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The aortic nerve-sympathetic reflex in the rat
Journal of the Autonomic Nervous System, 13 (1985) 65-79 Elsevier
65
JAN 00434
The aortic nerve-sympathetic reflex in the rat Y o s h i n o b u N u m a o , M a m o r u Siato, N a o h i t o T e r u i a n d M o m o r u K u m a d a Institute of Basic Medical Sciences, The University of Tsukuba, lbaraki- ken 305 (Japan) (Received July 10th, 1984) (Revised version received December 13th, 1984) (Accepted January 2nd, 1985)
Key words: rat's aortic nerve - renal nerve activity - cardiac nerve activity - inhibitory component - superior laryngeal nerve
Abstract The effects of stimulation of aortic nerve A- and C-fibers on the renal and cardiac sympathetic nerve activities in anesthetized and immobilized Sprague-Dawley rats were investigated. A separate aortic nerve was found in 46 rats (90%) out of 51. Activation of A- and C-fiber groups, alone or in combination, resulted in an inhibition of renal and cardiac nerve activities. However, an excitatory component preceding the inhibitory component, representing the reflex response to stimulation of non-barosensory afferent fibers contained in the carotid sinus or aortic nerve, was never observed. This results provides electrophysiological evidence supporting the view that the rat's aortic nerve does not contain a significant amount of functionally active non-barosensory afferents. As with the aortic nerve relfex in the rabbit and cat, the sympatho-inhibitory action of C-fibers was more powerful and longer-lasting than that of A-fibers. Furthermore, the C-fiber reflex was elicited at stimulus frequencies as low as 2 Hz. N o significant difference was found between the reflex response of cardiac and renal nerves. On the other hand, stimulation of the superior laryngeal nerve, which constitutes an important pathway carrying arterial baroreceptor fibers, caused a reflex sympathetic response typically consisting of excitatory and inhibitory components. Thus, the rat's aortic nerve provides a useful experimental means to activate selectively central neural structures associated with barosensory afferents and to elicit the reflex response homologous to that in the arterial baroreceptor reflex in rabbits and cats.
Correspondence: M. Kumada, Institute of Basic Medical Sciences, The University of Tsukuba, lbaraki-ken 305, Japan. 0165-1838/85/$03.30 ci3 1985 Elsevier Science Publishers B.V. (Biomedical Division)
66
Introduction It had long been thought that, in the rat, a separate aortic nerve was not present in most instances [11,20,22]. In fact, afferent fibers from the aortic arch baroreceptors variously course through the vagus, cervical sympathetic and recurrent and superior layrngeal nerves in this animal species [2,11,20]. A recent series of studies by Sapru and co-workers [28,29], however, demonstrated that, in certain strains of rats, a separate aortic nerve is regularly found and that functionally the aortic nerve of rats appears to consist exclusively of barosensory fibers. Considering an ever increasing number of studies on the central cardiovascular control conducted in rats, an anatomically well-defined pure barosensory nerve originating from arterial baroreceptors provides an useful experimental means to activate the central neural structures involved in the arterial baroreceptor reflex. In the present study, we correlated activation of aortic nerve A- a n d / o r C-fibers of the rat with the reflexly evoked response of the sympathetic postganglionic discharges and arterial pressure. The renal and cardiac sympathetic nerve activities were chosen as indices of the reflex efferent response. Special attention was paid to examine whether or not the rat's aortic nerve contained non-barosensory fibers such as chemosensory fibers in the cat [6] or nociceptive fibers in the rabbit [24]. If such fibers were present to a significant degree in the aortic nerve, the reflexly evoked sympathetic response would exhibit an excitatory component [6,24]. We also investigated the reflex effect of stimulation of the superior laryngeal nerve on the sympathetic nerve activity, since that nerve constitutes an important afferent pathway carrying barosensory signals from the aortic arch region and can elicit, on electrical stimulation, a pattern of hemodynamic responses identical to that of the arterial baroreceptor reflex [2,11].
Materials and Methods
Preparation of animals Experiments were performed on 55 mate Sprague-Dawley rats (Doken Lab., Ibaraki-ken, Japan) of 10-14 week-old with body weight between 300 and 480 g. The body weight of 10 and 14 week-old male rats of this strain, according to the supplier's data, was 329 + 24 g and 420 + 38 g (mean + S.D.), respectively. Each animal was anesthetized with an intraperitoneal administration of urethane (initial dose 800 mg/kg). Supplemental injections (80 m g / k g ) were given then necessary to maintain adequate anesthesia. After insertion of tracheal, arterial and venous cannulas, the animal was paralyzed with pancuronium bromide (initially 2.5 mg/kg, i.v., thereafter 1.2 m g / k g / h ) and artificially ventilated with oxygen by a Harvard respiratory pump. Body temperature was maintained at 37 ° + 0.5°C by a thermostatically regulated heating pad connected to a rectal thermistor probe. Measurement of cardiovascular and sympathetic nerve activities Instantaneous and mean arterial pressure and heart rate were monitored con-
67 tinously in all experiments. Arterial pressure was recorded from the iliac artery by a polyethylene catheter inserted through the femoral artery and connected to a transducer (Nihon Kohden MPU-0.5). Heart rate was computed from the arterial pressure pulse by a tachometer (Nihon Kohden AT-600G). The left renal nerve was approached retroperitoneally through a left flank incision, and was prepared for recording from near the renal artery. The right inferior (or stellate) branch of the cardiac nerve was approached from the upper thorax through a mid-sternal incision. That branch was identified as the major branch originating from the right stellate ganglion which proceeded caudally along the superior vena cava to the base of the heart. Communicating fibers from the vagus nerve, when visible, were removed. In preparing the cardiac nerve, the pleura and pericardium remained unopened. The central cut end of the nerve was placed on a pair of bipolar platinum electrodes connected to an amplifier (Nihon Kohden AVB-8). The output of the amplifier was displayed on an oscilloscope (Tektronix 5113). The lower and higher cut-off frequencies of the recording system were 100 and 3000 Hz, respectively. The multifiber sympathetic nerve activity (SNA) was usually rectified, and passed through a low-pass filter (time constant 20 ms). The evoked SNA was averaged during 16-32 successive sweeps with a data processing computer (Nihon Kohden ATAC-350) and displayed on an x-y recorder (Watanabe 4401). Because of the proximity of stimulating and recording electrodes, the evoked response recorded from the cardiac nerve was usually contamined with artifacts. Such artifacts preceding the evoked response were eliminated by a gate circuit designed and constructed by ourselves. The circuit switched off the input to the amplifier for 0.3-0.6 ms after the onset of the stimulating pulse and effectively eliminated the stimulus artifact without significantly distorting the evoked response. In quantitating the evoked response of the sympathetic nerve, we took into consideration temporal as well as individual variations of the background level of sympathetic discharges. The 'Normalized Response Magnitude' defined below was used for this purpose [24]. When SNA decreased below the prestimulus control level, the area below the control SNA was calculated and then divided by SNA integrated over the 1-s period immediately prior to nerve stimulation. The negative sign was attached when SNA decreased below the control level. When SNA increased above the prestimulus control level, the area above control SNA was divided by SNA integrated over the 1-s period prior to nerve stimulation. The positive sign was attached in this case. Electrical stimulation of the aortic ner~,e Aided by a dissecting microscope, the left aortic nerve was identified at its junction with the superior laryngeal nerve. The aortic nerve was then carefully separated from the adjacent cervical sympathetic nerve, dissected free of the surrounding connective tissue, and "transected 1 2 cm peripherally. The distal end of the nerve was placed across a pair of platinum stimulating electrodes spaced 2 - 5 m m apart. Electrical stimuli were square-wave pulses of 0.1 ms duration delivered to the animal from a pulse generator (Nihon Kohden SEN 7103) through an isolation
68 unit. The stimulus frequencies were between 1 and 300 Hz and the stimulus intensity usually ranged from 0.2 to 30 V. We did not record the afferent nerve activity and the reflex sympathetic response to aortic nerve stimulation simultaneously, because this maneuver often damaged the aortic nerve. Therefore, the afferent nerve activity was examined in the same animal after studying the reflex reponse first. To record the aortic nerve discharge, another pair of platinum electrodes was attached to the nerve several millimeters centrally, and the distal end of the nerve was electrically stimulated as explained above. In case where we failed to find a separate left aortic nerve, the same procedures were repeated on the right side to prepare the right aortic nerve. In 5 rats, the left superior laryngeal nerve was prepared for afferent stimulation. The central cut end of the nerve, dissected for 4 - 6 m m from its point of emergence between the larynx and cricothyroid muscles, was stimulated to elicit the reflex responses.
