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.


RESULTS
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
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.
Center for Cell-dynamics and Department of Physics, Korea University, Seoul, Republic of Korea.
Jin Hee
JH
Hong
Cheol Hong
CH
Min
Byeongha
B
Jeong
Tomoyoshi
T
Kojiya
Eri
E
Morioka
Takeharu
T
Nagai
Masayuki
M
Ikeda
Kyoung J
KJ
Lee
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002940:Circadian Rhythm; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D008297:Male; D051379:Mice; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D013493:Suprachiasmatic Nucleus / Q000378:metabolism*; D014966:Xanthenes / Q000494:pharmacology
D000814:Aniline Compounds; D002614:Chelating Agents; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium