Research Article: Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Date Published: March 10, 2010

Publisher: Public Library of Science

Author(s): Jin Hee Hong, Cheol Hong Min, Byeongha Jeong, Tomoyoshi Kojiya, Eri Morioka, Takeharu Nagai, Masayuki Ikeda, Kyoung J. Lee, Shin Yamazaki.

Abstract: 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 “Ca2+ spikes” (i.e., [Ca2+]c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms.

We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ 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 Ca2+ 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 Ca2+ spikes was increased to 13∼14%. Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]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 Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN.

Partial Text: The circadian clock system governs various daily rhythms in physiological activities, from cellular autonomic activities to animal behaviors. The key component of this system is the suprachiasmatic nucleus (SCN) [1]–[3]. The SCN is composed of a pair of nuclei, each of which is comprised of 8,000 neurons approximately [4], [5]. One key property of SCN neurons is the circadian oscillation in their spontaneous action potential (AP) firing rates [6]–[9]. Recent studies have shown that intracellular circadian oscillations are the result of transcription-translation feedback loops arising from multiple “clock genes” [1], [10]. Yet, no one has shown how clock gene products modulate AP firing frequencies in SCN neurons.