Intracellular calcium and survival of tadpole forebrain cells in anoxia

Abstract

SUMMARY The frog brain survives hypoxia with a slow loss of energy charge and ion homeostasis. Because hypoxic death in most neurons is associated with increases in intracellular calcium ([Ca2+]i), we examined the relationship between [Ca2+]i and survival of a mixed population of isolated cells from the forebrain of North American bullfrog Rana catesbeiana tadpoles. Forebrain cells from stage V-XV tadpoles were isolated by enzymatic digestion and loaded with one of three different calcium indicators (Fura-2, Fura 2-FF and BTC) to provide estimates of [Ca2+]i accurate at low and high[Ca2+]i. Propidium iodide (PI) fluorescence was used as an indicator of cell viability. Cells were exposed to anoxia (100%N2) and measurements of [Ca2+]i and cell survival made from 1 h to 18 h. Intracellular [Ca2+] increased significantly after 3-6 h anoxia (P<0.05), regardless of the type of Ca2+ indicator used; however, there were substantial differences in the measurements of [Ca2+]i with the different indicators, reflecting their varying affinities for Ca2+. Resting[Ca2+]i was approximately 50 nmol l-1 and increased to about 9-30 μmol l-1 after 4-6 h anoxia. The significant increase in [Ca2+]i during anoxia was not associated with significant increases in cell death, with 85-95% survival over this time period. Cells exposed to anoxia for 18 h, or those made anoxic for 4-6 and reoxygenated for 12 h to 16 h, had survival rates greater than 70%,but survival was significantly less than normoxic controls. These results indicate that large increases in [Ca2+]i are not necessarily associated with hypoxic cell death in vertebrate brain cells.

Blunted Neuronal Calcium Response to Hypoxia in Naked Mole-Rat Hippocampus

“…Non-mammalian models of hypoxia tolerance include some fishes, frogs, and turtles [55]. Interestingly, forebrain cells from tadpoles [56] and cortical slices from turtles [55] show increases in internal calcium during severe but survivable hypoxia without showing the cell damage characteristic of mammalian neurons exposed to high calcium.…”

Decreased NR1 phosphorylation and decreased NMDAR function in hibernating Arctic ground squirrels

“…However, excitotoxic injury depends on the Ca 2+ source (Tymianski et al,1993) as well as Ca 2+ concentration (Hartley et al,1993; Eimerl and Schramm,1994; Lu et al,1996). Ca 2+ influx through NMDAR is more toxic than that through other sources (Tymianski et al,1993; Hedrick et al,2005). Thus, although Glu‐induced [Ca 2+ ] i was similar in slices from hAGS and ibeAGS, the contribution of NMDAR to Glu‐induced increase in [Ca 2+ ] i was less in slices from hAGS, which is consistent with enhanced protection from oxygen nutrient deprivation and NMDA (Ross et al,2006).…”

Magnolol Reduces Glutamate-Induced Neuronal Excitotoxicity and Protects against Permanent Focal Cerebral Ischemia Up to 4 Hours

“…The level of [Ca 2+ ](i) were measured on a single cell fluorimeter [26], [27]. Briefly, neuronal cultures were incubated with 3 µM fura 2-acetoxymethylester (Fura-2 AM) and 10 µM ionomycin in a standard buffer (composition in mM: NaCl, 140; KCl, 3.5; KH 2 PO 4 , 0.4; Na 2 HPO 4 , 1.25; CaCl 2 , 2.2; MgSO 4 , 2; glucose, 10; HEPES, 10, pH 7.3) for 30 min, followed by incubation in dye-free standard buffer for 30 min and, then, the addition of vehicle or magnolol (0.01, 0.1, or 1 µM) for 20 min and the exposure of glutamate (300 µM).…”