Calcium dependence of damage to mouse motor nerve terminals following oxygen/glucose deprivation

Abstract

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca2+ contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca2+] indicator demonstrated an increase in intra-terminal [Ca2+] following OGD onset. When OGD was terminated within 20–30 min of the increase in resting [Ca2+], these changes were sometimes reversible; in other cases [Ca2+] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22 min OGD and 120 min reoxygenation (32.5 °C). Stress-induced motor terminal degeneration was Ca2+-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8 mM Ca2+, but increased to 81% when Ca2+ was absent. Removal of Ca2+ only during OGD was more protective than removal of Ca2+ only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca2+ entry into motor terminals, including voltage-dependent Ca2+ channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na+/Ca2+ exchanger (KB-R7943). Inhibition of a Ca2+-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca2+-dependent, and that inhibition of Ca2+ entry or Ca2+-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.

Calpains mediate axonal cytoskeleton disintegration during Wallerian degeneration

“…However, we cannot exclude the possibility that denervation is a “byproduct” of axonal degeneration, but this is less likely given that NMJ degeneration is detected earlier than that of sciatic nerve axons. Nevertheless, our data is consistent with published results demonstrating morphological protection of NMJs by pharmacologic calpain inhibitors in an ex vivo model of anti-ganglioside antibody-mediated motor nerve terminal injury (O'Hanlon et al, 2003) and in vitro model of ischemia (Talbot et al, 2012). The importance of calpains in these different injury models suggests that calpain-mediated degeneration is an important mechanism of synaptic pathology in many diseases.…”

Morphological and functional changes in innervation of a fast forelimb muscle in SOD1-G85R mice

“…Morphological assays of endplate occupancy used techniques similar to those described in Talbot et al (2011), as illustrated in Fig. 1B–D.…”

Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon

“…Extensor digitorum longus (EDL) muscle preparations from transgenic mice whose motor neurons express yellow fluorescent protein (YFP) were exposed to simulated ischemia/reperfusion (Talbot et al, 2012). Pre-treatment with calpain inhibitor VI [N-(4-fluorophenylsulphonyl)-L-valyl-L-leucinal] preserves neuromuscular junction (NMJ) innervation, assessed by YFP fluorescence.…”

“…Motor nerve terminals are more sensitive to ischemia than axons and muscle fibers (Mäkitie and Teräväinen, 1977; Tömböl et al, 2002). EDL preparations were harvested from transgenic mice expressing YFP in motor neurons and their axons (Talbot et al, 2012). Chelating bath Ca 2+ or adding pharmacologic inhibitors for plasma membrane NCX, P/Q-type Cav channels, L-type Cav channels, or Na + ,K + ,2Cl − co-transporter reduces the degeneration of EDL terminals, as assessed by YFP fluorescence, 120 min post-OGD.…”