Hypothermia reduces calcium entry via the N-methyl-D-aspartate and ryanodine receptors in cultured hippocampal neurons

Abstract

Hypothermia is a powerful neuroprotective method when induced following cardiac arrest, stroke, and traumatic brain injury. The physiological effects of hypothermia are multifaceted and therefore a better knowledge of its therapeutic targets will be central to developing innovative combination therapies to augment the protective benefits of hypothermia. Altered neuronal calcium dynamics have been implicated following stroke, status epilepticus and traumatic brain injury. This study was therefore initiated to evaluate the effect of hypothermia on various modes of calcium entry into a neuron. Here, we utilized various pharmacological agents to stimulate major routes of calcium entry in primary cultured hippocampal neurons. Fluorescent calcium indicator Fura-2AM was used to compare calcium ratio under normothermic (37 °C) and hypothermic (31 °C) conditions. The results of this study indicate that hypothermia preferentially reduces calcium entry through N-methyl-D-aspartate receptors and ryanodine receptors. Hypothermia, on the other hand, did not have a significant effect on calcium entry through the voltage-dependent calcium channels or the inositol tri-phosphate receptors. The ability of hypothermia to selectively affect both N-methyl-D-aspartate receptors and ryanodine receptors-mediated calcium systems makes it an attractive intervention for alleviating calcium elevations that are present following many neurological injuries.

Copyright © 2012 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Hypothermia did not affect Ca2+ entry through VDCCs. (A) Prior to stimulation of VDCCs with high potassium solution, hippocampal neurons from both treatment groups displayed similar 340/380 baseline ratios of 0.27±0.01 in the 31°C group and 0.27±0.02 in the 37°C group (black bars). When stimulated with 37°C high potassium solution 340/380 ratios peaked to 0.54±0.02. Stimulation with 31°C high potassium resulted in a similar 340/380 peak ratio of 0.51±0.01 (grey bars). *P<0.001 compared to 37°C baseline, one way ANOVA followed by post-hoc Tukey test, n=6 plates per condition. (B) Representative pseudocolor images obtained from baseline neuron (left panel), neuron stimulated with 37°C high potassium (top right panel), and neuron stimulated with 31°C high potassium (bottom right panel). Neurons from both groups exhibited elevated [Ca2+]i upon 37°C and 31°C stimulation.

Fig. 2

Hypothermia reduced NMDA receptor-dependent Ca2+ entry. (A) Prior to stimulation with 50 μM glutamate, hippocampal neurons exhibited similar 340/380 baseline ratios of 0.30±0.01 for neurons in the 31°C group and 0.31±0.02 for neurons in the 37°C group (black bars). Upon stimulation with 37°C glutamate, 340/380 ratios peaked to 0.636±0.04, which is significantly elevated compared to baseline. When neurons were stimulated with 31°C glutamate, 340/380 ratios peaked to 0.38±0.01 (grey bars). *P<0.001 compared to baseline; #P<0.001 between 31°C and 37°C peaks, one way ANOVA followed by post-hoc Tukey test, n=7 plates per condition. (B) Representative pseudocolor images obtained from baseline neuron (left panel), neuron stimulated with 37°C glutamate (top right panel), and neuron stimulated with 31°C glutamate (bottom right panel). Neurons exhibited elevated [Ca2+]i upon 37°C stimulation and a diminished response to 31°C stimulation.

Fig. 3

The role of NMDA receptor subtypes on [Ca2+]i upon glutamate stimulation. Prior to glutamate stimulation, the average baseline ratio value was 0.38±0.01. When stimulated with 50 μM glutamate, ratio values increased to 1.09±0.09. When neurons were stimulated with glutamate (50 μM) in the presence of GluN2B selective antagonist Ro 8-4304 (10 μM) 340/380 ratios increased to 0.94±0.08, which were not significantly different from glutamate alone conditions. When stimulated with GluN2A selective antagonist TCN-213, 340/380 ratios increased to 0.52±0.04, which were significantly lower than control glutamate levels. *P<0.001 compared to glutamate, one-way ANOVA followed by post-hoc Dunnett’s test, n=7 plates per condition.

Fig. 4

Hypothermia did not affect IP3 receptor-mediated Ca2+ entry. (A) Prior to bradykinin-mediated stimulation of IP3 receptors, neurons from 31°C and 37°C treatment groups displayed similar 340/380 baseline ratios of 0.27±0.01 and 0.27±0.01, respectively (black bars). Stimulation with 37°C bradykinin resulted in a peak 340/380 ratio of 0.65±0.03. Similarly, when stimulated with 31°C bradykinin, 340/380 ratios peaked to 0.60±0.01 (grey bars). *P<0.001 compared to 37°C baseline, one way ANOVA followed by post-hoc Tukey test, n=5 plates per condition. (B) Representative pseudocolor images obtained from baseline neuron (left panel), neuron stimulated with 37°C bradykinin (top right panel), and neuron stimulated with 31°C bradykinin (bottom right panel). Neurons in both groups exhibited elevated [Ca2+]i upon stimulation with 37°C and 31°C bradykinin.

Fig. 5

Hypothermia reduced ryanodine receptor-mediated Ca2+ induced Ca2+ release. (A) Prior to caffeine-mediated RyR stimulation, baseline 340/380 ratio values from both treatment groups were not significantly different with values of 0.28±0.01 in the 31°C group and 0.34±0.01 in the 37°C group (black bars). Upon stimulation with 37°C caffeine, 340/380 ratios peaked to 0.72±0.05. When stimulated with 31°C caffeine, neurons exhibited a diminished response with a peak ratio of 0.44±0.04 (grey bars). *P<0.001 compared to 37°C baseline, **P<0.05 compared to 37°C baseline, #P<0.001 between 31°C and 37°C peaks, one way ANOVA followed by post-hoc Tukey test, n=10 plates per group. (B) Representative pseudocolor images obtained from baseline neuron (left panel), neuron stimulated with 37°C caffeine (top right panel), and neuron stimulated with 31°C caffeine (bottom right panel). Neurons exhibited elevated [Ca2+]i upon 37°C stimulation and a diminished response to 31°C stimulation.