Recording of neural firing from single-unit muscle sympathetic nerve activity (MSNA) is a new strategy offering information about the frequency of pure sympathetic firing. However, it is uncertain whether and when single-unit MSNA would be more useful than multiunit MSNA for analysis of various physiological stresses in humans. In 15 healthy subjects, we measured single-unit and multiunit MSNA before and during handgrip exercise at 30% of maximum voluntary contraction for 3 min and during the Valsalva maneuver at 40 mmHg expiratory pressure for 15 s. Shapes of individual single-unit MSNA were proved to be consistent and suitable for further evaluation. Single-unit and multiunit MSNA exhibited similar responses during handgrip exercise. However, acceleration of neural firing determined from single-unit MSNA became steeper than multiunit MSNA during the Valsalva maneuver. During the Valsalva maneuver, unlike handgrip exercise, the distribution of multiunit burst between 0, 1, 2, 3, and 4 spikes was significantly shifted toward multiple spikes within a given burst (P < 0.05). These results indicated that evaluation of single-unit MSNA could provide more detailed and accurate information concerning the role and responses of neuronal discharges induced by various physiological stresses in humans, especially amid intense sympathetic activity.
- handgrip exercise
- Valsalva maneuver
since microneurography first was performed in humans (8, 19), this technique has become well established in assessment of peripheral sympathetic activity. Previous studies using microneurography have examined the frequency of muscle sympathetic nerve activity (MSNA) bursts derived from multiunit recordings, which cannot determine whether every unit within a burst has a vasoconstrictor role, because the multiunit burst is an aggregate of many individual action potentials. The amplitude and area of the burst can be measured to evaluate the relative changes in the MSNA within recording sessions. However, because the amplitude and area of integrated sympathetic bursts are highly influenced by the position of the recording electrode, the actual sizes of bursts cannot be compared between individual subjects (1, 16, 17). From this point of view, the method of evaluating precise sympathetic nerve activity has become very important.
Recently, a technique of recording a single-unit MSNA from awake supine subjects has been developed (9). This approach clearly can select individual vasoconstrictor units, eliminating the possibility of analyzing activity from fibers that might not be innervating vascular smooth muscle. Thus addition of single-unit MSNA analysis permits a more detailed examination of sympathetic nerve activity. Pure sympathetic activity has been measured by single-unit MSNA in healthy subjects and patients with congestive heart failure at rest (2–7, 9–11). As opposed to the resting state, very little is known about the effects of physiological stresses on sympathetic firing characteristics in humans. It is also uncertain whether recording of single-unit MSNA would have an advantage over multiunit MSNA in such an analysis.
We presently used single-unit MSNA to determine the frequency of pure sympathetic vasoconstrictor firing and to accurately assess physiological responses of sympathetic excitation. The purpose of this study was to examine whether single-unit MSNA was useful in evaluating sympathetic nerve activity during physiological stresses and whether different physiological stresses could alter the responses of single-unit and multiunit MSNA. In addition, we evaluated whether the analysis of single-unit MSNA was more accurate and informative than that of multiunit MSNA.
Data were obtained from 10 male and 5 female subjects ranging in age from 18 to 70 yr (mean 33.5 ± 4.6 yr). The experimental protocol and its purpose were explained in detail to each subject, and informed consent was obtained. All subjects were nonsmokers of average physical fitness, and none took any regular medication. The study protocol was approved by the ethical panel of the Graduate School of Medical Science, Kanazawa University.
All experiments were carried out in a quiet, electrically shielded room with the subject in a supine position. Heart rate was determined from a continuous electrocardiogram. Arterial pressure was recorded continuously from the radial artery using a noninvasive tonometry monitoring system (JENTOW-7700; Nihon Colin, Komaki, Japan). Postganglionic MSNA was recorded from the left peroneal nerve, as previously described (9, 13, 24). Briefly, with the subject in a comfortable supine position, the common peroneal nerve was located by palpation and electrical stimulation via the skin surface. A high-impedance tungsten microelectrode with a shaft diameter of 5 μm and an impedance of 9–12 MΩ (type 25–5-1; Frederic Haer) was inserted percutaneously into a motor fascicle of the nerve. The electrode was adjusted until spontaneous pulse-synchronous sympathetic burst activities could be recorded. For the obtaining of single-unit MSNA from unprocessed action potential, more adjustment of an electrode was needed until the position in which a large unitary discharge within the multiunit sympathetic burst could be recorded (2, 3, 9). In this study, the single-unit and multiunit MSNA were simultaneously evaluated in the same electrode position.
