Dantrolene protects motoneurons in culture against AMPA receptor mediated excitotoxicity

Chronic excitotoxicity induced by overstimulation of calcium permeable AMPA receptors (AMPAR) was shown to be a key factor in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). The presence of AMPAR, however, is also necessary for neuronal development. AMPAR may exert such bimodal influence on neuronal survival by altering intracellular calcium homeostasis through control of calcium induced calcium release from the endoplasmic reticulum (ER). The objective of this investigation was to determine a protective effect of the selective ryanodine receptor blocker dantrolene against AMPA/Kainate-induced excitotoxicity in cultured motoneurons. Ventral horn (VH) neurons and dorsal horn (DH) neurons harvested from E14 rat lumbar spinal cord were cultured for 13 days and then incubated for 24 hours with either kainate (30 mM), dantrolene (30 mM) or a combination of both (each 30 mM). SMI32-antibodies against the non-phosphorylated neurofilament-H/M, and tubulin-βIII- antibodies were used to estimate the number of motoneurons and total neuron counts (mean +/- S.E.M.). Total neuron counts remained equal in each treatment condition (240+/-17 VH, n=36; 438+/-35,2 DH, n=36). In VH control condition the percentage of motoneurons in relation to total neuron count was 27%+/-3% (n=9). Incubation with kainate significantly reduced the percentage of motoneurons to 14%+/-2% (n=8), p<0.01. Simultaneous application of kainate and dantrolene significantly recovered the percentage of motoneurons to 27%+/-3% (n=9, p<0.01). Dantrolene alone increased the percentage of motoneurons present to 40%+/-7% (n=7, p<0.05). This represented a significant increase compared to the kainate condition (p<0.01) and a trend compared to the control condition (p=0.07). The DH preparations contained a low percentage of motoneurons (5.9%+/-2.1%, n=12) where no significant changes due to KA and dantrolene treatment were observed. Calcium permeable AMPAR activate ryanodine receptors of the endoplasmic reticulum which causes a spread of the calcium signal along the surface of the ER. The resulting calcium induced calcium release (CICR) leads to a decrease of ER calcium concentration and to calcium uptake into mitochondria, thereby controlling protein folding and energy metabolism. In our preparation dantrolene exerted a significant neuroprotective effect against kainate induced excitotoxicity, and in kainate-free conditions where physiological AMPAR activation can contribute to motoneuron death. Dantrolene may positively influence excitotoxic motoneuron degeneration by lowering energy metabolism and protein folding rates which seem to be pathologically increased in ALS.