Results
Afferent discharges of the aortic nerve In 42 out of 51 rats we used, an aortic nerve was found as a separate strand on the left side. Of the 9 animals in which a separate left aortic nerve was absent, the right aortic nerve could be isolated in 4 cases. Thus, in over 90% of the animals, an anatomically separate aortic nerve could be isolated and stimulated. As the intensity of the single pulse stimulus to the aortic nerve was increased, 2 groups of deflections regularly appeared in the action potential recorded proximally from the same nerve (Fig. 1). Conduction velocities of the 2 deflections at 37°C, calculated as the latency-to-onset divied by the distance between the stimulating and recording electrodes, were 18.1 + 3.8 m / s (n = 13) and 0.93 + 0.41 m / s (n = 15), respectively. The temperature coefficient of 1.6 was used for the calculation [24,32]. The two groups thus represent activity of the aortic nerve A- and C-fibers. Conduction velocity for the A-fibers, calculated as the latency-to-peak divided by the distance was 8.0 _+ 1.1 m / s (n = 15). The same calculation was not made for the C-fibers, since the afferent activity quite often consisted of multiple deflections of comparable sizes. We subsequently investigated the relation between the afferent fiber group activated by graded stimulation and the consequent response indicated by sympathetic nerve activity and arterial pressure. Reflex sympathetic nerve response evoked by stimulation of the aortic nerve Stimulation of the aortic nerve with a pulse train of 100 Hz elicited inhibition of the renal and cardiac nerve discharges, when the stimulus intensity was slightly above the threshold of A-fibers (Figs. 1 and 2). The response, termed the inhibitory component, was augmented in magnetude and duration as stimulus intensity was increased. With recruitment of aortic nerve C-fibers, a further increase in that component was brought about in both remal and cardiac nerve responses. This
69
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Fig. l. Correlation between the compound action potential of the aortic nerve (left panel) and the evoked response of the renal nerve (right panel) to graded electrical stimulation of the aortic nerve. To elicit the compound action potential in the left panel (a e), a single rectangular pulse was given distally to the central cut end of the aortic nerve at the moment indicated by the small triangles in each record. It was averaged over 32 successive sweeps produced every 1 s. The duration of the pulse was fixed at 0.1 ms, while its amplitude was varied between 0.35 V to 30 V as shown in the.upper right corner of the tracings. Two major peaks corresponding to activation of aortic nerve A- and C-fibers are seen in d and e. When such stimulating pulses were applied repetitively to the aortic nerve at the frequency of 100 Hz, the evoked renal nerve response corresponding to the left panel was obtained (f-j). The stimulus train, given once every 20 s for 32 times, lasted for 1 s as shown by horizontal bars at the top of each tracing.
increase was partly ascribable to a prolongation of the silent period, and partly to a subsequent weak but long-lasting inhibition of the sympathetic nerve activity (Fig. 2). With the fixed stimulus frequency of 100 Hz, no marked difference was observed in the magnitude of the reflex inhibition between the renal and cardiac nerves. At times, a sudden and transient increase in the sympathetic activity was observed on cessation of the stimulus train (Fig. lg and h). The onset latency of the inhibitory component was 65 ± 35 ms (mean + S.D.; n = 15) with the renal nerve and 37 ± 25 ms (n = 7) with the cardiac nerve. In none of 51 rats we examined, activation of A- a n d / o r C-fibers employing various combinations of stimulus parameters resulted in an excitatory component preceding the inhibitory component (Fig. 1). Such an excitatory component, observed in the aortic nerve-sympathetic reflex in cats [6] and rabbits [24], is known to originate from non-barosensory fibers. The absence of the excitatory component in the aortic nerve reflex of the rat is electrophysiological evidence indicating that
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Fig. 2. Effect of different intensities of electrical stimulation of the aortic nerve on the inhibitory component of renal (upper panel) and cardiac (lower panel) nerve activity: The stimulus consisted of a 1s train of square pulses. Duration and frequency of pulses were 0.1 ms and 100 Hz, respectively. Intensities of stimulation were altered as indicated by the abscissa. The ordinate represents reflex responses expressed as the Normalized Response Magnitude. Thick broken lines are the mean values of responses, whereas thin solid lines represent responses in individual experiments. At the top of each illustration is shown the range of thresholds of aortic nerve A- and C-fibers. Data were obtained from experiments in 5 rats for each nerve. f u n c t i o n a l l y the rat's aortic nerve consists exclusively of b a r o s e n s o r y fibers. It should be n o t e d that s t i m u l a t i o n of the aortic nerve never elicited excitation of the cardiac nerve discharge. This result d e m o n s t r a t e s that the evoked response of the cardiac nerve in our experiments reflects activity of the sympathetic b u t n o t vagal fibers efferent to the heart. H a d there b e e n a significant a m o u n t of f u n c t i o n a l l y active vagal fibers in our cardiac nerve p r e p a r a t i o n , a n excitatory c o m p o n e n t would have been elicited [15].
Frequency characteristics of the aortic nerve-sympathetic reflex W h e n the aortic nerve A-fibers were excited m a x i m a l l y without activating C-fibers, a m i n u t e i n h i b i t o r y c o m p o n e n t appeared b o t h in the renal a n d cardiac nerve responses at the frequency of 20 Hz. The i n h i b i t o r y c o m p o n e n t increased in size as the stimulus frequency was increased a n d reached a n e a r m a x i m u m at 100 Hz (Fig.
