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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1 G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1 G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

We characterized toxin-insensitive calcium currents expressed by acutely dissociated embryonic dorsal root ganglion neurons. In the presence of 3 microM omega-conotoxin-GVIA, 3 microM nitrendipine and either 500 nM omega-agatoxin-IVA or 500 nM omega-conotoxin-MVIIC to inhibit N-, L- and P/Q-type currents, respectively, all neurons expressed two residual currents: a T-type and another which we referred to as toxin-resistant current. The toxin-resistant current (i) consisted of an inactivating and a sustained components, (ii) had a threshold of activation and a steady-state inactivation comprised between that of the T-type current and that of the other high-voltage-activated currents, (iii) had the same permeability for barium and calcium used as charge carriers, (iv) was highly sensitive to both cadmium and nickel; and (v) was insensitive to 500 microM amiloride which abolished the T-type at this concentration. The properties of the toxin-resistant current are very similar to those of the currents expressed in oocytes following injection of alpha(1E) subunits which we demonstrated to be present in these neurons. Therefore a component of the toxin-resistant current calcium channels in sensory neurons may be closely related to those calcium channels formed by alpha(1E) subunits.

Analysis of neuronal development has emphasized the importance of voltage-activated Ca2+ currents during the initial period of differentiation. We investigated non-N, non-L Ba2+ currents through Ca2+ channels in freshly dissociated large diameter embryonic mouse dorsal root ganglion neurons using the whole-cell patch-clamp technique. Two types of omega-agatoxin IVA-sensitive currents were clearly distinguished at embryonic day 13: a sustained P-type current blocked selectively at 30 nM (IC50 = 3nM) and an inactivating Q-type current blocked in the range 50-500 nM (IC50 = 120nM). The P-type Ca2+ current disappeared at day 15 whereas the Q-type Ca2+ current increased two- to three-fold during the same embryonic period. In contrast, the contribution of the non-L, non-N, omega-agatoxin IVA-resistant current (R-type) was constant during this developmental span. In conclusion, our results clearly show that P- and Q-type Ca2+ currents are differentially expressed during ontogenesis in large diameter dorsal root ganglion neurons. The developmental change, which occurs during the period of target innervation, could be related to specific key events such as natural neuron death and onset of synapse formation.