Repetitive Nerve Stimulation

The use of repetitive nerve stimulation (RNS) dates back to the late 1800s, when Jolly made visual observations of muscle movement that occurred after nerve stimulation. Although his initial studies were done with submaximal stimuli and mechanical rather than electrical measurements were made, Jolly noted a decrementing response following RNS in patients with myasthenia gravis and correctly concluded that the disorder was peripheral.

Subsequently, RNS has been refined and validated as one of the most useful electrodiagnostic (EDX) tests in the evaluation of patients with suspected neuromuscular junction (NMJ) disorders. RNS should be performed whenever there is a possible diagnosis of myasthenia gravis, Lambert–Eaton myasthenic syndrome, or botulism. It also should be considered in any patient who presents with fatigability, proximal weakness, dysphagia, dysarthria, or ocular abnormalities, which are clinical symptoms and signs suggestive of a possible NMJ disorder.

In the EDX laboratory, the effects of RNS are studied on the compound muscle action potential (CMAP), with analysis of any decremental or incremental response forming the basis of the study. Understanding these responses requires knowledge of normal NMJ physiology and the effects of repetitive stimulation on a single NMJ and its associated muscle fiber. That knowledge can be used in the EDX laboratory to accurately predict the effect of RNS on the CMAP, both in normal subjects and in patients with NMJ disorders.

Normal Neuromuscular Junction Physiology

The NMJ essentially forms an electrical–chemical–electrical link between nerve and muscle ( Figure 6–1 ). The chemical neurotransmitter at the NMJ is acetylcholine (ACH). ACH molecules are packaged as vesicles in the presynaptic terminal in discrete units known as quanta; each quantum contains approximately 10,000 molecules of ACH. The quanta are located in three separate stores. The primary, or immediately available store consists of approximately 1000 quanta located just beneath the presynaptic nerve terminal membrane. This store is immediately available for release. The secondary, or mobilization store consists of approximately 10,000 quanta that can resupply the primary store after a few seconds. Finally, a tertiary, or reserve store of more than 100,000 quanta exists far from the NMJ in the axon and cell body.

When a nerve action potential invades and depolarizes the presynaptic junction, voltage-gated calcium channels (VGCCs) are activated, allowing an influx of calcium. The infusion of calcium starts a complicated interaction of many proteins that ends in the release of ACH from the presynaptic terminal. The greater the calcium concentration inside the presynaptic terminal, the more quanta are released. ACH then diffuses across the synaptic cleft and binds to ACH receptors (ACHRs) on the postsynaptic muscle membrane. The postsynaptic membrane is composed of numerous junctional folds, effectively increasing the surface area of the membrane, with ACHRs clustered on the crests of the folds. The binding of ACH to ACHRs opens sodium channels, resulting in a local depolarization, the endplate potential (EPP). The size of the EPP is proportional to the amount of ACH that binds to the ACHRs.

In a process similar to the generation of a nerve action potential, if the EPP depolarizes the muscle membrane above threshold, an all-or-none muscle fiber action potential is generated and propagated through the muscle fiber. Under normal circumstances, the EPP always rises above threshold, resulting in a muscle fiber action potential. The amplitude of the EPP above the threshold value needed to generate a muscle fiber action potential is called the safety factor . In the synaptic cleft, ACH is broken down by the enzyme acetylcholinesterase, and the choline subsequently is taken up into the presynaptic terminal to be repackaged into ACH.

During slow RNS (2–3 Hz) in normal subjects, ACH quanta are progressively depleted from the primary store, and fewer quanta are released with each successive stimulation. The corresponding EPP falls in amplitude, but because of the normal safety factor, it remains above threshold to ensure generation of a muscle fiber action potential with each stimulation. After the first few seconds, the secondary (mobilization) store begins to replace the depleted quanta with a subsequent rise in the EPP.

The physiology of rapid RNS (10–50 Hz) in normal subjects is more complex. Depletion of quanta from the presynaptic terminal is counterbalanced not only by the mobilization of quanta from the secondary store but also by the accumulation of calcium. Normally, it takes about 100 ms for calcium to be actively pumped out of the presynaptic terminal. If RNS is rapid enough so that new calcium influx occurs before the previously infused calcium has been fully pumped out, calcium accumulates in the presynaptic terminal, causing an increased release of quanta. Normally, this accumulation of calcium predominates over depletion, leading to an increased number of quanta being released and a correspondingly higher EPP. However, the result is the same as with any other EPP above threshold: an all-or-none muscle fiber action potential is generated.