3). O n s i m u l t a n e o u s activation of aortic nerve A- a n d C-fibers, the sympatho-in_hibitory response was o b t a i n e d at frequencies above 1 Hz. The response was graded with
71
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Fig. 3. Stimulus frequency-response characteristics for the inhibitory component of renal (upper panel) and cardiac (lower panel) nerve activity by electrical stimulation of the aortic nerve. The inhibitory component was expressed in terms of the Normalized Response Magnitude. Stimulus frequencies were indicated by the abscissa. Attached to each characteristic curve is the fiber,group(s) activated to elicit the response. The stimulus consisted of a 1 s train of square wave pulses. Intensity and duration of the pulse were 1-2 V and 0.l ms, respectively,to activate aortic nerve A-fibers alone, or 20 V and 0.1 ms to activate A- and (.'-fibers in combination. Vertical lines are S.D. Data were obtained from 5 rats for each nerve activity.
regard to stimulus frequency a n d reached a near m a x i m u m at 100 Hz (renal nerve) or 200 Hz (cardiac nerve). I n terms of the N o r m a l i z e d Response M a g n i t u d e , s y m p a t h o - i n h i b i t o r y responses of cardiac a n d renal nerves were c o m p a r a b l e (Fig. 3). However, the m a x i m a l response of the cardiac nerve tended to be greater than that of the renal nerve, although the difference was statistically insignificant ( P < 0.2; S t u d e n t ' s t-test). N a m e l y , with the stimulus frequency of 200 Hz a n d intensity of 20 V, the reflex i n h i b i t i o n of the renal a n d cardiac nerve activities was 2.4 ___0.7 ( m e a n + S.D.; n = 5) a n d 2.9 + 0.9 (n = 5), respectively. Inhibition curve
A frequently used way to express the e x c i t a b i l i t y - r e c o v e r y property of a symp a t h o - i n h i b i t o r y reflex system is to apply c o n d i t i o n i n g a n d test stimuli to the system, while recording the efferent activity [18,24,32]. The s y m p a t h o - i n h i b i t o r y effect of aortic nerve A- a n d C-fibers was c o m p a r e d in 4 rats by m e a n s of this m e t h o d to o b t a i n the i n h i b i t i o n curve (Fig. 4). T h e c o n d i t i o n i n g stimulus consisted of a train of
72
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Fig. 4. Inhibitory curve of the aortic nerve reflex mediated by A-fibers (left panel) or A- and C-fibers (right panel). The conditioning stimulus was a train of pulses given to the aortic nerve to excite A-fibers (amplitude of pulse 2 V, duration of pulse 0.1 ms and frequency 100 Hz) or A- and C-fibers (amplitude 20 V, duration 0.1 ms and frequency 20 Hz). The duration of the pulse train was 1 s for the former and 0.9 s for the latter. Test stimulation was 2 pulses having the amplitude of 100/~A, duration of 0J2 ms and pulse interval of 10 ms. The stimulus site was 2 mm anterior to the obex, 1 mm lateral to the mid-sagittal plane and 0.75 mm below the dorsal surface of the brainstem. It was within the medullary reticular pressor area: The interval between conditioning and test stimuli was varied up to 2 s. The upper drawings consist of superimposed tracings of renal nerve activity elicited by test stimuli given at intervals indicated by small dots. In the lower drawings, the reflex response of renal nerve activity is plotted as a function of time interval.
pulses, given to the aortic nerve, with the d u r a t i o n of 1 s or a little less than that. A silent period of renal sympathetic discharges lasting a b o u t 1 s was p r o d u c e d by the stimulus train. Test s t i m u l a t i o n was 2 pulses delivered through a u n i p o l a r electrode placed in the medullary reticular pressor area [1,5] from the exposed dorsal surface of the lower b r a i n s t e m . This area encompassed the n u c l e u s reticularis parvocellutaris as well as passing catecholaminergic fibers originating from cells in the ventrolateral m e d u l l a [27]. W h e n the aortic nerve A-fibers alone were activated by a 100 Hz train of pulses, the i n h i b i t o r y effect was observed d u r i n g the c o n d i t i o n i n g stimulus. The excitability, however, recovered within 0.4 s after cessation of the stimulus a n d there usually followed a period of e n h a n c e d excitability (Fig. 4). The aortic nerve was then stimulated b y a 20 Hz train of pulses with a stimulus intensity above the threshold for C-fibers. A t this stimulus frequency, the reflex response a t t r i b u t a b l e to the aortic nerve A-fibers was m i n i m a l [24]. The i n h i b i t o r y effect of C-fibers assessed in this way was more powerful t h a n that of A-fibers a n d outlasted the c o n d i t i o n i n g stimulus. Even 1 s after cessation of the c o n d i t i o n i n g stimulus, the reflex sympathetic response to the test stimulus was partially suppressed. These differences
73 between barosensory A- and C-fibers with respect to their sympatho-inhibitory e f f e c t a r e q u i r e s i m i l a r t o t h o s e i n t h e a o r t i c n e r v e r e f l e x of t h e r a b b i t [24] o r c a t [25].
Depressor response to actiuation of aortic nerve A- and C-fibers The effect of electrical stimulation of the rat's aortic nerve on hemodynamic and respiratory variables has already been described systematically by Sapru, Gonzalez a n d K r i g e r [28]. T h e y v a r i e d s t i m u l u s p a r a m e t e r s o v e r a w i d e r a n g e , w h i l e r e c o r d i n g responses of arterial pressure, heart rate and respiratory movement. In the present AORTIC NERVE POTENTIAL
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Fig. 5. Correlation between the compound action potential of the aortic nerve (left panel) and the depressor response (middle and right panels) to graded electrical stimulation of the aortic nerve. The compound action potential was elicited by a single rectangular pulse having the duration of 0.1 ms and 5 levels of stimulus intensity shown in the upper right corner of the tracings. Graded depressor responses were obtained by applying such stimulating pulses to the aortic nerve at the frequency of 20 Hz (middle panel S or 100 Hz (right panel). The stimulus train lasted for 10 s as shown by horizontal bars at the top of middle and right panels. Fig. 6. Stimulus intensity-response characteristics (upper panel) and stimulus frequency response characteristics (lower panel) for the depressor response elicited by electrical stimulation of the aortic nerve. The response was expressed as change in arterial pressure from the prestimulus level. Stimulus consisted of a 10 s train of square pulses of 0.1 ms duration. In the upper panel, the stimulus frequency was 100 Hz (filled circles) or 20 Hz (open circles) and the stimulus intensity was indicated by abscissa. The range of thresholds of aortic A- and C-fibers was shown at the top of the illustration. Data were obtained from 13 rats. In the lower panel, the stimulus intensity was 1-2 V to activate A-fibers near maximally without activating C-fibers (filled circles) or 10-30 V to elicit a maximal depressor response due to simultaneous activation of A- and C-fibers (open circles). Stimulus frequency was indicated by the abscissa. Data were obtained from 11 rats.
74
study, we correlated activation of aortic nerve A- and C-fibers to their depressor action, when the nerve was stimulated by a 10 s train of pulses of 0.1 ms duration at frequencies of 20 and 100 Hz (Figs. 5 and 6A). Because the animals were immobilized by pancuronium bromide, an agent known to posses a vagolytic action [31], the depressor response described here was predominatly due to sympathetic inhibition. Actually, the reflex decrease in heart rate was mostly less than 40 beats/rain. At stimulus intensities above the threshold of A-fibers, a sizeable depressor response was elicited by a 100 Hz stimulus train. Recruitment of C-fibers resulted in a minor additional depressor response at that stimulus frequency. Thus, the depressor reponse to a stimulus train of 100 Hz reflected predominantly the relfex action of aortic nerve A-fibers. On the other hand, at a fixed stimulus frequency of 20 Hz, only a minimal depressor response was evoked by aortic nerve A-fibers. The response, however, was markedly augmented on activation of C-fibers, even though A-fibers were already maximally excited. As was demonstrated by experiments in which activation of aortic nerve A-fibers of the rabbit was suppressed by anodal block [24], a possible contribution of A-fibers to the reflex response was expected to be small over the range of stimulus frequency below 20 Hz. It is most likely that the depressor response to a stimulus train of 20 Hz here predominantly reflected the reflex action of aortic nerve C-fibers. The effect of stimulus frequency on the depressor response was summarized in the lower panel of Fig. 6. When the A-fibers were excited near maximally without activating C-fibers, the depressor response was elicited at stimulus frequencies above 10 Hz. The response was increased in magnitude as the stimulus frequency was raised and reached a maximum at 150 Hz. On simultaneously activation of A- and C-fibers, a depressor response was elicited at frequencies as low as 2 Hz and the maximal response was attained at 100 Hz. Considering a minimal contribution of aortic nerve A-fibers on the baroreceptor reflex over a stimulus frequency range below 20 Hz (see above), the reflex depressor response of C-fibers of the rat, like those of rabbits [9,24] and cats [10,19], appear to operate over a stimulus frequency range lower than that of A-fibers.
