Repetitive Nerve Stimulation

Normal Neuromuscular Junction Physiology

The NMJ essentially forms an electrical-chemical-electrical link between nerve and muscle ( Fig. 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 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 quanta 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 (MFAP) is generated and propagated through the muscle fiber. Under normal circumstances, the EPP always rises above the threshold, resulting in an MFAP. The amplitude of the EPP above the threshold value needed to generate an MFAP 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 the threshold to ensure generation of an MFAP 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 MFAP 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 an MFAP. 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 an MFAP. In pathologic conditions where baseline EPP is below threshold and an MFAP is not generated, rapid RNS may increase the number of quanta released, resulting in a larger EPP, so that threshold is reached. An MFAP 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 1–2 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 MFAP, and the CMAP can best be illustrated with the following three examples ( Fig. 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), an MFAP 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, enough 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 the EPP always stays above threshold (15 mV), resulting in the consistent generation of an MFAP ( Fig. 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 because 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 MFAPs not being generated ( Fig. 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).

Stimulus	n	m	EPP	MFAP	CMAP
1	1000	20	4	−	Low
2	980	19.6	3.9	−	Decrement
3	960	19.2	3.8	−	Decrement
4	940	18.8	3.7	−	Decrement
5	920	19.2	3.8	−	Decrement (repair)

In this next example, the number of quanta in the immediately available store (n) is normal, and the EPP is normal for the number of quanta released (m) . What is abnormal is the number of ACH quanta released (m) and the baseline EPP. In Lambert-Eaton myasthenic syndrome, the calcium concentration in the presynaptic terminal is reduced, due to an antibody attack on the VGCCs. Thus, the probability of release (p) falls dramatically, along with a decrease in the number of quanta released. There still is depletion, although it is not as marked as in normal or postsynaptic disorders. Simply because so few quanta are released, the subsequent amount of depletion cannot be as great. In this example, because the EPP is below threshold at baseline, an MFAP is never generated ( Fig. 6.2C ). Thus, the baseline CMAP is low in amplitude because many muscle fibers do not reach threshold due to inadequate release of quanta after a single stimulus at baseline. With slow RNS, there is also further decrement of the CMAP because subsequent stimuli result in further loss of MFAPs. Just as in postsynaptic disorders, 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). Note that in some presynaptic disorders, the baseline EPP may be low but still above threshold, resulting in a reduced safety factor. In this situation, an MFAP initially may be generated but then fails to be generated as the EPP falls below threshold with slow RNS.

Repetitive Nerve Stimulation in the Electromyography Laboratory

RNS is easy to learn and to perform and requires no special equipment. However, it is poorly tolerated in some patients and is prone to a number of important technical problems that, if not recognized and corrected, can influence its reliability, validity, and, therefore, its value. The earlier discussion pertained to single endplate and individual MFAPs. During RNS in the electromyography (EMG) laboratory, all measurements are made on the CMAP, the sum of the individual MFAPs generated in a muscle. Thus it is assumed that the CMAP amplitude and area are proportional to the number of muscle fibers activated. In normal subjects, the EPP is affected by both slow and rapid RNS. However, in both cases, the EPP always stays above threshold, resulting in consistent generation of MFAPs. Thus in normal subjects, CMAPs generated following either slow or rapid RNS do not change significantly in amplitude or area.

In NMJ disorders, if the normal EPP safety factor is reduced, slow RNS will cause a depletion of quanta and reduce the amplitude of the EPP. If the EPP of some muscle fibers falls below threshold, those MFAPs will not be generated, and the number of individual MFAPs will decline. This provides the basis for the decremental CMAP response to slow RNS seen in the EMG laboratory ( Fig. 6.3A ). As the number of individual MFAPs declines, a decrement of the CMAP amplitude and area occurs. This decrement reflects fewer EPPs reaching threshold and fewer individual MFAPs contributing to the CMAP.

In NMJ disorders in which some EPPs are below threshold at baseline (usually the presynaptic disorders), rapid RNS can be used to facilitate the EPP. If subthreshold EPPs can be brought above threshold, MFAPs will be generated where they had not been present previously, and the number of individual MFAPs will increase. This provides the basis for the incremental CMAP response to rapid RNS seen in the EMG laboratory. As the number of individual MFAPs increases, an increment of CMAP amplitude and area occurs ( Fig. 6.4 ). This increment reflects more EPPs reaching threshold and more individual MFAPs contributing to the CMAP. Incremental responses that are >100% (i.e., double in value) in response to rapid RNS are not unusual in presynaptic NMJ disorders.