Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to motor neuron degeneration, alteration in neuromuscular junctions (NMJs), muscle atrophy, and paralysis. To investigate the NMJ functionality in ALS we tested, in vitro, two innervated muscle types excised from SOD1(G93A) transgenic mice at the end-stage of the disease: the Soleus, a postural muscle almost completely paralyzed at that stage, and the diaphragm, which, on the contrary, is functional until death. To this aim we employed an experimental protocol that combined two types of electrical stimulation: the direct stimulation and the stimulation through the nerve. The technique we applied allowed us to determine the relevance of NMJ functionality separately from muscle contractile properties in SOD1(G93A) animal model. Functional measurements revealed that the muscle contractility of transgenic diaphragms is almost unaltered in comparison to control muscles, while transgenic Soleus muscles were severely compromised. In contrast, when stimulated via the nerve, both transgenic muscle types showed a strong decrease of the contraction force, a slowing down of the kinetic parameters, as well as alterations in the neurotransmission failure parameter. All together, these results confirm a severely impaired functionality in the SOD1(G93A) neuromuscular junctions.

Figure 1

Example of a complete stimulation protocol showing the force values recorded from a wild-type Soleus. The muscle is initially stimulated with four single pulses (Tw), delivered alternatively one directly and one through the nerve. Subsequently, a series of eight pulse trains are delivered to the specimen at the indicated frequency, alternatively directly and through the nerve to measure the force-frequency curves. The protocol ends up with two fatigue paradigms: the first delivered at the firing frequency (35 Hz) and the second at tetanic frequency (80 Hz). Insets show stimulation pulses and force response curves on a magnified time scale

Figure 2

Twitch response properties of control (WT) and transgenic (SOD) Soleus muscles stimulated directly (M) and through the nerve (N). Shown are the mean ± SEM of the TTP (a), the ½RT (b) and the dF/dt (c). SOD is for SOD1^G93A. * and **: p < 0.05 and p < 0.01 vs. WT M; ^# and ^###: p < 0.05 and p < 0.001 vs. WT N; ⁺⁺⁺: p < 0.001 vs. SOD M

Figure 3

Twitch response properties of control (WT) and transgenic (SOD) diaphragm muscles stimulated directly (M) and through the nerve (N). Shown are the mean ± SEM of the TTP (a), the ½RT (b) and the dF/dt (c). SOD is for SOD1^G93A. ^#: p < 0.05 vs. WT N; ⁺ and ⁺⁺: p < 0.05 and p < 0.01 vs. SOD M

Figure 4

Relationship between specific force and frequency of stimulation from single pulse to tetanus, for Soleus (a) and diaphragm (b) specimens. M indicates direct membrane stimulation, N indicates nerve stimulation. For both muscle types, the SOD M curve was significantly different from the WT M and the SOD N curve was significantly lower than the SOD M. Values are mean ± SEM. SOD is for SOD1^G93A. ** and ***: p < 0.01 and p < 0.001 vs. WT M; ⁺⁺⁺: p < 0.001 vs. SOD M

Figure 5

Analysis of intratetanic fatigue computed for Soleus (a) and diaphragm (d) muscles. Force values measured with nerve stimulation at the beginning of the protocol allows for a reliable IF measurement for both SOD1^G93A Soleus (b) and diaphragm (e). At the end of the protocol the force generated by transgenic Soleus drops down near zero (c) therefore, from this point on, the values of IF expresse noise. Thus, statistical analysis has been performed taking into account only the first 8 min of stimulation, as indicated by the dotted line in (a), were the contraction forces are higher than 5 mN. On the contrary, forces generated by diaphragm specimens allow for a good IF measurement until the end of the protocol (f). Values are mean ± SEM. SOD is for SOD1^G93A. ^# and ^##: p < 0.05 and p < 0.01 vs. WT N

Figure 6

Neurotransmission failure computed at the tetanic frequency of 80 Hz for Soleus (a) and 100 Hz for diaphragm (b) specimens. NF values for Soleus muscle should be considered reliable only until the 8th min of stimulation, as indicated by the dotted vertical line (a), where the forces generated by the muscle specimens were higher than 5 mN. Values are mean ± SEM. SOD is for SOD1^G93A. *: p < 0.05 vs. WT

Figure 7

Neurotransmission failure computed at the firing frequency of 35 Hz for Soleus (a) and diaphragm (b). Values are mean ± SEM. SOD is for SOD1^G93A. ***: p < 0.001 vs. WT