The electrodes were connected to a preamplifier at a gain of 1,000 and to an amplifier at a gain of 70. The signal was fed through a band-pass filter (500–3,000 kHz) and a resistance-capacitance integrating circuit with a time constant of 0.1 s to produce a mean voltage neurogram. The signal was displayed on an oscilloscope (Neuropack 2; Nihon Kohden, Tokyo) and stored on a digital audio tape recorder (DT120RT; SONY, Tokyo) at 12 kHz. During off-line analysis, the multiunit MSNA was identified on the basis of its relationship to cardiac activity in the integrated nerve recording. In the unprocessed nerve recording, a single-unit MSNA was carefully checked by inspection of its morphology using Sony PC Scan II software (Sony, Tokyo). We used four criteria for identifying a single-unit MSNA: occurrence in pulse-synchronous bursts of multiunit discharge, large negative spike amplitude, consistent morphology, and superimposability using an expanded time scale (4, 9, 15). For each subject, multiunit MSNA and single-unit MSNA were expressed as the number per minute and the number per 100 heartbeats. Additionally, single-unit MSNA was represented as the distribution of the burst with multiple single spikes because single-unit MSNA can increase within a given cardiac interval (2, 3, 5, 9, 10).
After placement of the microelectrode for MSNA recording, subjects were allowed to rest for 15 min. Baseline values for heart rate, arterial pressure, multiunit MSNA, and single-unit MSNA were recorded for 5 min. All subjects performed a sustained handgrip exercise at 30% of maximal voluntary contraction for 3 min. After a 10-min interval, the Valsalva maneuver was performed at an expiratory pressure of 40 mmHg for 15 s. We recorded all parameters for 1 min before the end of handgrip exercise and for 15 s during phase II of the Valsalva maneuver.
Values are expressed as means ± SE. Student's paired t-test was used for paired comparisons. Polynomial regression analysis was employed to determine the relationship between single-unit MSNA and multiunit MSNA. All calculations were performed with a personal computer using the StatView-J 5.0 statistical software package (Abacas Concepts, Berkeley, CA). P values below 0.05 were considered to indicate statistical significance.
The examples of single-unit and multiunit MSNA at rest and during handgrip exercise and the Valsalva maneuver are presented, respectively, in Figs. 1, 2, and 3. The single-fiber activity occurred as pulse-synchronous multiunit burst discharges as shown in Figs. 1A, 2A, and 3A. Figures 1B, 2B, and 3B demonstrate one or multiple single-unit MSNA within a multiunit burst on the expanding time scale of Figs. 1A, 2A, and 3A, respectively. In this study, the maximum spikes of single-unit MSNA within one multiunit burst were three spikes during handgrip exercise and four spikes during the Valsalva maneuver. Figures 1C1, 2C, and 3C show recordings of single-unit MSNA activity that were isolated from bursts of Figs. 1B, 2B, and 3B, respectively. The shape of single-unit MSNA had small differences caused by the electrical noise level. However, other different unit activities (Fig. 1C2) could be obviously distinguished from single-unit MSNA as shown in Fig. 1C1, and triphasic action potential spike of single-unit MSNA during physiological stresses was almost consistent, as superimposed in Figs. 1D, 2D, and 3D. Similar results were observed in all subjects.
The effects of handgrip exercise and the Valsalva maneuver on all cardiovascular variables are summarized in Table 1. Handgrip exercise significantly increased heart rate, arterial pressure, and multiunit and single-unit MSNA compared with baseline. We excluded one subject who could not perform to a pressure of 40 mmHg for the evaluation of the Valsalva maneuver. The Valsalva maneuver significantly increased heart rate, multiunit MSNA, and single-unit MSNA, while arterial pressure decreased.
Figure 4, A and B, displays the absolute changes of the multiunit and the single-unit MSNA during handgrip exercise. Handgrip exercise increased burst frequency and single-unit MSNA frequency by an average of 8.6 ± 1.1 bursts/min and 8.2 ± 1.1 spikes/min, respectively (Fig. 4A). Similarly, burst incidence and single-unit MSNA (spikes/100 heartbeats) were increased during handgrip exercise (9.0 ± 1.4 bursts/100 heartbeats, 8.4 ± 1.2 spikes/100 heartbeats, respectively; Fig. 4B). However, the differences in changes were not significant between single-unit and multiunit MSNA. In contrast, as shown in Fig. 4, C and D, the Valsalva maneuver significantly increased absolute changes of both single-unit MSNA per minute and per 100 heartbeats (58.9 ± 4.3 spikes/min and 71.5 ± 6.0 spikes/100 heartbeats, respectively) compared with those of multiunit MSNA (29.4 ± 1.9 bursts/min and 33.0 ± 3.1 bursts/100 heartbeats, respectively).
