INTRODUCTION

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RECORDINGS OF MULTIFIBER renal sympathetic nerve discharge show a rhythmic bursting multispike activity that is coupled to the cardiac cycle, being more prominent in diastole and less prominent in systole. Each burst is diamond shaped with the amplitude of the initial and terminal spikes being less than those in the middle of the burst. Each burst or peak may be characterized by its duration, height, and frequency. Measurement of mean integrated voltage of renal sympathetic nerve discharge over short time intervals, although providing a time-averaged measurement of total voltage, does not provide quantitative information on these characteristics (duration, height, frequency) of the individual peaks in synchronized renal sympathetic nerve discharge (15, 16). It has been determined that these characteristics of the individual peaks are defined by different aspects of neuroanatomic control with peak height being determined by the number of active renal nerve fibers (19), whereas peak frequency reflects the inherent generation of discharges by the central nervous system and their modulation by baroreceptor inputs (18). Additional studies indicate that increases and decreases in mean integrated voltage of renal sympathetic nerve activity are closely coupled to and dependent on parallel changes in the peak height of synchronized renal sympathetic nerve discharge, reflecting increases and decreases in the number of active renal nerve fibers, respectively (3, 4, 6, 10). Thus increases and decreases in mean integrated voltage of renal sympathetic nerve activity involves the recruitment of previously silent renal nerve fibers and the silencing of active renal nerve fibers, respectively. Because renal sympathetic nerve fibers are not a homogeneous population (7), this allows for the recruitment and derecruitment of groups of renal sympathetic nerve fibers that might be functionally specific, i.e., fibers that separately innervate and influence the individual effectors within the kidney, the vasculature, the tubules, and the juxtaglomerular granular cells.

When studying the influence of renal sympathetic nerve activity on renal function, investigators have generally used constant-voltage electrical stimulation to apply stimuli that were square wave in nature to a multifiber renal sympathetic nerve preparation, consisting of single pulses of known duration, amplitude, and frequency (reviewed in Ref. 5). By means of this approach, considerable information has been acquired concerning the renal sympathetic neural control of renal function. Results using direct electrical renal sympathetic nerve stimulation have been in qualitative agreement with those using reflex stimuli to physiologically activate renal sympathetic nerve fibers with regard to the control of the renal circulation, renal tubular sodium, and water reabsorption and renin secretion rate. This qualitative agreement has supported the validity of using results from artificial square-wave stimulation in understanding the physiological effects of renal sympathetic nerve activity on renal function. However, quantitative comparison has been difficult because of substantial differences in experimental design involved in these two quite different approaches.

Digital methods allow the construction of an external electrical stimulus pattern that faithfully reproduces that recorded in vivo from a multifiber renal sympathetic nerve preparation. This study compares the effects of multifiber renal sympathetic nerve stimulation using these computer-generated stimuli with those using the described standard square-wave paradigm on renal hemodynamic and excretory function in the anesthetized rat.

	METHODS

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Sprague-Dawley rats of either sex, weighing between 250 and 300 g, were used for all experiments. They were maintained in individual cages for a minimum of 1 wk before experimentation and were given normal rat chow and tap water as drinking fluid.

Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). An endotracheal tube was placed, and the rats were allowed to breath spontaneously. The right jugular vein and carotid artery were catheterized for administration of supplemental doses of anesthetic and isotonic saline at 50 µl/min and measurement of arterial pressure, respectively.

The left renal nerves were isolated, dissected, and placed on a bipolar silver wire stimulating electrode, and the nerve-electrode preparation was embedded in Wacker Sil-Gel. The left renal nerves were cut proximal to the electrode, assuring that the only electrical activity that passed to the left kidney was derived from the stimulating electrode. For measurement of renal blood flow (RBF), an electromagnetic flow probe was placed on the left renal artery. The left ureter was catheterized with a PE-50 catheter. For measurement of inulin clearance [taken as glomerular filtration rate (GFR)], inulin was administered as an intravenous priming dose and was added to the sustaining infusion of isotonic saline in sufficient quantity to establish and maintain a plasma inulin concentration of ~75 mg/100 ml. After surgical preparation, a 60-min equilibration period was allowed to elapse.

