As a typical model to study the development and function of the NMJ [43 (link)–45 (link)], diaphragm muscle was dissected with special care to preserve phrenic nerve connectivity. Isolated nerve–muscle preparations were immersed in Ringer’s solution and maintained at 26 °C.
One hemidiaphragm was used as a treatment, and the other served as its paired untreated control. All treatments were performed ex vivo. Muscles were stimulated through the phrenic nerve at 1 Hz, which allows the maintenance of different tonic functions without depleting synaptic vesicles, for 30 min using the A-M Systems 2100 isolated pulse generator (A-M System) as in previous studies [38 (link)–40 (link)]. We designed a protocol of stimulation that preserves the nerve stimulation and the associated neurotransmission mechanism. This method prevents other mechanisms associated with non-nerve-induced (direct) muscle contraction [46 –48 (link)]. To verify muscle contraction, a visual checking was done. Two main experiments were performed to distinguish the effects of synaptic activity from those of muscle activity (Fig. 1).

Presynaptic stimulation (Ctrl versus ES): to show the impact of the synaptic activity, we compared presynaptically stimulated muscles whose contraction was blocked by μ-CgTx-GIIIB with nonstimulated muscles also incubated with μ-CgTx-GIIIB to control for nonspecific effects of the blocker.

Contraction (ES versus ES + C): to estimate the effect of nerve-induced muscle contraction, we compared stimulated/contracting muscles with stimulated/noncontracting muscles whose contraction was blocked by μ-CgTx-GIII. By comparing the presynaptic stimulation with or without postsynaptic activity, we separate the effect of contraction. However, one should consider that postsynaptic contraction experiments also contain presynaptic activity.

Design of experimental treatment for the study of effects of presynaptic activity and nerve-induced muscle contraction. μ-CgTx-GIIIB, μ-conotoxin GIIIB

In the experiments that needed only stimulation without contraction, μ-CgTx-GIIIB was used (see “Reagents”). Nevertheless, before immersing these muscles in μ-CgTx-GIIIB, a visual checking of the correct contraction of the muscle was done [39 (link)].
Furthermore, to assess the effect of PKA blocking, three different experiments have been performed:

To estimate the effect of PKA inhibition under synaptic activity, we compared presynaptically stimulated muscles whose contraction was blocked by μ-CgTx-GIIIB with and without H-89: ES versus ES + H-89.

To show the impact of the PKA inhibition under muscle contraction, we compared stimulating and contracting muscles with and without H-89: (ES + C) versus (ES + C) + H-89.

To demonstrate if degradation or redistribution along the axon is involved, the diaphragm muscle was dissected with special care to preserve phrenic nerve connectivity. We compared stimulating and contracting muscles with and without protease inhibitor (Prot.Inh.) cocktail 1% (10 μl/ml; Sigma, Saint Louis, MO, USA): (ES + C) versus (ES + C) + Prot.Inh.

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