Diaphragm Neuromuscular Transmission Failure in a Mouse Model of an Early-Onset Neuromotor Disorder.

The spa transgenic mouse displays spasticity and hypertonia that develops during the early postnatal period, with motor impairments that are remarkably similar to symptoms of human cerebral palsy. Previously, we observed that spa mice have fewer phrenic motor neurons innervating the diaphragm muscle (DIAm). We hypothesize that spa mice exhibit increased susceptibility to neuromuscular transmission failure (NMTF) due to an expanded innervation ratio. We retrogradely-labeled phrenic motor neurons with rhodamine and imaged them in horizontal sections (70 µm) using confocal microscopy. Phrenic nerve-DIAm strip preparations from wildtype and spa mice were stretched to optimal length, and force was evoked by phrenic nerve stimulation at 10, 40 or 75 Hz in 330 ms duration trains repeated each s (33% duty cycle) across a 120 s period. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in hematoxylin and eosin stained strips. Compared to wildtype, spa mice had over two-fold greater NMTF during the first stimulus train that persisted throughout the 120 s period of repetitive activation. In both wildtype and spa mice, NMTF was stimulation-frequency dependent. There was no difference in neuromuscular junction morphology or the total number of DIAm fibers between wildtype and spa mice, however there was an increase innervation ratio (39%) in spa mice. We conclude that early-onset developmental neuromotor disorders impair the efficacy of DIAm neuromuscular transmission, likely to contribute to respiratory complications.