Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne Ca 2+ mediates axotomy-induced necrosis and apoptosis of satellite glial cells remote from the transection site in the isolated crayfish mechanoreceptor Andrey Khaitin, Mikhail Rudkovskii, Anatoly Uzdensky ⁎ Laboratory of Molecular Neurobiology, Academy of Biology and Biotechnology, Southern Federal University, Rostov-on-Don 344090, Russia ARTICLE INFO Keywords: Axotomy Glia Ca 2+ Calcium homeostasis Necrosis Apoptosis ABSTRACT Severe nerve injury such as axotomy induces neuron degeneration and death of surrounding glial cells. Using a crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glia, we showed that axotomy not only mechanically injures glial cells at the transection location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. We studied Ca 2+ role in spontaneous or ax- otomy-induced death of remote glial cells. Stretch receptors were isolated using the original technique that kept the neuron connected to the ventral cord ganglion (control preparations). Using Ca 2+ -sensitive fluorescence probe fluo-4, we showed Ca 2+ accumulation in neuronal perikarion and glial envelope. Ca 2+ gradually accu- mulated in glial cells after axotomy. In saline with triple Ca 2+ concentration the axotomy-induced apoptosis of glial cells increased, but spontaneous or axotomy-induced necrosis was unexpectedly reduced. Saline with 1/ 3[Ca 2+ ], oppositely, enhanced glial necrosis. Application of ionomycin, CdCl 2 , thapsigargin, and ryanodine showed the involvement of Ca 2+ influx through ionic channels in the plasma membrane, inhibition of en- doplasmic reticulum Ca 2+ -ATPase, and Ca 2+ release from endoplasmic reticulum through ryanodine receptors in axotomy-induced glial necrosis. Apoptosis of glial cells surrounding axotomized neurons was promoted by ionomycin and thapsigargin. Possibly, other Ca 2+ sources such as penetration through the plasma membrane contributed to axotomy-induced apoptosis and necrosis of remote glial cells. Thus, modulating different path- ways that maintain calcium homeostasis, one can modulate axotomy-induced death of glial cells remote from the transection site. 1. Introduction Traumatic cerebral or spinal cord injury are among main causes of people's death and disability especially in young and middle age men (Hill et al., 2016; Kobeissy, 2015). Axon transection, or axotomy, occurs not only during spine or brain trauma, but also in wounds, or during surgery. Axotomy leads either to degeneration of the damaged neuron, or to axon regeneration and restoration of its connections with other neurons, muscle fibers, and other targets. In order to treat the con- sequences of nerve injury, the balance between neurodegeneration and neuroprotection processes in damaged nerves should be rapidly shifted to suppression of neuronal death. Unfortunately, reliable neuroprotec- tors with proven efficiency are not found yet. So, deeper and compre- hensive study of molecular processes that occur after axon disruption is required. Intercellular neuroglial interactions provide the integrity of the nervous tissue and its resistance to harmful impacts. Glial cells play a considerable role in maintaining neuronal survival and regeneration after axon injury (Aldskogius and Kozlova, 1998; Giaume et al., 2013; Whiteside, 1998). Glia damage was shown to suppress the neuronal functions and induce death of neurons (Largo et al., 1996). On the other hand, nerve injury induced death of surrounding Schwann cells (Kopp et al., 1997). Localized injury of the soma in the crayfish mechan- oreceptor neuron by a focused laser beam enhanced apoptosis of sur- rounding glial cells, thus suggesting the anti-apoptotic influence of the neuronal body on glial cells (Kolosov and Uzdensky, 2006). Never- theless, the mechanisms of axotomy-induced lesion and death of glial cells have not been studied in detail (Verkhratsky and Butt, 2013). https://doi.org/10.1016/j.mcn.2017.12.004 Received 21 August 2017; Received in revised form 5 November 2017; Accepted 5 December 2017 ⁎ Corresponding author at: Laboratory of Molecular Neurobiology, Academy of Biology and Biotechnology, Southern Federal University, 194/1 Stachky prospekt, Rostov-on-Don 344090, Russia. E-mail address: auzd@yandex.ru (A. Uzdensky). Abbreviations: AT, axotomized preparation; CICR, calcium-induced calcium release; CsA, cyclosporin A; CSR, crayfish stretch receptor; MRN, mechanoreceptor neuron; Int, intact preparation; Im, ionomycin; MPTP, mitochondrial permeability transition pore; Rya, ryanodine; Tg, thapsigargin; VNC, ventral nerve cord Molecular and Cellular Neuroscience 88 (2018) 7–15 Available online 08 December 2017 1044-7431/ © 2017 Elsevier Inc. All rights reserved. T