Reflex response to stimulation of the superior laryngeal nerve The rat's superior laryngeal nerve carries part of the afferent barosensory signal from the aortic arch region [2]. That nerve may even constitute a significant afferent pathway for the aortic baroreceptor reflex, since the reflex cardiovascular changes are greatly attenuated by its transection [11]. Furthermore, by a suitable choice of stimulus parameters, stimulation of the superior laryngeal nerve produces a pattern of hemodynamic responses identical to that of the arterial baroreceptor r e f l e x [ l l ]. We therefore examined in 5 rats whether or not stimulation of the superior laryngeal nerve elicited a reflex sympathetic response identical to that following aortic nerve stimulation. Throughout these experiments, the intensity and duration of the stimulating pulse was fixed at 15 V and 0.5 ms, respectively. When the superior laryngeal nerve was stimulated over a range of stimulus frequencies between 1 and 100 Hz, the reflex reponse of the renal and cardiac nerves was typically a mixture of inhibitory and excitatory components (Fig. 7). Although
75 !
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e~ 1 /~.~ ] 2 / , v 0.5 s e c Fig. 7. Effect of electrical stimulation of the superior laryngeal nerve on the cardiac nerve activity, when the stimulus frequency was fixed at 6 levels between 1 and 100 Hz as shown at each specimen record. Stimulus consisted of a 1 s train of square wave pulses of 15 V intensity and 0.5 ms duration. The cardiac nerve activity was averaged over 32 successive sweeps produced every 20 s. Stimulation is applied at the time indicated by small triangles or horizontal bars at the top of each tracing.
the sequence of excitatory and inhibitory components depended on individuals, s, the stimulus parameters employed and depth of anesthesia, the excitatory component, which consisted wither of multiple spikes or a single deflection, usually appeared prior to or in the latter part of the inhibitory component. At frequencies below 10 Hz, the inhibitory component tended to be greater than the excitatory component. In conclusion, the reflex sympathetic responses to stimulation of the aortic and superior laryngeal nerves were not identical.
Discussion The present paper correlated, for the first time, the renal and cardiac sympathetic discharges to activation of aortic nerve A- and C-fibers in the rat. Our findings are summarized as follows. Electrical stimulation of aortic nerve A- a n d / o r C-fibers of the rat reflexly evoked inhibition of renal and cardiac nerve activities. In contrast with stimulation of the carotid sinus or aortic nerve of cats [6] and rabbits [24], the excitatory component preceding the inhibitory component was never elicited. With respect to the regional non-uniformity of the reflex response, cardiac and renal nerves responded in a similar manner. As observed in another animal species, the sympatho-inhibitory action of aortic nerve C-fibers was more powerful and longerlasting thatn that of A-fibers. Furthermore, the C-fiber reflex could be elicited at lower stimulus frequencies. On the other hand, the reflex sympathetic response to stimulation of the superior laryngeal nerve was typically a mixture of excitatory and inhibitory components.
76
In the present study, the fastest conduction velocity of the aortic nerve was calculated to be 18.0 + 3.8 m / s and 0.9 _+ 0.4 m / s for A- and C-fibers, respectively. Although the velocity of C-fibers is in agreement with the result of other studies [4,9,34], that of A-fibers calculated from the latency-to-onset or latency-to-peak is generally smaller than those observed in cats (3-40 m / s [7,8,26]) or rabbits (5-4(I m / s [8,9,21]). However, in normotensive and spontaneously hypertensive strains of Wistar rats, a result comparable to ours was reported by Brown et al. [4]. Namely, the conduction velocity of myelinated aortic baroreceptor fibers in these strains of rats ranged between 3 and 22.5 m / s . Thus, the conduction velocity of the rat's aortic nerve A-fibers appears to fall within the velocity range of the slow conducting myelinated fibers of the arterial baroreceptor nerve of the cat and rabbit. With respect to the sensory modality of fibers comprising the rat's aortic nerve. Sapru and Krieger [29] reported that the discharge of that nerve was not affected by an intra-arterial administration of stimulants of arterial chemoreceptors such as sodium cyanide or lobeline HC1. Since electrical stimulation of the aortic nerve elicited decreases in the arterial pressure, heart rate and phrenic nerve activity [28], they concluded that the rat's aortic nerve consisted of barosensory afferents with few, if any, functional chemosensory fibers [28]. In our study, stimulation of the aortic nerve resulted exclusively in inhibition of renal and cardiac sympathetic discharges. As mentioned earlier, this finding provides electrophysiological evidence for the lack of functionally active non-barosensory afferents in the rat's aortic nerve. As we have demonstrated previously [24], averaging the evoked sympathetic response enables us to detect even a tiny excitatory component that might have been overlooked in the presence of a dominating inhibitory component. We are aware that the present electrophysiological approach cannot detect the effect of aberrant vagal fibers innnervating cardiopulmonary stretch receptors other than aortic arch baroreceptors such as those found in the rabbit's aortic nerve [33]. Activation of such fibers, if contained in the rat's aortic nerve, might account for part of the inhibition of sympathetic discharges [3], although, to our knowledge, no physiological and morphological evidence positively supports this possibility. We were able to correlate activity of aortic nerve A- and C-fibers with reflex responses of renal and cardiac ne~e. In agreement to the aortic nerve reflex in rabbits [9,24] and cats [19,25], C-fibers exerted depressor and sympatho-inhibitory actions more powerful and longer-lasting than that of A-fibers. It should be mentioned in this connection that, judging from stimulus parameters employed, Sapru, Gonzalez and Krieger activated in most of their experiments both A- and C-fibers simultaneously [28]. A characteristic feature of the sympathetic nervous system is its regional non-univormity of the reflex reponse to various types of stimuli [14]. To mild arterial hypoxia [12,13] or to a stretch of the arterial wall [17], for example, the activity of renal and cardiac sympathetic nerves responds in the opposite direction. On electrical stimulation of the aortic nerve in our study, the activity of the renal and cardiac nerves was diminished in a similar manner, although the maximal response of the cardiac nerve tended to be greater than that of the renal nerve. The result may
77
correspond to the observation by Ninomiya et al. in the cat [23] that the gain of the sympathetic nerve response in the arterial baroreceptor reflex was slightly greater with the cardiac nerve than the renal nerve. It seems likely that the cardiac nerve of the rat can respond more to the arterial baroreceptor inputs than the renal nerve. We demonstrated that stimulation of the superior laryngeal nerve resulted in an evoked response on renal and cardiac nerves typically consisting of both excitatory and inhibitory components. This finding seems to be reasonable, since the superior laryngeal nerve contains myelinated as well as non-mylinated afferent fiber groups innervating the mucous membrane of the upper larynx [2,26]. Somatic and visceral afferents of different fiber groups or sensory modalities are expected to elicit either excitation or inhibition of the sympathetic nerve activity [18,30]. Thus, the superior laryngeal nerve may substitute the aortic nerve in so far as to be able to produce an identical pattern of hemodynamic responses on electrical stimulation with appropriate combinations of stimulus parameters [11,16]. The two nerves, however, are quite distinct with respect to their reflex effect on the sympathetic nerve activity. Our study, together with those by Sapru and co-workers [28,29], indicates that the rat's aortic nerve is the most useful nerve preparation known to date to selectively activate central neural structures associated with barosensory (presumably arterial baroreceptor) afferents and to study the neural mechanism of the arterial baroreceptot reflex.