Computer Generated Stimuli

View larger version (36K): [in this window] [in a new window]   Fig. 1.   A: example of square-wave pattern; B: example of sinc-wave pattern; C: example of hybrid (diamond)-wave pattern, square · sinc. For same integrated voltage, either amplitude or duration of diamond wave will be twice that of square wave.

Except where specifically indicated, the diamond- and square-wave stimuli were constructed with a sample size of 150, corresponding to the approximate number of fibers often contained within a renal nerve bundle (3, 7). The duration of the diamond- or square-wave stimulus was 150 ms, which corresponds to the average duration of burst discharges as determined with the sympathetic peak detection algorithm for analysis of synchronized renal sympathetic nerve discharge recorded in vivo (3, 4, 17). The frequency at which the diamond- or square-wave stimulus was delivered was 1 Hz. The integrated voltage delivered was calculated as amplitude × duration for the square-wave and as 0.5 (amplitude × duration) for the diamond-wave stimulus; the units were V · ms. For the same integrated voltage, either the amplitude or the duration of the diamond wave will be twice that of the square wave.

Experimental Protocols

Diamond-wave stimuli: amplitude vs. duration. This protocol evaluated the effects on RBF of varying the level of integrated voltage in diamond-wave stimuli by changes in either duration or amplitude. Amplitude was varied between 1 and 1.5 V, and duration was varied between 150, 300, and 450 ms. The left renal nerves were stimulated for 30 s. The integrated voltages delivered were 300, 450, and 675 V · ms.

Diamond- vs. square-wave stimuli: RBF, GFR, and urinary sodium excretion. This protocol compared the effect of diamond- vs. square-wave stimuli delivered at the same integrated voltage (subvasoconstrictor) on RBF, GFR, and urinary sodium excretion (U_NaV). The diamond wave was generated with an amplitude in either the uniform white noise or sinc patterns that was just beneath the threshold for a decrease in RBF. A square wave of identical integrated voltage was generated by selecting the amplitude in the uniform white noise pattern. After two consecutive 10-min control urine collection periods, the diamond wave was applied, and two consecutive 10-min experimental urine collection periods were made. Then the diamond wave was stopped and two consecutive 10-min recovery urine collection periods were made. After a 15-min interval, the sequence of control, experimental, and recovery urine collection periods was repeated with the square wave during the experimental period. In half the rats, the order of stimulation patterns was reversed.

Continuous vs. intermittent stimulation: fixed train length. The purpose of this protocol was to compare the effects of continuous vs. intermittent stimulation on RBF. Square-wave pulses of 0.2 ms duration and 15 V in amplitude were administered at 1, 3, 5, and 10 Hz in a continuous mode (pulses equally spaced throughout the 1-s time interval) and at 2, 6, 10, and 20 Hz in an intermittent mode in which the pulses were given as a 0.5-s train at the onset of each 1-s time interval. Thus the intermittent and continuous stimuli were applied at the same overall number of pulses per second. In the intermittent stimulation all the pulses were given in the first 0.5 s of each 1-s time interval, such that the second 0.5 s of each 1-s time interval was free of pulses. The left renal nerves were stimulated for 30 s. The amplitude of 15 V and the duration of 0.2 ms were chosen to mimic commonly used (renal) sympathetic nerve stimulation parameters in which supramaximal voltage (amplitude) is applied at a fixed duration with the frequency being the independent variable (5).

Continuous vs. intermittent stimulation: rest period. This protocol compared the effects on RBF of continuous stimulation at 5 Hz vs. intermittent stimulation at various frequencies while keeping the number of pulses delivered in each 1-s time interval constant at 5 Hz. The frequencies of intermittent stimulation were 6, 7, 8, 9, 10, and 20 Hz; to deliver the same number of pulses (i.e., 5 Hz) in the 1-s time interval, these frequencies were applied for 833, 714, 625, 556, 500, and 250 ms, respectively, out of each 1-s time interval. Thus there was a reciprocally varying amount of time within the 1-s time interval that was free of pulses ("rest time"), i.e., 167, 286, 375, 444, 500, and 750 ms. The left renal nerves were stimulated for 30 s.

