density. (E) KCl elevation boosted IPSC frequency in high-density but not low-density cultures. (F) Inward sodium currents evoked by voltage steps from −80 mV to 0 mV in neurons grown at high and low density. (G) Peak inward sodium current amplitude ± SEM in neurons grown at high density (n = 8) and low density (n = 12).

Fig. 3.

Circadian rhythms in a fully isolated SCN neuron. A single, representative SCN neuron (white arrow) recorded before (A) and after (B) removal of all other cells. (C) PER2-driven bioluminescence from this neuron had an average period of ≈27.7 h for the last 5 days after isolation (black arrow). This SCN neuron expressed neither AVP nor VIP.

Fig. 4.

Compared with SCN neurons plated at high density, only a subset of nearly-isolated SCN neurons maintain circadian firing rate rhythms. (A) Firing rate traces over 4 days from representative neurons plated and recorded on multielectrode arrays at extremely low density show unstable circadian rhythms (top 2 traces, Left) compared with the more precise circadian patterns in high-density cultures (top 3 traces, Right). Arrhythmic firing patterns were more common in low-density cultures (bottom 2 traces, Left) than in high-density cultures (bottom trace, Right). (B) The proportion of circadian (black) and arrhythmic (white) SCN neurons from low-density cultures compared with high-density cultures. Note that firing rate, like PER2, rhythms were intrinsic to a small fraction of neurons in low-density cultures.

Fig. 5.

Repeated TTX treatment revealed switching in circadian behavior of SCN neurons. (A) Long-term recordings of PER2::LUC expression from 4 representative SCN cells in an explant before, during, and between 2 6-day TTX treatments (red and green bars). All cells showed drastically reduced PER2::LUC expression, and few cells were circadian in either the first or second TTX administration. (B) Approximately 13% (n = 24 of 190 recorded cells in 2 cultures) remained rhythmic during both treatments, whereas 38% became rhythmic or lost rhythmicity in the second TTX treatment.