Acknowledgements We thank Miss M. Yoshikubo and Miss T. Maeda for technical assistance and Mrs. K. Nagaoka for the help in preparing the manuscript. This work was supported by Grants from Ministries of Education, Science and Culture, and Health and Welfare of Japan.
References 1 Alexander, R.S., Tonic and reflex functions of medullary sympathetic cardiovascular center, J. Neurophysiol., 9 (1946) 205-217. 2 Andrew, B.L., A laryngeal pathway for aortic baroreceptor impulses, J. Physiol. (Lond.), 125 (1954) 352-360. 3 Brown, A., Cardiac reflexes. In R.M. Berne, N. Sperelakis and S.R. Geiger (Eds.), Handbook of Physiology, Section 2; The Cardiovascular System, Vol. I; The Heart, American Physiological Society, MD, 1979, pp. 677-689. 4 Brown, A.M., Saum, W.R. and Tuley, F.H., A comparison of aortic baroreceptor discharge in normotensive and spontaneously hypertensive rats, Circulat. Res., 39 (1976) 488-496. 5 Dampney, R.A.L., Kumada, M. and Reis, D.J., Central neural mechanisms of cerebral ischemic response. Characterization, effect of brainstem and cranial nerve transections, and simulation by electrical stimulation of restricted regions of medulla oblongata in rabbit, Circulat. Res., 44 (1979) 48 62. 6 DeGroat, W.C. and Lalley, P.M., Reflex sympathetic firing in response to electrical stimulation of the carotid sinus nerve in the cat, Brain Res., 80 (1974) 17-40.
78 7 Donoghue, S. Garcia, M., Jordan D. and Spyer, K.M., Identification and brain-stem projections t,l aortic baroreceptor afferent neurons in nodose ganglion of cats and rabbits, J. Physiol. (Lond.), 322 (1982) 337-352. 8 Douglas, W.W. and Ritchie, J.M., Cardiovascular reflexes produced by electrical excitation of non-medullated afferents in the vagus, carotid sinus and aortic nerves, J. Physiol. (Lond.), 134 (1956~ 167-178. 9 Douglas, W.W., Ritchie J.M. and Schaumann, W., Depressor reflexes from medullated and nonmedullated fibres in the rabbit's aortic nerve, J. Physiol. (Lond.), 132 (1956) 187-198. 10 Douglas, W.W. and Schaumann, W., A study of the depressor and pressor components of the cat's carotid sinus and aortic nerves using electrical stimuli of different intensities and frequencies. J. Physiol. (London), 132 (1956) 173-186. 11 Faber, J.E. and Brody, M.J., Reflex hemodynamic response to superior laryngeal nerve stimulation ~n the rat, J. auton. Nerv. Syst., 9 (1983) 607-622. 12 lriki, M. and Kozawa, E., Renal sympathetic baroreflex during normoxia and during hypoxia in conscious and in anesthetized rabbits, Pfl~ger's Arch. ges. Physiol., 398 (1983) 23-26. 13 Iriki, M., Riedel, W. and Simon, E., Patterns of differentiation in various sympathetic efferents induced by changes of blood gas composition and by central thermal stimulation in anesthetized rabbits, Jpn. J. Physiol, 22 (1972) 585-602. 14 Iriki, M. and Simon, E., Regional differentiation of sympathetic efferents. In M. Ito, N. Tsukahara, K. Kubota and Y. Yagi (Eds.) Integrative Control Functions of the Brain, Vol. I, Kodansha, Tokyo/Elsevier, Amsterdam, 1978, pp. 221-238. 15 Iriuchijima, J. and Kumada, M., Activity of single vagal fibers efferent to the heart, Jpn. JII Physiol, 14 (1964) 479-487. 16 lriuchijima, J. and Kumada, M., On the cardionhibitory reflex originating from the superior laryngeal nerve, Jpn. J. Physiol., 18 (1968) 453-461. 17 Karim, F., Kidd, C., Malpus, C.M. and Penna, P.E., The effects of stimulation of the left arterial receptors on sympathetic efferent nerve activity, J. Physiol. (Lond.), 227 (1972) 243-260. 18 Koizumi, K. and Brooks, C. McC., The integration of autonomic system reactions: a discussion of autonomic reflexes, their control and their association with somatic reactions, Ergebn. Physiol., 67 (1972) 1-68. [19]Koizumi, K. and Sato, A., Reflex activity of single sympathetic fibres to skeletal muscle produced by electrical stimulation of somatic and vago-depressor afferent nerves in the cat, Pflt~ger's ARch. ges. Physiol., 332 (1972) 283-301. 20 Krieger, E.M. and Marseillan, R.F., Aortic depresssor fibers in the rat: an electrophysiological study, Amer. J. Physiol., 205 (1963) 771-774. 21 Kumada, M. and Nakajima, H., Field potentials evoked in rabbit brainstem by stimulation of the aortic nerve, Amer. J. Physiol., 223 (1972) 575-582. 22 McCnbben, J.W., Masson, G.M.C. and Page, I.H., Aortic depressor nerves of the rat, Arch. int. Pharmacodyn., 114 (1958) 303-306. 23 Ninomiya, I., Nisimura, N. and Irisawa, H., Sympathetic nerve activity to the spleen, kidney, and heart in response to baroceptor input, Amer. J. Physiol., 221 (1971) 1346-1351. 24 Numao, Y., Saito, M., Terui, N. and Kumada, M., Physiological and pharmacological properties of the three subsystems constituting the aortic nerve-renal sympathetic reflex in rabbits, J. auton. Nerv. Syst., 9 (1983) 36!-380. 25 Oberg, B., Kendrick, E., Thor~n P. and Wennergren, G., Reflex cardiovascular responses to graded stimulations of low-and high-thresholds afferents in the carotid sinus and aortic nerves in the cat, Acta physiol, scand., 113 (1981) 129-137. 26 Paintal, A.S. Vagal sensory receptors and their reflex effects, Physiol. Rev., 53 (1973) 159-227. 27 Ross, C.A., Ruggiero, D.A., Park. D.H. Joh, T.H., Sved, A.F., Fernandez-Partial, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effecto of electrical or chemical stimulation of the area containing CI adrenaline neurons on arterial pressure, heart rate, and plasma catechalamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 28 Sapru, H.N., Gonzalez, E. and Krieger, A.J., Aortic nerve stimulation in the rat: cardiovascular and respiratory responses, Brain Res. Bull, 6 (1981) 393-398.