Figure 5 shows the distribution of multiunit bursts ranging from 0 to 4 spikes during physiological stresses. Handgrip exercise increased the frequency of multiunit and single-unit MSNA compared with baseline, while the distribution of bursts between 0, 1, 2, and 3 spikes did not differ between baseline and handgrip exercise (20.6 ± 2.5, 58.7 ± 2.2, 13.4 ± 1.4, and 3.4 ± 0.8%, respectively, in baseline; 24.5 ± 2.1, 55.3 ± 1.2, 13.6 ± 1.4, and 4.5 ± 1.0%, respectively, during handgrip exercise). During the Valsalva maneuver, unlike handgrip exercise, the distribution of multiunit bursts between 0, 1, 2, 3, and 4 spikes was significantly shifted toward multiple spikes within a given burst (12.9 ± 1.9, 35.1 ± 6.2, 30.6 ± 3.4, 16.8 ± 2.8, and 4.7 ± 1.8%, respectively; P < 0.05 compared with baseline). Thus the Valsalva maneuver increased not only burst frequency but also single-unit MSNA frequency within a burst.
The relationships between single-unit and multiunit MSNA are illustrated in Fig. 6. A significant correlation was evident between burst incidence and single-unit MSNA (spikes/100 heartbeats), as assessed by polynomial regression analysis. With mild sympathetic hyperactive state (<40 spikes/100 heartbeats), the value of single-unit MSNA was the same as that of multiunit MSNA. However, with marked sympathetic activity, the value of single-unit MSNA was greater than that of multiunit MSNA.
This is the first report to examine the effects of physiological stresses on single-unit MSNA in humans. The points of this investigation were as follows. First, physiological stresses significantly increased not only multiunit burst but also single-unit MSNA. Second, different stresses increased single-unit MSNA to result in different patterns of single-spike firing within sympathetic burst. For instance, the distribution of multiunit bursts changed to favor multiple spikes during the Valsalva maneuver but remained unchanged during handgrip exercise. Finally, during marked sympathetic activation, the absolute values of single-unit MSNA increased more than those of multiunit MSNA. In contrast, the absolute values of single-unit and multiunit MSNA did not differ during mild sympathetic activation. These results suggest that the single-unit MSNA analysis could be useful for evaluating sympathetic responses during various physiological stresses. Accordingly, the measurement of the single-unit MSNA may provide detailed information about neuron discharge that is more sensitive than the multiunit MSNA, especially during marked sympathetic activation.
We know of no previous reports describing the shape of single-unit MSNA during various physiological stresses, such as handgrip exercise and the Valsalva maneuver. We used four criteria for identifying a single-unit MSNA: occurrence in pulse-synchronous bursts of multiunit discharge, large negative spike amplitude, consistent morphology, and superimposability using an expanded time scale (4, 9). Single vasoconstrictor fibers have been reported previously in patients with congestive heart failure at rest and during cardiac dysarrhythmias. In these cases, the firing was followed by a transient fall in blood pressure, and morphological consistency was demonstrated in the shape of single-fiber spikes (2). In our subjects, the shape of a single-unit MSNA was maintained during both physiological stresses (Figs. 1, 2, and 3). Thus the single-unit MSNA could be reliable for evaluating sympathetic neural activity both at rest and under physiological stresses.
MSNA evoked by physiological stresses were mediated by central command, the feedback mechanisms via the afferent nerves (group III and IV fibers), and arterial and cardiopulmonary baroreflex (12, 14, 20). In our study, the response of single-unit MSNA was significantly augmented by physiological stresses. The sympathetic firing impulses contributed to augmentation of heart rate and arterial pressure through release of the neurotransmitter norepinephrine, whose plasma concentrations have been shown to correlate positively with multiunit MSNA (21). Analysis of norepinephrine spillover has shown a strong relationship between muscle sympathetic drive to muscle, heart (22), and kidney (23). However, it has been uncertain whether the release of neurotransmitter could be correlated with single-unit MSNA. Previous reports demonstrated that single-unit MSNA firing frequency was related to R-R interval and to blood pressure (9, 18). These results suggested that the augmentation of sympathetic vasoconstrictor unit firing could play an important role in increasing release of neurotransmitter.
