Traditionally, multiunit MSNA is quantified by counting the number of bursts during a specified period of time and 100 heart beats [2]. As multiunit MSNA is mostly regulated by arterial baroreceptors, burst occurrence is synchronized with the cardiac interval. Pulse synchronous bursts are a specific feature of MSNA, which is different from skin sympathetic nerve activity and sudomotor or vasomotor nerve activity.

First limitation of analysis of multiunit MSNA is that counting multiunit MSNA does not increased over heart rate. Namely, under conditions of augmented sympathetic excitation, including heart failure, essential hypertension, or obstructive sleep apnea, multiunit MSNA is near the maximum response level to sympathoexcitatory stimulation (i.e., 100 bursts per 100 heart beats). To break through this limitation, total MSNA and/or normalized amplitude measurements have been utilized. Total MSNA is calculated as the product of the burst rate and the burst amplitude per minute, with all amplitudes normalized to the maximum amplitude [2]. The normalization process of absolute burst amplitudes has been shown to be a reproducible variable [4]. This approach assumes that the burst of the greatest amplitude reflects maximal recruitment of active neurons for that particular recording site. However, this approach cannot be used to compare subjects or to compare the same subject on different occasions, when the intervention changes the burst amplitude (i.e., shifts the distribution), because the normalization procedure will eliminate the change [5]. In addition, total multiunit MSNA cannot distinguish between changes in sympathetic nerve firing due to the recruitment of additional single-unit vasoconstrictor neurons and that due to an increase in firing rate of already active single-unit fibers.

The other limitation of multiunit MSNA is that multiunit MSNA is composed of many single-unit firing action potentials that might include functionally different firing responses to physiological stress. In the analysis of multiunit MSNA recordings during physiological stress, different firing responses cannot be observed because multiunit MSNA is calculated with all action potentials by integrating the mean of all voltages within 0.1 s. In an animal model using efferent renal sympathetic preparations, DiBona [6] showed that specific nerve firing frequencies and discrete afferent inputs exert important electrolyte, humoral, and vascular regulatory responses that are functionally distinct.

Macefield et al. [3] refined single-unit MSNA analysis. This technically demanding method requires obtaining high signal to noise ratio and adjustment of the tungsten electrode until a large unitary discharge can be observed in a raw nerve action potential signal to discriminate a single-unit action potential. The two different points in recording single-unit MSNA compared with multiunit MSNA are that (1) data sampling from raw action potentials is needed to be over 10 Hz because spike interval is near 1 ms and (2) using high-impedance microelectrode (>10 mΩ) because of the reduction in electrical noise levels.

In offline analysis, spike detection software is necessary to analyze the data of single-unit MSNA from raw nerve action potentials. Several software are utilized in the recent year. We usually use Spike2 software (ver.5, Cambridge Electronics Design, Cambridge, UK) and/or Power Lab recording system (Model ML 785/8SP; ADInstruments, Bella Vista, Australia).

To identify single-unit MSNA, candidate single units were selected by isolating large identifiable unitary spikes in the raw neurogram within a distinct discharge amplitude range. Confirmation that these action potentials originated from a single fiber were made by established criteria: (1) spike synchronization with a multiunit burst, (2) triphasic spike morphology with the main phase being negative, and (3) superimposition of candidate action potentials with minimal variation using abovementioned software.

Several points were needed in obtaining excellent single-unit spikes. However, the technique provides an estimate of single-unit firing properties in relation to the number of active firings and/or the recruitment of fibers from central or reflex effects. Using this technique, additional measurements can be obtained with regard to the mean firing frequency, the firing probability (the percentage of cardiac intervals in which a unit fires), and the percentage of spikes a unit generates per cardiac interval.

Single-unit MSNA describes three possible scenarios to explain an increase in sympathetic outflow: (1) an increase in overall mean spike firing frequency without an increase in the rate of multiple firings per cardiac interval, (2) an increase in the firing frequency by multiple spike firing within one cardiac interval, and (3) an increase in the previous silent neuron active [7–9].

We demonstrated that single-unit MSNA can also be recorded during periods of physiological stress (e.g., handgrip [HG] exercise and the Valsalva maneuver) and that reflex sympathoexcitation could be attributed to changes in the frequency of single-unit spike firing within each multiunit sympathetic burst in healthy subjects. In particular, the firing of multiple spikes within one cardiac interval was significantly augmented during the Valsalva maneuver [10]. Multiunit MSNA is near the maximum response level to sympathoexcitatory stimulation (i.e., 100 bursts per 100 heart beats). However, single-unit MSNA can increase firing frequency even within one cardiac interval. In Fig. 6.2, during mild sympathetic activation (40 spikes/100 heartbeats), results for single-unit MSNA were similar to those for multiunit MSNA. However, during intense sympathetic activation, single-unit MSNA was greater than multiunit MSNA. This finding indicates that single-unit MSNA may overcome multiunit MSNA with regard to quantifying sympathetic nerve activity especially during intense sympathoexcitation state. Macefield et al. [11] demonstrated that single-unit MSNA tends to fire only once per cardiac interval even under conditions associated with elevated sympathetic nerve activity such as heart failure. These results suggest that single-unit MSNA has the capacity to increase multiple spike firing within one cardiac interval in a state of intense sympathoexcitation.