79 29 Sapru, H.N. and Krieger, A.J,, Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol.: Respirat. Environ. Exercise Phsiol., 42 (1977) 344-348. 30to, A. and Schmidt, R.F., Samatic reflexes: afferent fibers, central pathways, discharge characteristics, Physiol. Rev., 53 (1973) 916-947. 31 Son, S.L. and Waud, D.R., A vagolytic action of neuromuscular blocking agents at the pacemaker of the isolated guinea pig atrium, Anesthesiology, 48 (1978) 191-194. 32 Terui, N., Numao, Y., Kumada, M. and Reis, D.. Identification of the primary afferent fiber group and adequate stimulus initiating the trigeminal depressor response, J. auton. Nerv. Syst, 4 (1981) 1-16. 33 Thor6n, P. and Jones, V., Characteristics of aortic baroreceptor C-fibres in the rabbit, Acta physiol. scand., 99 (1977) 448-456. 34 Thor6n, P., Saum, W.R. and Brown, A.M., Characteristics of aortic baroreceptors with non-medullated afferents in the rat, Circular. Res., 40 (1977) 231-237.
65
JAN 00434
The aortic nerve-sympathetic reflex in the rat Y o s h i n o b u N u m a o , M a m o r u Siato, N a o h i t o T e r u i a n d M o m o r u K u m a d a Institute of Basic Medical Sciences, The University of Tsukuba, lbaraki- ken 305 (Japan) (Received July 10th, 1984) (Revised version received December 13th, 1984) (Accepted January 2nd, 1985)
Key words: rat's aortic nerve - renal nerve activity - cardiac nerve activity - inhibitory component - superior laryngeal nerve
Abstract The effects of stimulation of aortic nerve A- and C-fibers on the renal and cardiac sympathetic nerve activities in anesthetized and immobilized Sprague-Dawley rats were investigated. A separate aortic nerve was found in 46 rats (90%) out of 51. Activation of A- and C-fiber groups, alone or in combination, resulted in an inhibition of renal and cardiac nerve activities. However, an excitatory component preceding the inhibitory component, representing the reflex response to stimulation of non-barosensory afferent fibers contained in the carotid sinus or aortic nerve, was never observed. This results provides electrophysiological evidence supporting the view that the rat's aortic nerve does not contain a significant amount of functionally active non-barosensory afferents. As with the aortic nerve relfex in the rabbit and cat, the sympatho-inhibitory action of C-fibers was more powerful and longer-lasting than that of A-fibers. Furthermore, the C-fiber reflex was elicited at stimulus frequencies as low as 2 Hz. N o significant difference was found between the reflex response of cardiac and renal nerves. On the other hand, stimulation of the superior laryngeal nerve, which constitutes an important pathway carrying arterial baroreceptor fibers, caused a reflex sympathetic response typically consisting of excitatory and inhibitory components. Thus, the rat's aortic nerve provides a useful experimental means to activate selectively central neural structures associated with barosensory afferents and to elicit the reflex response homologous to that in the arterial baroreceptor reflex in rabbits and cats.
Correspondence: M. Kumada, Institute of Basic Medical Sciences, The University of Tsukuba, lbaraki-ken 305, Japan. 0165-1838/85/$03.30 ci3 1985 Elsevier Science Publishers B.V. (Biomedical Division)
66
Introduction It had long been thought that, in the rat, a separate aortic nerve was not present in most instances [11,20,22]. In fact, afferent fibers from the aortic arch baroreceptors variously course through the vagus, cervical sympathetic and recurrent and superior layrngeal nerves in this animal species [2,11,20]. A recent series of studies by Sapru and co-workers [28,29], however, demonstrated that, in certain strains of rats, a separate aortic nerve is regularly found and that functionally the aortic nerve of rats appears to consist exclusively of barosensory fibers. Considering an ever increasing number of studies on the central cardiovascular control conducted in rats, an anatomically well-defined pure barosensory nerve originating from arterial baroreceptors provides an useful experimental means to activate the central neural structures involved in the arterial baroreceptor reflex. In the present study, we correlated activation of aortic nerve A- a n d / o r C-fibers of the rat with the reflexly evoked response of the sympathetic postganglionic discharges and arterial pressure. The renal and cardiac sympathetic nerve activities were chosen as indices of the reflex efferent response. Special attention was paid to examine whether or not the rat's aortic nerve contained non-barosensory fibers such as chemosensory fibers in the cat [6] or nociceptive fibers in the rabbit [24]. If such fibers were present to a significant degree in the aortic nerve, the reflexly evoked sympathetic response would exhibit an excitatory component [6,24]. We also investigated the reflex effect of stimulation of the superior laryngeal nerve on the sympathetic nerve activity, since that nerve constitutes an important afferent pathway carrying barosensory signals from the aortic arch region and can elicit, on electrical stimulation, a pattern of hemodynamic responses identical to that of the arterial baroreceptor reflex [2,11].
Materials and Methods
Preparation of animals Experiments were performed on 55 mate Sprague-Dawley rats (Doken Lab., Ibaraki-ken, Japan) of 10-14 week-old with body weight between 300 and 480 g. The body weight of 10 and 14 week-old male rats of this strain, according to the supplier's data, was 329 + 24 g and 420 + 38 g (mean + S.D.), respectively. Each animal was anesthetized with an intraperitoneal administration of urethane (initial dose 800 mg/kg). Supplemental injections (80 m g / k g ) were given then necessary to maintain adequate anesthesia. After insertion of tracheal, arterial and venous cannulas, the animal was paralyzed with pancuronium bromide (initially 2.5 mg/kg, i.v., thereafter 1.2 m g / k g / h ) and artificially ventilated with oxygen by a Harvard respiratory pump. Body temperature was maintained at 37 ° + 0.5°C by a thermostatically regulated heating pad connected to a rectal thermistor probe. Measurement of cardiovascular and sympathetic nerve activities Instantaneous and mean arterial pressure and heart rate were monitored con-
67 tinously in all experiments. Arterial pressure was recorded from the iliac artery by a polyethylene catheter inserted through the femoral artery and connected to a transducer (Nihon Kohden MPU-0.5). Heart rate was computed from the arterial pressure pulse by a tachometer (Nihon Kohden AT-600G). The left renal nerve was approached retroperitoneally through a left flank incision, and was prepared for recording from near the renal artery. The right inferior (or stellate) branch of the cardiac nerve was approached from the upper thorax through a mid-sternal incision. That branch was identified as the major branch originating from the right stellate ganglion which proceeded caudally along the superior vena cava to the base of the heart. Communicating fibers from the vagus nerve, when visible, were removed. In preparing the cardiac nerve, the pleura and pericardium remained unopened. The central cut end of the nerve was placed on a pair of bipolar platinum electrodes connected to an amplifier (Nihon Kohden AVB-8). The output of the amplifier was displayed on an oscilloscope (Tektronix 5113). The lower and higher cut-off frequencies of the recording system were 100 and 3000 Hz, respectively. The multifiber sympathetic nerve activity (SNA) was usually rectified, and passed through a low-pass filter (time constant 20 ms). The evoked SNA was averaged during 16-32 successive sweeps with a data processing computer (Nihon Kohden ATAC-350) and displayed on an x-y recorder (Watanabe 4401). Because of the proximity of stimulating and recording electrodes, the evoked response recorded from the cardiac nerve was usually contamined with artifacts. Such artifacts preceding the evoked response were eliminated by a gate circuit designed and constructed by ourselves. The circuit switched off the input to the amplifier for 0.3-0.6 ms after the onset of the stimulating pulse and effectively eliminated the stimulus artifact without significantly distorting the evoked response. In quantitating the evoked response of the sympathetic nerve, we took into consideration temporal as well as individual variations of the background level of sympathetic discharges. The 'Normalized Response Magnitude' defined below was used for this purpose [24]. When SNA decreased below the prestimulus control level, the area below the control SNA was calculated and then divided by SNA integrated over the 1-s period immediately prior to nerve stimulation. The negative sign was attached when SNA decreased below the control level. When SNA increased above the prestimulus control level, the area above control SNA was divided by SNA integrated over the 1-s period prior to nerve stimulation. The positive sign was attached in this case. Electrical stimulation of the aortic ner~,e Aided by a dissecting microscope, the left aortic nerve was identified at its junction with the superior laryngeal nerve. The aortic nerve was then carefully separated from the adjacent cervical sympathetic nerve, dissected free of the surrounding connective tissue, and "transected 1 2 cm peripherally. The distal end of the nerve was placed across a pair of platinum stimulating electrodes spaced 2 - 5 m m apart. Electrical stimuli were square-wave pulses of 0.1 ms duration delivered to the animal from a pulse generator (Nihon Kohden SEN 7103) through an isolation
68 unit. The stimulus frequencies were between 1 and 300 Hz and the stimulus intensity usually ranged from 0.2 to 30 V. We did not record the afferent nerve activity and the reflex sympathetic response to aortic nerve stimulation simultaneously, because this maneuver often damaged the aortic nerve. Therefore, the afferent nerve activity was examined in the same animal after studying the reflex reponse first. To record the aortic nerve discharge, another pair of platinum electrodes was attached to the nerve several millimeters centrally, and the distal end of the nerve was electrically stimulated as explained above. In case where we failed to find a separate left aortic nerve, the same procedures were repeated on the right side to prepare the right aortic nerve. In 5 rats, the left superior laryngeal nerve was prepared for afferent stimulation. The central cut end of the nerve, dissected for 4 - 6 m m from its point of emergence between the larynx and cricothyroid muscles, was stimulated to elicit the reflex responses.
