Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

BACKGROUND: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms. METHODOLOGY/PRINCIPAL FINDINGS: We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%. CONCLUSIONS/SIGNIFICANCE: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.

Cholecystokinin-A receptors regulate photic input pathways to the circadian clock.

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.