Multiple spike firing of single-unit MSNA during one cardiac interval is thought as one of the important observations that is obtained from the analysis of single-unit MSNA. In a previous study in animal model, acute irregular and rapid nerve stimulation have been shown to evoke a greater effector organ response than regular stimulation through increased norepinephrine release in anesthetized rats [12]. In humans, Lambert et al. [13] reported that the incidence of multiple firing was associated with cardiac norepinephrine spillover. These results suggest that the firing of these instantaneous multiple spikes are thought to influence strong effector organ responses. Recent research has shown that a resting high heart failure firing frequency and/or incidence (percentage) of multiple spikes is related to cardiovascular risk factors, including hypertension [14], type 2 diabetes mellitus [15], obstructive sleep apnea [16], panic disorder [17], myocardial infarction [18], and congestive heart failure [10, 19]. The multiple firing frequency of single-unit MSNA within one cardiac interval may be involved in disease progression.

Augmented sympathetic nerve activity is a characteristic feature of heart failure. Excessive sympathetic activation under resting conditions has been shown to increase from the early stages of the disease, and elevated levels of sympathetic nerve activity are correlated with a poor prognosis [20–23]. Sympathetic activity plays an essential role in maintaining blood pressure in acute heart failure, but excessive sympathetic activity in chronic heart failure has deleterious effects on the heart, including beta receptor downregulation [24], cardiac myocyte apoptosis [25], and calcium overload [26].

Although sympathetic nerve activity is difficult to assess in clinical settings, the assessment of sympathetic nerve activity is considered important in human heart failure. Since the development of microneurography, the increased response of multiunit MSNA was considered to indicate elevated central sympathetic nerve activity to the peripheral vascular bed. Augmented multiunit MSNA and its mechanism in healthy human subjects were reported previously [27]; however, there was a controversy with regard to the differences in reflex response of multiunit MSNA to handgrip exercise between heart failure patients and age-matched healthy controls [28–30].

It is likely that the responses of multiunit MSNA are dependent on disease severity, race, sex, and intensity of exercise. However, these controversial results may be attributable to the limitations of multiunit MSNA. In chronic heart failure, the level of multiunit MSNA is nearly maximal at rest, so multiunit MSNA cannot increase further. That is, multiunit MSNA cannot increase above 100 bursts/100 heartbeats because of pulse synchrony.

Thus, as mentioned above, single-unit MSNA analysis is useful for determining actual sympathetic neural firing within one cardiac interval. We demonstrated that the percentage of multiple single-unit spikes within one cardiac interval was increased during handgrip exercise in chronic heart failure patients compared to healthy subjects, although the response of multiunit MSNA was not significantly different between the two groups [19] (Fig. 6.3). These results suggest that single-unit MSNA responses contributed to the exaggerated sympathoexcitation measured during exercise in chronic heart failure patients. The instantaneous firing frequency within one cardiac interval may increase the peripheral vascular tone, which might contribute to exercise intolerance.

Arrhythmia is a common complication of chronic heart failure caused by arrhythmogenic substrates [31]. Several studies indicated that a large multiunit MSNA burst occurred during premature ventricular contraction (PVC) [32] and atrial fibrillation (AF) [33, 34] in heart failure patients. The low diastolic pressure induced by these arrhythmic conditions unloads arterial baroreceptors and evokes a larger and longer multiunit MSNA burst [35, 36]. However, only counting multiunit MSNA could cause the actual level of sympathetic nerve activity to be underestimated, because a large sympathetic activity burst could produce prolonged sympathetic inhibition. In a human study, frequent PVC and AF were recognized as exclusion criteria for evaluating sympathetic outflow by multiunit MSNA analysis.

The mechanism underlying augmentation of the sympathetic nerve activity in heart failure has been assumed to involve a disorder of arterial baroreceptors. However, recent observations indicated that arterial baroreceptor function is preserved, maintaining appropriate blood pressure in heart failure [37]. Elam et al. [38] demonstrated the instantaneous augmentation of multiple single-unit firings following premature ventricular contraction (PVC) in heart failure patients. Frequent PVC is thought to induce moderate and severe left ventricular dysfunction [39]. Elimination of PVC with catheter ablation has been reported to improve cardiac dysfunction [40].

Cardiac norepinephrine spillover was reported to be related to multiple spike firing of single-unit MSNA within one cardiac interval [13]. Although the mechanisms of reduced left ventricular contraction induced by PVC remain unclear, multiple spike firing of single-unit MSNA is considered to cause the progression of heart dysfunction by instantaneous norepinephrine release to the heart.

As mentioned above, the analysis of multiunit MSNA in AF is not without limitations in that a prolonged irregular ventricular response would cause a large burst followed by prolonged sympathetic inhibition. The results of previous assessments of sympathetic nerve activity in acute paroxysmal AF patients using multiunit MSNA are controversial. Grassi et al. [33] used multiunit MSNA to assess sympathetic nerve activity during AF and sinus rhythm (SR) in patients with paroxysmal AF and observed a reduction in multiunit MSNA during AF. In contrast, Wasmund et al. [34] found significant augmentation of multiunit MSNA during AF, which was induced by right atrial pacing. Recently, we analyzed the single-unit MSNA frequency in heart failure patients with AF [41]. Multiunit MSNA in heart failure patients with AF was decreased compared to that in heart failure patients with SR. However, the single-unit MSNA in heart failure patients with AF was significantly greater than that in heart failure patients with SR. Moreover, the incidence of multiple firing of single-unit MSNA within one cardiac interval was augmented in heart failure + AF patients compared to heart failure + SR patients, and it shifted toward multiple firing spikes of single-unit MSNA in heart failure patients with AF. It has been suggested that not only does heart failure lead to a predisposition to AF, but AF may also facilitate and worsen the development of heart failure. The coexistence of these cardiac disorders produces a vicious cycle, which leads to advanced pump failure in heart failure patients [42].