Data analysis. For the renal artery flow probe, mechanical zero was determined by transient occlusion of the renal artery distal to the flow probe. The flow probe was calibrated in situ by making timed collection of blood emerging from the transected renal artery. RBF responses were calculated as both the maximum steady-state response (magnitude, unrelated to time) and area under the curve (AUC; integrated, magnitude × time). The results with the two methods were similar for all protocols except those involving comparisons of continuous and intermittent stimulation (Continuous vs. intermittent stimulation: fixed train length and Continuous vs. intermittent stimulation: rest period). Urine volume was determined gravimetrically assuming specific density of 1. Inulin and sodium were measured in plasma and urine by an anthrone colorimetric method and lithium internal standard flame photometry, respectively. GFR was taken as the clearance of inulin, C_in = (U_in · V/P_in), where C_in represents inulin clearance, U_in and P_in represent the concentrations of inulin in urine and plasma, respectively, and V is urinary flow rate. Urinary sodium excretion is represented by U_NaV, where U_Na is the urine sodium concentration.

Statistical analysis. Results were analyzed with ANOVA for repeated measurements followed by post hoc testing with the Student-Newman-Keuls test to identify within and between group differences (24). Differences were taken as statistically significant when P < 0.05. Data in text, tables, and figures are presented as means ± SE.

	RESULTS

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Analysis of results by sex showed there to be no significant difference between male and female rats.

Diamond- vs. Square-Wave Stimuli

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of diamond- vs. square-wave stimuli at identical steps of integrated voltage delivered on renal blood flow (RBF). Stimuli were 150 ms in duration, delivered at a frequency of 1 Hz; renal nerves were stimulated for 30 s at each step. Circles represent diamond-wave stimuli generated by changes in the noise pattern () or in the sinc pattern (). , square-wave stimuli generated by changes in the noise pattern. Data are means ± SE, n = 10. * P < 0.05 for square-wave stimuli vs. both diamond-wave stimuli.

Diamond-Wave Stimuli: Amplitude vs. Duration

Diamond- vs. Square-Wave Stimuli: GFR, Urinary Flow Rate, and Sodium Excretion

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Renal hemodynamic and urinary sodium excretion (UNaV) response to diamond- vs. square-wave stimulation at identical values of integrated voltage (40 ± 2 V · ms) that was subthreshold for decreases in RBF. Data are means ± SE for RBF and glomerular filtration rate (GFR) and values for individual rats for UNaV; n = 12. C, control period; S, stimulation period; R, recovery period.

Continuous vs. Intermittent Stimulation: Fixed Train Length

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects on RBF of intermittent vs. continuous stimulation with square-wave stimuli at same number of pulses. Data are means ± SE, n = 16. AUC, area under the curve. * P < 0.05 for continuous vs. intermittent.

Continuous vs. Intermittent Stimulation: Rest Period Basal

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of variations in rest time on RBF response to intermittent stimulation compared with continuous stimulation. Square-wave stimuli at 5 pulses/s. Data are means ± SE, n = 14. Data are presented as %change in AUC RBF for intermittent stimulation vs. continuous stimulation with number of pulses being constant at 5/s (C5). All points are significantly different (P < 0.05) from no (0%) change.

	DISCUSSION

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The major finding of the current study is that, at constant integrated voltage over time, the pattern of renal sympathetic nerve stimulation has a substantial influence on the renal functional responses observed. Not surprisingly, the pattern of synchronized renal sympathetic nerve discharge as observed in in vivo recordings from multifiber renal sympathetic nerve preparations produces alterations in renal function that are different from those observed with the commonly employed square-wave pattern of renal sympathetic nerve stimulation. The findings from intermittent stimulation with the dependence of the magnitude of the response on the duration of the rest time suggest that issues related to exhaustion of the neurotransmitter release mechanism within the renal sympathetic nerve terminals are important determinants of the magnitude of the overall renal functional response.