Results
Afferent discharges of the aortic nerve In 42 out of 51 rats we used, an aortic nerve was found as a separate strand on the left side. Of the 9 animals in which a separate left aortic nerve was absent, the right aortic nerve could be isolated in 4 cases. Thus, in over 90% of the animals, an anatomically separate aortic nerve could be isolated and stimulated. As the intensity of the single pulse stimulus to the aortic nerve was increased, 2 groups of deflections regularly appeared in the action potential recorded proximally from the same nerve (Fig. 1). Conduction velocities of the 2 deflections at 37°C, calculated as the latency-to-onset divied by the distance between the stimulating and recording electrodes, were 18.1 + 3.8 m / s (n = 13) and 0.93 + 0.41 m / s (n = 15), respectively. The temperature coefficient of 1.6 was used for the calculation [24,32]. The two groups thus represent activity of the aortic nerve A- and C-fibers. Conduction velocity for the A-fibers, calculated as the latency-to-peak divided by the distance was 8.0 _+ 1.1 m / s (n = 15). The same calculation was not made for the C-fibers, since the afferent activity quite often consisted of multiple deflections of comparable sizes. We subsequently investigated the relation between the afferent fiber group activated by graded stimulation and the consequent response indicated by sympathetic nerve activity and arterial pressure. Reflex sympathetic nerve response evoked by stimulation of the aortic nerve Stimulation of the aortic nerve with a pulse train of 100 Hz elicited inhibition of the renal and cardiac nerve discharges, when the stimulus intensity was slightly above the threshold of A-fibers (Figs. 1 and 2). The response, termed the inhibitory component, was augmented in magnetude and duration as stimulus intensity was increased. With recruitment of aortic nerve C-fibers, a further increase in that component was brought about in both remal and cardiac nerve responses. This
69
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Fig. l. Correlation between the compound action potential of the aortic nerve (left panel) and the evoked response of the renal nerve (right panel) to graded electrical stimulation of the aortic nerve. To elicit the compound action potential in the left panel (a e), a single rectangular pulse was given distally to the central cut end of the aortic nerve at the moment indicated by the small triangles in each record. It was averaged over 32 successive sweeps produced every 1 s. The duration of the pulse was fixed at 0.1 ms, while its amplitude was varied between 0.35 V to 30 V as shown in the.upper right corner of the tracings. Two major peaks corresponding to activation of aortic nerve A- and C-fibers are seen in d and e. When such stimulating pulses were applied repetitively to the aortic nerve at the frequency of 100 Hz, the evoked renal nerve response corresponding to the left panel was obtained (f-j). The stimulus train, given once every 20 s for 32 times, lasted for 1 s as shown by horizontal bars at the top of each tracing.
increase was partly ascribable to a prolongation of the silent period, and partly to a subsequent weak but long-lasting inhibition of the sympathetic nerve activity (Fig. 2). With the fixed stimulus frequency of 100 Hz, no marked difference was observed in the magnitude of the reflex inhibition between the renal and cardiac nerves. At times, a sudden and transient increase in the sympathetic activity was observed on cessation of the stimulus train (Fig. lg and h). The onset latency of the inhibitory component was 65 ± 35 ms (mean + S.D.; n = 15) with the renal nerve and 37 ± 25 ms (n = 7) with the cardiac nerve. In none of 51 rats we examined, activation of A- a n d / o r C-fibers employing various combinations of stimulus parameters resulted in an excitatory component preceding the inhibitory component (Fig. 1). Such an excitatory component, observed in the aortic nerve-sympathetic reflex in cats [6] and rabbits [24], is known to originate from non-barosensory fibers. The absence of the excitatory component in the aortic nerve reflex of the rat is electrophysiological evidence indicating that
70 A RENAL NERVE ACTIVITY c o
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Fig. 2. Effect of different intensities of electrical stimulation of the aortic nerve on the inhibitory component of renal (upper panel) and cardiac (lower panel) nerve activity: The stimulus consisted of a 1s train of square pulses. Duration and frequency of pulses were 0.1 ms and 100 Hz, respectively. Intensities of stimulation were altered as indicated by the abscissa. The ordinate represents reflex responses expressed as the Normalized Response Magnitude. Thick broken lines are the mean values of responses, whereas thin solid lines represent responses in individual experiments. At the top of each illustration is shown the range of thresholds of aortic nerve A- and C-fibers. Data were obtained from experiments in 5 rats for each nerve. f u n c t i o n a l l y the rat's aortic nerve consists exclusively of b a r o s e n s o r y fibers. It should be n o t e d that s t i m u l a t i o n of the aortic nerve never elicited excitation of the cardiac nerve discharge. This result d e m o n s t r a t e s that the evoked response of the cardiac nerve in our experiments reflects activity of the sympathetic b u t n o t vagal fibers efferent to the heart. H a d there b e e n a significant a m o u n t of f u n c t i o n a l l y active vagal fibers in our cardiac nerve p r e p a r a t i o n , a n excitatory c o m p o n e n t would have been elicited [15].
Frequency characteristics of the aortic nerve-sympathetic reflex W h e n the aortic nerve A-fibers were excited m a x i m a l l y without activating C-fibers, a m i n u t e i n h i b i t o r y c o m p o n e n t appeared b o t h in the renal a n d cardiac nerve responses at the frequency of 20 Hz. The i n h i b i t o r y c o m p o n e n t increased in size as the stimulus frequency was increased a n d reached a n e a r m a x i m u m at 100 Hz (Fig.
