Translational Electrophysiology in Neuromuscular Disease Models: Putting Action Potentials to Work

By Aaron Fantina-Woblistin, PhD
Research & Development Officer, Scantox

When a promising ALS therapy fails in Phase II, the retrospective analysis often raises a familiar question: were the preclinical endpoints that drove the program forward actually measuring what matters to patients—the survival and function of motor units? In most neuromuscular programs, the primary readouts are still relatively macroscopic: motor behavior, strength, survival, histopathology, and fluid biomarkers. These endpoints are indispensable, but they don’t directly report on the core functional unit we’re trying to preserve—the motor unit and its ability to generate and propagate action potentials.

Electrophysiology gives us access to that level of resolution. By measuring stimulation-driven responses in vivo, we can quantify axonal integrity, neuromuscular junction performance, and motor unit loss in the same framework that neurologists apply in the clinic. The goal of our recent work at Scantox has been to build a practical, reproducible electrophysiology platform in neuromuscular disease models that delivers genuinely translational endpoints, rather than just mechanistic snapshots.

Why Focus on Action Potentials?

Action potential-based measures offer three advantages that matter in drug development:

• Mechanism-function linkage: They directly measure the physiology you’re trying to preserve
• Early sensitivity: They detect dysfunction before behavioral compensation masks deficits
• Clinical alignment: They use the same conceptual framework neurologists apply in EMG and nerve conduction studies

Rather than rehashing EMG fundamentals, it is more useful to highlight what we actually extract from in vivo recordings:

Compound Muscle Action Potential (CMAP)
CMAP amplitude, onset and peak latency, and duration provide a compact summary of how effectively the motor nerve can recruit the target muscle. In practice, we treat CMAP as a combined measure of axonal transmission and neuromuscular junction function, with clear expectations about how it should evolve in a given model.

Motor Unit Number Estimation (MUNE)
Using incremental stimulation paradigms, we can estimate the number of functional motor units and track their loss over time. While MUNE carries known assumptions and variability, particularly in small rodents, it remains one of the most sensitive ways to detect early motor neuron degeneration when behavior is still near normal. In practice, this can enable detection of treatment effects earlier than RotaRod or grip strength alone, particularly when the primary pathology is motor unit loss.

Repeated Stimulation Paradigms
High- or moderate-frequency trains of stimuli stress the neuromuscular junction. Measuring the decrement in CMAP amplitude across pulses reveals fatigability and transmission failure that a single supramaximal pulse will not expose.

The key point is that these measures aren’t abstract electrophysiological curiosities—they parallel clinical practice and can be integrated directly into translational decision-making.

Building an Electrophysiology Platform in an ALS Mouse Model

To test the utility of this approach, we implemented a longitudinal electrophysiology package in the B6.SOD1-G93A mouse model of amyotrophic lateral sclerosis. The objective was straightforward: capture high-quality, clinically interpretable neuromuscular readouts across disease progression in a way that can be realistically deployed in discovery or preclinical studies.

Using a minimally invasive setup under isoflurane anesthesia with appropriate analgesia, we:

• Stimulated the sciatic nerve via subcutaneous needle electrodes placed near the sciatic notch
• Recorded CMAPs from the gastrocnemius muscle using subcutaneous recording and reference electrodes positioned at the muscle belly and distal tendon
• Implemented a standardized stimulation and acquisition protocol across all animals and time points

From this single preparation, we extracted CMAP, MUNE, and repeated-stimulation endpoints. The overall workflow was designed to be tractable for multi-cohort studies while still delivering sufficient signal-to-noise for longitudinal analysis. In our hands, experienced operators can complete full CMAP/MUNE/repeated-stimulation assessments in 15-20 minutes per animal, allowing longitudinal tracking of 30-40 animals per week with appropriate scheduling.

Longitudinal Case Study: B6.SOD1-G93A ALS Mice

In the white paper, we describe a study in which transgenic and wild type (WT) littermates were followed across several ages capturing pre-symptomatic, early symptomatic, and more advanced disease stages. Here, I’ll focus on what electrophysiology actually told us and how it complements other endpoints.

CMAP as a Marker of Axonal and Neuromuscular Function

Supramaximal sciatic stimulation elicited robust CMAPs in the gastrocnemius. Across time, several trends were consistent with progressive neuromuscular failure in SOD1-G93A mice:

• A progressive reduction in CMAP amplitude compared with WT controls, reflecting loss of functional motor units and/or impaired neuromuscular transmission
• Prolonged onset and peak latencies, consistent with slowing of conduction and desynchronized recruitment
• Broadening of CMAP duration, suggesting altered temporal summation of individual motor unit responses

In practical terms, this gives us a compact, reproducible measure of gross neuromuscular function that can be sampled at multiple time points without exhausting animals or study resources.

MUNE: Detecting Early Motor Unit Loss

Using incremental stimulation, we identified discrete stepwise increases in CMAP amplitude corresponding to recruitment of individual or small groups of motor units. From these, we derived:

• Average motor unit action potential (MUAP) amplitude
• Estimates of total motor unit number by dividing maximal CMAP amplitude by average MUAP

The value of this analysis was not theoretical—it materially sharpened our view of early disease stages. MUAP amplitude was significantly decreased throughout the experiment, indicating that each motor unit was recruiting fewer muscle fibers, while MUNE remained similar between groups. Taken together with the CMAP changes, these metrics revealed advanced neuromuscular degeneration at the earliest electrophysiology time point—occurring 4–5 weeks before RotaRod performance diverged from controls. In this dataset, however, the early diagnostic potential typically attributed to MUNE could not be fully appreciated, because CMAP had already deteriorated. For discovery teams, this underscores that therapies aimed at preserving motor units may exert effects that are detectable in these electrophysiological measures before behavioral readouts show clear differences, and that sampling earlier in the disease course would further enhance the value of MUNE.

At the same time, we observed the expected limitations: variability between animals, the impact of anesthesia depth, and the challenges of applying MUNE in late-stage animals with very low CMAP amplitudes. These aren’t reasons to avoid MUNE, but factors that need to be baked into study design and statistical planning.

Repeated Stimulation: Stressing the Neuromuscular Junction

We then applied repeated stimulation trains to probe neuromuscular junction capacity under stress. By comparing CMAP amplitudes across pulses within a train (pulse 1 vs 5 vs 10), we quantified the degree of decrement over a short time window.

In SOD1-G93A mice, we observed:

• A greater decrement in CMAP amplitude across the train than in WT controls
• An age-dependent worsening of this decrement, consistent with progressive neuromuscular junction vulnerability

Because repetitive nerve stimulation is widely used in human neuromuscular clinics, these preclinical results are straightforward to interpret and communicate across translational teams.