The initial protocol indicates that exogenously delivered stimuli that more faithfully represent what is observed spontaneously in the renal sympathetic nerves in vivo produce greater renal vasoconstrictor responses than the conventionally applied square-wave stimulus pattern, when the two stimuli are delivered at identical integrated voltage. Thus it is likely that the diamond-wave pattern results in a greater release of neurotransmitter than the square-wave pattern. Furthermore, although the integrated voltage (seen by the nerve preparation as current) is an important determinant of the amount of neurotransmitter released, it appears likely that the nature of the wave pattern that conveys this voltage also contributes quantitatively to this process. In the canine gracilis muscle and the porcine spleen, stimulating the nerves either with impulses derived from a recording of the normal irregular sympathetic discharge to human skeletal muscle or with conventional continuous regular impulses indicated that the vasoconstrictor responses and the overflow of norepinephrine were influenced by the pattern as well as the frequency of sympathetic nerve stimulation (12, 21).

Within the diamond-wave pattern, increasing integrated voltage by increasing either the duration or the peak voltage of the pattern produced different RBF responses. At each level of peak voltage, the decreases in RBF were similar at each duration. However, at each duration, the decreases in RBF were enhanced to a similar degree by increasing the peak voltage, i.e., amplitude modulation. By analogy with analysis of synchronized renal sympathetic nerve discharge using the sympathetic peak detection algorithm (17), increases in duration reflect a greater degree of asynchrony among a fixed number of active fibers, whereas increases in peak voltage reflect an increase in the number of active fibers via recruitment of previously silent fibers. Although increased asynchrony among a fixed number of active fibers would not be predicted to produce a greater renal vasoconstrictor response, an increase in the overall number of active fibers would be expected to result in a greater renal vasoconstrictor response.

The functional effect of these differences in wave pattern is not limited to the renal vasculature. At similar levels of integrated voltage wherein neither diamond- nor square-wave pattern affected RBF or GFR, U_NaV was decreased by diamond-wave pattern but was unaffected by square-wave pattern, indicating a direct and selective (nonvascular) effect on renal tubular sodium reabsorption. These results suggest that the amounts of neurotransmitter released by the two patterns of stimulation, although subthreshold for renal vasoconstriction or decreases in GFR, were sufficiently different to be expressed as differential effects on renal tubular sodium reabsorption. These results confirm the view that the stimulus-response curve for renal tubular sodium reabsorption and U_NaV lies to the left of that for RBF (5).

Comparison of intermittent and continuous stimulation at the same number of pulses per second demonstrated that intermittent stimulation produced faster (smaller time constant) and larger renal vasoconstrictor responses. Because the intermittent stimulation used a frequency that was twice that of continuous stimulation but was delivered over the initial 0.5 s of each 1-s interval (i.e., same number of pulses per second), the difference between the renal vasoconstrictor responses to intermittent and continuous stimulation was relatively constant over the entire frequency range. This also highlights the fact that a shift from continuous to intermittent discharge in vivo would be expected to augment the renal vasoconstrictor responses even in the lower physiological range of discharge frequencies, e.g., 0 to 6-8 Hz. Others have demonstrated that intermittent stimulation (bursts) produces faster and greater vasoconstrictor responses than continuous stimulation in cat skeletal muscle (1), rabbit hindquarters (2), and rat stomach (22). High-frequency intermittent stimulation has been reported to result in a greater release of norepinephrine from selected arteries (nonrenal) than continuous stimulation (11). Neuropeptide Y is preferentially released from the kidney by high-frequency intermittent stimulation of the porcine renal sympathetic nerves (20).

