Analysis of Circadian Rhythm Using a Novel SCN-Specific Cre Transgenic Mouse Line




Chang, Alexander S.

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The neurons that make up the suprachiasmatic nucleus (SCN) temporally organize behavior into circadian cycles of activity and rest. When dissociated, these neurons individually oscillate with various period, phase, and amplitude. These conflicting results can be reconciled if inter-neuronal networking in the SCN is required for a consolidated behavioral circadian rhythm. To test this hypothesis, a novel SCN-specific Cre transgenic mouse line, named NMS-Cre, was developed by inserting a bicistronic Cre expression cassette at the 3’-untranslated region of the Neuromedin S (NMS) gene. By crossing NMS-Cre line to a lox-STOP-lox diphtheria toxin receptor line, behavioral circadian rhythm was disrupted upon intraperitoneal injection of diphtheria toxin. A histological examination showed that diphtheria toxin injection eliminated ~85% of NMS-Cre containing neurons at the SCN. Next, I generated NMS-Cre mediated Bmal1 conditional knockout animals to study behavioral rhythm output when most of the SCN neurons are without a molecular oscillator. The NMS-Cre(+);Bmal1flox/flox animals have essentially normal circadian rhythm of locomotor activity. Then, I generated NMS-Cre mediated Vesicular GABA Transporter (VGAT) conditional knockout animals because GABA has long been suspected to play a role in behavioral circadian rhythm. The NMS-Cre;VGATflox/flox animals performed normally in behavioral circadian rhythm parameter such as free-running period, robustness, and phase response curve. These in vivo data demonstrated a model that intra-SCN neuronal network is required for behavioral circadian rhythm, and can be a conduit that mediates molecular clock outputs from a small number of SCN neurons. Despite the fact that virtually all SCN neurons are GABAergic, GABA is an unlikely transmitter for this intra-SCN networking. Finally, NMS knockout animals have a well-consolidated behavioral circadian rhythm. However, when subjected to photic phase advancement, NMS knockout animals shifted their activity onset time quicker than wild type control animals. In situ hybridization results ruled out that an altered response to light stimuli or dampened molecular clock oscillation in the SCN as the cause for the rapid phase shift. NMS knockout animals switching from constant illumination to constant dark environment are unable to return to the typical less than twenty-four hour free-running period. Therefore, NMS is involved in a circadian pacemaker function.

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