www.elsevier.com/locate/brainres Available online at www.sciencedirect.com Research Report AMPA receptor activation causes preferential mitochondrial Ca 2þ load and oxidative stress in motor neurons Dinesh C. Joshi, Bhanu P. Tewari, Mahendra Singh, Preeti G. Joshi, Nanda B. Joshi n Department of Biophysics National Institute of Mental Health and Neuro Sciences, Bangalore, 560 029, India article info Article history: Accepted 22 April 2015 Keywords: Motor neurons Amyotrophic lateral sclerosis AMPA receptors Excitotoxicity Voltage gated calcium channels Calcium signaling Mitochondrial calcium Oxidative stress abstract It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca 2þ loading. Earlier we have reported that in motor neurons a generic rise in [Ca 2þ ] i does not always lead to mitochondrial Ca 2þ loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca 2þ regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca 2þ ] m in both motor and other spinal neurons that was sustained up to the end of recording time of 30 min. The rise in [Ca 2þ ] m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca 2þ ] m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca 2þ ] m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na þ /Ca 2þ exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca 2þ sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na þ /Ca 2þ exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both http://dx.doi.org/10.1016/j.brainres.2015.04.042 0006-8993/& 2015 Elsevier B.V. All rights reserved. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; EMEM, Eagle's minimal essential medium; HEPES, N-2-hydroxyethyl piperazine-n-2 ethanesulphonic acid; [Ca 2þ ] i , Intracellular calcium; Ca 2þ ] m , Mitochondrial calcium; HEt, Dihydroethidium; ROS, Reactive oxygen species; GSH, Glutathione; ALS, Amyotrophic lateral sclerosis; MN, Motor neurons; OSN, Other spinal neurons. n Corresponding author. Fax: þ91 80 2656 4830. E-mail addresses: joshi.nanda@gmail.com, nbjoshi@nimhans.kar.nic.in (N.B. Joshi). brain research ] ( ]]]] ) ]]] – ]]] Please cite this article as: Joshi, D.C., et al., AMPA receptor activation causes preferential mitochondrial Ca 2þ load and oxidative stress in motor neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.04.042