Tetrodotoxin prevents motor unit enlargement after partial denervation in rat hindlimb muscles

Abstract

Findings that increased neuromuscular activity significantly reduced sprouting in partially denervated muscles prompted this present study to determine if the converse is true, namely that reduced activity promotes sprouting and motor unit (MU) enlargement. Partial denervation of rat hindlimb muscles by either the L4 or L5 spinal root avulsion resulted in extensive denervation (> 80 %) in tibialis anterior (TA) and medial gastrocnemius (MG) muscles, and moderate denervation (~50 %) in soleus (SOL) and plantaris (PL) muscles. The partially denervated muscles were then subjected to a 4 week programme of normal caged activity or TTX-induced neuromuscular inactivity. At 1 month, measurement of MU enlargement and quantification of sprouting were evaluated, respectively, by electrophysiological and histochemical means. Analysis of electrophysiological data showed that MU forces were significantly increased in both extensively and moderately denervated muscles 1 month after partial denervation and normal cage activity and that neuromuscular activity blockade by TTX completely abolished the MU enlargement in these partially denervated muscles. Histochemical analysis of sprouting revealed that the number of sprouts was significantly increased after partial denervation and normal cage activity, particularly after extensive denervation. TTX-induced neuromuscular inactivity dramatically reduced the number of sprouts and increased the number of free endplates in the extensively but not the moderately denervated muscles. These data demonstrate that a reduction in neuromuscular activity mediated by presynaptic blockade of neural action potentials reduces MU enlargement in partially denervated muscles by reducing axonal sprouting.

Figure 2

Percentage frequency histogram plotted on a linear scale of innervated muscle fiber CSAs in partially denervated TA muscle of rats experiencing normal caged activity (▪) as compared with partially denervated muscles with TTX treatment (□)

Figure 1. Frequency histograms of twitch force in partially denervated TA and MG muscles

A and B, percentage frequency histograms plotted on a linear scale and C-F, cumulative frequency histograms, plotted on logarithmic scale, of MU twitch force distributions in partially denervated tibialis anterior (TA; A, C and E) and medial gastrocnemius (MG; B, D and F) muscles of rats experiencing normal caged activity (○) as compared with control muscles (▪), and partially denervated muscles with tetrodotoxin (TTX) treatment (•). With TTX treatment, MU twitch forces after normalization for muscle fiber CSA were significantly reduced as shown by the leftward shift of the MU force distributions (P < 0.05). In A and B, the vertical arrows denote the mean values of the motor unit forces of partially denervated muscles (▪) and partially denervated muscles with TTX treatment (□).

Figure 3. Frequency histograms of twitch forces in partially denervated plantaris and soleus muscles

A and B, percentage frequency histograms plotted on a linear scale and C-F, cumulative frequency histograms, plotted on logarithmic scale, of MU twitch force distributions in partially denervated plantaris (PL; A, C and E) and soleus (SOL; B, D and F) muscles of rats experiencing normal caged activity (○) as compared with control muscles (▪), and partially denervated muscles with TTX treatment (•). The detrimental effect of TTX treatment was seen as a leftward shift of the MU force (P < 0.05). In A and B, the vertical arrows denote the mean values of the motor unit forces of partially denervated muscles (▪) and partially denervated muscles with TTX treatment (□).

Figure 4. Numbers of sprouts and free endplates in partially denervated and control plantaris and soleus muscles

A, mean (± s.e.m.) number of sprouts per 100 innervated endplates, and B, number of free endplates per 100 endplates of contralateral controls (horizontal line; n = 38), moderately denervated (PD < 75 %), and extensively denervated (PD >75 %) PL and SOL muscles after normal caged activity (▪; n = 7 for PD < 75 % and n = 4 for PD>75 %) and TTX treatment (; n = 7 for PD < 75 % and n = 5 for PD>75 %). The dotted horizontal lines denote the s.e.m. values for the contralateral control data. TTX treatment did not have a significant effect on sprouting for PD < 75 %, but dramatically reduced the number of sprouts and hence increased the number of free endplates for PD >75 %.

Figure 5. Higher power combined Ag-AChE histochemical photomicrographs of 100 μm thick cryostat longitudinal sections of partially denervated and control muscles

A, contralateral control and B and C, extensively denervated PL muscles (>75 % PD) after normal caged activity (B), and TTX treatment (C). TTX blockade of neuromuscular activity dramatically reduced the incidence of sprouts and further increased the amount of free endplates. Both motor endplates and muscle fibres became atrophic. Filled arrows indicate sprouts and open arrows indicate free endplates.