The distribution of the firing pattern of single-unit MSNA during one cardiac interval has been described in healthy subjects (2, 3, 11). Multiunit MSNA is comprised of the various neuron firing units around the recording electrode. Some multiunit MSNA existed without appearance of a large unitary action potential, reflecting limitations of the recording process. In simultaneous recording of single-unit and multiunit MSNA, we also examined the distribution of bursts with 0 spikes to evaluate the burst that was only composed of other different unit firings. However, the additional consideration with our data concerning percentages of cardiac intervals in which neurons generated 1, 2, 3, or 4 spikes at rest were 79, 18, 4.8, and 0%, respectively, at rest, similar to findings in a previous investigation (3).
The most important advantage of single-unit MSNA analysis would be the ability to demonstrate multiple spikes firing within the burst. A previous study demonstrated that the measurement of single-unit MSNA provided both quantitative and qualitative discharge characteristics (10). In this study, we found that the Valsalva maneuver, but not handgrip exercise, affected the quality of multiunit bursts. Elam and Macefield (2) analyzed the effect of premature beats on single-unit MSNA in patients with congestive heart failure, finding the firing frequency of vasoconstrictor fibers to be augmented after premature beats together with the frequency of multiple firing per burst (i.e., 2 spikes being more common than 1 within a burst). Sympathetic excitation is known to result from premature beats that reduced arterial baroreceptor restraint because of the resulting fall in arterial blood pressure. The Valsalva maneuver affects arterial barorecepter in a manner similar to premature beats (1). These reports were in accord with our results that the Valsalva maneuver changed not only firing frequency, but also the firing pattern of single-unit MSNA.
Measurements of burst amplitude and area of integrated multiunit sympathetic firing were developed to resolve some problem in recording multiunit MSNA. However, these parameters are highly dependent on the position of the recording electrode, precluding the comparison of the actual sizes of bursts between different subjects (1, 16, 17). Although single-unit MSNA is more difficult to record than multiunit MSNA, such a measurement of the mean frequency of a single neuronal unit reflects the true central sympathetic output to the periphery better than the method that includes other recruited neural units (6). Nevertheless, the considerable advantages and benefits of recording single-unit MSNA have not yet been discussed. In this study, we demonstrated that the response of single-unit MSNA was more logical and responsive to physiological stresses than that of multiunit MSNA, especially during intense sympathetic activation. These findings suggested that single-unit MSNA analysis might be more sensitive in various physiological stresses than multiunit MSNA, which has an upper frequency limit of 100 per 100 heartbeats.
Several different mechanisms have been proposed to explain short-term sympathoexcitatory responses in humans (2, 3, 10). The first mechanism is an increase in the firing frequency of vasoconstrictor fibers that are already active. The second mechanism is an increase in multiple firing within a burst. The last mechanism is the recruitment of previously silent fibers. Although we could not identify mechanisms for increasing single-unit MSNA during each stress, we could characterize discharge behavior by intensity of sympathetic excitation. According to our data, responses of both single-unit and multiunit MSNA were similar during mild sympathetic activation. However, the frequency of single-unit MSNA increased much more than that of multiunit MSNA during marked sympathetic excitation. The multiunit MSNA must be comprised of both single-unit MSNA and other different and recruitment units. It is most likely that the firing pattern within one burst should be dependent on the remaining interval in the cardiac cycle because sympathetic firing could not increase during diastole in healthy subjects. Accordingly, the firing pattern of single-unit MSNA during one cardiac interval should not change with mild to moderate increase of burst incidence (such as with handgrip exercise), in which the recruitment of silent fibers and the increase of firing frequency can compensate the vasoconstriction enough for peripheral demands. However, the firing pattern of single-unit MSNA should shift toward multiple spikes within a burst during high burst incidence (such as the Valsalva maneuver), in which burst frequency is not able to increase further.
Our human subjects were healthy and had low resting sympathetic nerve activity. In subjects with high levels of sympathetic nerve activity at rest, future examination is anticipated. A previous report demonstrated that the firing pattern did not change in congestive heart failure, which is characterized by high sympathetic burst at rest (10). If sympathetic outflow at baseline is chronically activated, the change of the firing pattern would be induced during light exercise and the slope of the relationship between single-unit and multiunit MSNA might shift upward.
In conclusion, the frequency of single-unit MSNA increased much more than that of multiunit MSNA during marked sympathetic activation, whereas response of both single-unit and multiunit MSNA was similar during mild sympathetic activation. These results suggest that single-muscle vasoconstrictor fiber activity during physiological stresses is more sensitive in evaluation of sympathetic nerve activity than multiunit MSNA
We thank Dr. T. Mano (Tokai Central Hospital) and Dr. S. Toma (Dept. of Neurology, Yumura Hot Spring Hospital) for invaluable help with development of the microneurographic technique.
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- Copyright © 2006 by the American Physiological Society