Greater norepinephrine release is but one possible mechanism to explain the greater effectiveness of diamond- vs. square-wave stimuli and of intermittent vs. continuous stimulation. It is possible that decreased norepinephrine reuptake and/or degradation might contribute to the enhanced renal vasoconstrictor responses observed. With respect to reuptake, it is known that this is an extremely rapid process that is voltage sensitive (9). Norepinephrine uptake increases markedly at negative potentials. The implication is that noradrenergic synapses clear more rapidly at hyperpolarized potentials than at depolarized potentials. It could be predicted that depolarization of the synaptic terminal would slow the rate of norepinephrine uptake, resulting in an increased concentration and/or prolonged duration of norepinephrine in the synaptic cleft. Thus diamond-wave stimuli or intermittent stimulation could influence the voltage profile of the synapse in such a way as to favor an increased norepinephrine concentration or prolonged norepinephrine duration in the synaptic cleft, resulting in an enhanced renal vasoconstrictor response.

Folkow (8) demonstrated that 10 Hz continuous stimulation produced nearly maximal constrictor responses of the cat skeletal muscle vasculature, whereas the physiological range of the average discharge frequency of sympathetic nerve fibers was between 0 and 6-8 Hz. In response to activation of somatic afferent reflexes in cats, the maximal reflex-induced firing frequency of single pre- and postganglionic fibers was <20 Hz (13, 23). Thus these data indicate that the maximum firing frequency of sympathetic postganglionic fibers is approximately twice the average firing frequency observed under basal physiological conditions.

In these cat experiments (8), stimulation at supramaximal frequencies (15-20 Hz) for ~2-3 min resulted in declining vasoconstrictor responses that could be restored by temporarily stopping stimulation or lowering the stimulation frequency into the physiological range (0 to 6-8 Hz). This was taken as a manifestation of exhaustion of the neurotransmitter release mechanism that could be restored by a short period of complete (no stimulation) or relative rest (slower stimulation rate). These findings suggested the hypothesis that there was an optimal relationship between the fraction of each time interval during which stimulation occurred and that during which there was no stimulation (rest). Herein, it was found that there was increasing augmentation of the renal vasoconstrictor response to intermittent stimulation compared with continuous stimulation over the range of rest periods from 167 to 500 ms (16.7-50% of the total 1-s time interval) with a maximum plateau value of ~55% at 500 ms. It should be noted that this maximum rest period of 500 ms is 2.5 times the rest period between pulses during continuous stimulation at 5 Hz (i.e., 200 ms). Furthermore, at a rest period similar to that of continuous stimulation at 5 Hz (i.e., 200 ms) intermittent stimulation augmented the renal vasoconstrictor response by nearly 35%.

With use of the microneurographic technique to examine the discharge characteristics of single vasoconstrictor fibers in man, it was observed that individual vasoconstrictor units discharged in only 21% of cardiac cycles with an overall mean frequency of 0.47 Hz (14). When calculated from cardiac cycles in which a single unit fired more than once, the mean within-burst firing rate was 18.8 ± 2.5 Hz with occasional maximal instantaneous frequencies above 50 Hz being observed. It has been suggested that these higher frequency intermittent firing rates may optimize the contractile responses of vascular smooth muscle and be engaged under circumstances where responses that are more rapid and of greater magnitude are required, e.g., to defend circulatory integrity. From the current studies comparing intermittent with continuous stimulation, not only would the faster and greater vasoconstrictor responses be beneficial but also the optimization of the rest time with less likelihood of exhaustion of the neurotransmitter release mechanism coupled with enhanced release of neurotransmitter.

The renal nerves were cut proximal to the stimulating electrode, thus removing tonic efferent renal sympathetic nerve discharge. As this was done acutely, it is not likely that there was renal hypersensitivity to norepinephrine. In the basal state, even after anesthesia and surgery, efferent renal sympathetic nerve activity is relatively low, as reflected by the fact that there was no significant change in renal blood flow after renal denervation. This makes it unlikely that the removal of tonic efferent renal sympathetic nerve activity significantly participated in the observed differences between diamond- and square-wave stimuli and between continuous and intermittent stimulation.

In summary, these results indicate that the pattern of renal sympathetic nerve stimulation (activity) significantly influences the rapidity, magnitude, and selectivity of the renal vascular and tubular responses.

Perspectives