Thus, the effects of slow and rapid RNS are very different at the molecular level, yet in normal subjects the result is the same: the consistent generation of a muscle fiber action potential. In pathologic conditions where the safety factor is reduced (i.e., baseline EPP is reduced but still above threshold), slow RNS will cause depletion of quanta and may drop the EPP below threshold, resulting in the absence of a muscle fiber action potential. In pathologic conditions where baseline EPP is below threshold and a muscle fiber action potential is not generated, rapid RNS may increase the number of quanta released, resulting in a larger EPP, so that threshold is reached. A muscle fiber action potential is then generated where one had not been present previously. These concepts form the basis of the decrements with slow RNS and increments with rapid RNS that are seen in NMJ disorders.

Physiologic Modeling of Repetitive Nerve Stimulation

RNS in normal subjects and patients with NMJ disorders can be modeled effectively by making the following three assumptions:

1

m = pn , where m represents the number of quanta released during each stimulation; p is the probability of release (effectively proportional to the concentration of calcium), typically approximately 0.2 in normal subjects; and n represents the number of quanta in the immediately available store (at baseline, approximately 1000 in normal subjects).
2

The mobilization store starts to replenish the immediately available store after 1 to 2 seconds.
3

Approximately 100 ms is required to pump calcium out of the presynaptic terminal. If stimulation occurs again sooner than 100 ms (i.e., stimulation rate >10 Hz), the calcium concentration increases, the probability of release of ACH quanta increases, and more quanta are released.

Modeling Slow Repetitive Nerve Stimulation

The effects of slow RNS on the EPP, the muscle fiber action potential (MFAP), and the CMAP can best be illustrated with the following three examples ( Figure 6–2A–C ):

3 Hz Repetitive Nerve Stimulation: Normal Subject

Stimulus	n	m	EPP	MFAP	CMAP
1	1000	200	40	+	Normal
2	800	160	32	+	No change
3	640	128	26	+	No change
4	512	102	20	+	No change
5	640	128	26	+	No change

In this first example, initially there are 1000 quanta in the immediately available store ( n ), and with each stimulation, 20% of the quanta are released. If the EPP is >15 mV (threshold in this example), a muscle fiber action potential is generated. Note the normal depletion of the immediately available store ( n ), the subsequent decline in the number of quanta released ( m ), and the corresponding fall in the EPP from the first to the fourth stimulation. During the second stimulation, only 160 quanta are released instead of the initial 200 because the number of quanta in the immediately available store has dropped to 800 (1000 minus the 200 released during the first stimulation), and subsequently 20% of the 800 is released. At the fifth stimulus, however, sufficient time has elapsed for the secondary or mobilization store to begin to resupply the primary store. The number of quanta in the immediately available store increases, with a corresponding increase in the number of ACH quanta released, resulting in a higher EPP. Note that at all times the EPP stays above threshold (15 mV), resulting in the consistent generation of a muscle fiber action potential ( Figure 6–2A ). In the EDX laboratory, these findings translate to normal baseline CMAPs with no change in amplitude, because action potentials are generated in all muscle fibers.

3 Hz Repetitive Nerve Stimulation: Postsynaptic Disorder (e.g., Myasthenia Gravis)

Stimulus	n	m	EPP	MFAP	CMAP
1	1000	200	20	+	Normal
2	800	160	16	+	Normal
3	640	128	13	−	Decrement
4	512	102	10	−	Decrement
5	640	128	13	−	Decrement (repair)

In this next example, the number of quanta in the immediately available store ( n ), the number of quanta released ( m ), and the depletion of quanta with slow RNS all are normal. The response to the quanta (i.e., the EPP) is abnormal, however. Whereas in normal subjects the release of 200 quanta generated an EPP of 40 mV, in this case the same number of quanta generates an EPP of only 20 mV. Accordingly, the safety factor is reduced. In myasthenia gravis, this occurs as a result of fewer ACHRs and, accordingly, less binding of ACH. The reduced safety factor, in conjunction with normal depletion of quanta, results in subsequent EPPs falling below threshold and their corresponding muscle fiber action potentials not being generated ( Figure 6–2B ). As the number of individual MFAPs declines, a decrement of CMAP amplitude and area occurs. This decrement reflects fewer EPPs reaching threshold and fewer individual MFAPs contributing to the CMAP. Often, after the fifth or sixth stimulus, the secondary stores are mobilized and no further loss of MFAPs occurs. This results in stabilization or sometimes slight improvement or repair of the CMAP decrement after the fifth or sixth stimulus, giving the characteristic “U-shaped” decrement (see later).

3 Hz Repetitive Nerve Stimulation: Presynaptic Disorder (e.g., Lambert-Eaton Myasthenic Syndrome)