3). O n s i m u l t a n e o u s activation of aortic nerve A- a n d C-fibers, the sympatho-in_hibitory response was o b t a i n e d at frequencies above 1 Hz. The response was graded with
71
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Fig. 3. Stimulus frequency-response characteristics for the inhibitory component of renal (upper panel) and cardiac (lower panel) nerve activity by electrical stimulation of the aortic nerve. The inhibitory component was expressed in terms of the Normalized Response Magnitude. Stimulus frequencies were indicated by the abscissa. Attached to each characteristic curve is the fiber,group(s) activated to elicit the response. The stimulus consisted of a 1 s train of square wave pulses. Intensity and duration of the pulse were 1-2 V and 0.l ms, respectively,to activate aortic nerve A-fibers alone, or 20 V and 0.1 ms to activate A- and (.'-fibers in combination. Vertical lines are S.D. Data were obtained from 5 rats for each nerve activity.
regard to stimulus frequency a n d reached a near m a x i m u m at 100 Hz (renal nerve) or 200 Hz (cardiac nerve). I n terms of the N o r m a l i z e d Response M a g n i t u d e , s y m p a t h o - i n h i b i t o r y responses of cardiac a n d renal nerves were c o m p a r a b l e (Fig. 3). However, the m a x i m a l response of the cardiac nerve tended to be greater than that of the renal nerve, although the difference was statistically insignificant ( P < 0.2; S t u d e n t ' s t-test). N a m e l y , with the stimulus frequency of 200 Hz a n d intensity of 20 V, the reflex i n h i b i t i o n of the renal a n d cardiac nerve activities was 2.4 ___0.7 ( m e a n + S.D.; n = 5) a n d 2.9 + 0.9 (n = 5), respectively. Inhibition curve
A frequently used way to express the e x c i t a b i l i t y - r e c o v e r y property of a symp a t h o - i n h i b i t o r y reflex system is to apply c o n d i t i o n i n g a n d test stimuli to the system, while recording the efferent activity [18,24,32]. The s y m p a t h o - i n h i b i t o r y effect of aortic nerve A- a n d C-fibers was c o m p a r e d in 4 rats by m e a n s of this m e t h o d to o b t a i n the i n h i b i t i o n curve (Fig. 4). T h e c o n d i t i o n i n g stimulus consisted of a train of
72
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STIMULUS INTERVAL ( s e e )
Fig. 4. Inhibitory curve of the aortic nerve reflex mediated by A-fibers (left panel) or A- and C-fibers (right panel). The conditioning stimulus was a train of pulses given to the aortic nerve to excite A-fibers (amplitude of pulse 2 V, duration of pulse 0.1 ms and frequency 100 Hz) or A- and C-fibers (amplitude 20 V, duration 0.1 ms and frequency 20 Hz). The duration of the pulse train was 1 s for the former and 0.9 s for the latter. Test stimulation was 2 pulses having the amplitude of 100/~A, duration of 0J2 ms and pulse interval of 10 ms. The stimulus site was 2 mm anterior to the obex, 1 mm lateral to the mid-sagittal plane and 0.75 mm below the dorsal surface of the brainstem. It was within the medullary reticular pressor area: The interval between conditioning and test stimuli was varied up to 2 s. The upper drawings consist of superimposed tracings of renal nerve activity elicited by test stimuli given at intervals indicated by small dots. In the lower drawings, the reflex response of renal nerve activity is plotted as a function of time interval.
pulses, given to the aortic nerve, with the d u r a t i o n of 1 s or a little less than that. A silent period of renal sympathetic discharges lasting a b o u t 1 s was p r o d u c e d by the stimulus train. Test s t i m u l a t i o n was 2 pulses delivered through a u n i p o l a r electrode placed in the medullary reticular pressor area [1,5] from the exposed dorsal surface of the lower b r a i n s t e m . This area encompassed the n u c l e u s reticularis parvocellutaris as well as passing catecholaminergic fibers originating from cells in the ventrolateral m e d u l l a [27]. W h e n the aortic nerve A-fibers alone were activated by a 100 Hz train of pulses, the i n h i b i t o r y effect was observed d u r i n g the c o n d i t i o n i n g stimulus. The excitability, however, recovered within 0.4 s after cessation of the stimulus a n d there usually followed a period of e n h a n c e d excitability (Fig. 4). The aortic nerve was then stimulated b y a 20 Hz train of pulses with a stimulus intensity above the threshold for C-fibers. A t this stimulus frequency, the reflex response a t t r i b u t a b l e to the aortic nerve A-fibers was m i n i m a l [24]. The i n h i b i t o r y effect of C-fibers assessed in this way was more powerful t h a n that of A-fibers a n d outlasted the c o n d i t i o n i n g stimulus. Even 1 s after cessation of the c o n d i t i o n i n g stimulus, the reflex sympathetic response to the test stimulus was partially suppressed. These differences
73 between barosensory A- and C-fibers with respect to their sympatho-inhibitory e f f e c t a r e q u i r e s i m i l a r t o t h o s e i n t h e a o r t i c n e r v e r e f l e x of t h e r a b b i t [24] o r c a t [25].
Depressor response to actiuation of aortic nerve A- and C-fibers The effect of electrical stimulation of the rat's aortic nerve on hemodynamic and respiratory variables has already been described systematically by Sapru, Gonzalez a n d K r i g e r [28]. T h e y v a r i e d s t i m u l u s p a r a m e t e r s o v e r a w i d e r a n g e , w h i l e r e c o r d i n g responses of arterial pressure, heart rate and respiratory movement. In the present AORTIC NERVE POTENTIAL
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Fig. 5. Correlation between the compound action potential of the aortic nerve (left panel) and the depressor response (middle and right panels) to graded electrical stimulation of the aortic nerve. The compound action potential was elicited by a single rectangular pulse having the duration of 0.1 ms and 5 levels of stimulus intensity shown in the upper right corner of the tracings. Graded depressor responses were obtained by applying such stimulating pulses to the aortic nerve at the frequency of 20 Hz (middle panel S or 100 Hz (right panel). The stimulus train lasted for 10 s as shown by horizontal bars at the top of middle and right panels. Fig. 6. Stimulus intensity-response characteristics (upper panel) and stimulus frequency response characteristics (lower panel) for the depressor response elicited by electrical stimulation of the aortic nerve. The response was expressed as change in arterial pressure from the prestimulus level. Stimulus consisted of a 10 s train of square pulses of 0.1 ms duration. In the upper panel, the stimulus frequency was 100 Hz (filled circles) or 20 Hz (open circles) and the stimulus intensity was indicated by abscissa. The range of thresholds of aortic A- and C-fibers was shown at the top of the illustration. Data were obtained from 13 rats. In the lower panel, the stimulus intensity was 1-2 V to activate A-fibers near maximally without activating C-fibers (filled circles) or 10-30 V to elicit a maximal depressor response due to simultaneous activation of A- and C-fibers (open circles). Stimulus frequency was indicated by the abscissa. Data were obtained from 11 rats.
74
study, we correlated activation of aortic nerve A- and C-fibers to their depressor action, when the nerve was stimulated by a 10 s train of pulses of 0.1 ms duration at frequencies of 20 and 100 Hz (Figs. 5 and 6A). Because the animals were immobilized by pancuronium bromide, an agent known to posses a vagolytic action [31], the depressor response described here was predominatly due to sympathetic inhibition. Actually, the reflex decrease in heart rate was mostly less than 40 beats/rain. At stimulus intensities above the threshold of A-fibers, a sizeable depressor response was elicited by a 100 Hz stimulus train. Recruitment of C-fibers resulted in a minor additional depressor response at that stimulus frequency. Thus, the depressor reponse to a stimulus train of 100 Hz reflected predominantly the relfex action of aortic nerve A-fibers. On the other hand, at a fixed stimulus frequency of 20 Hz, only a minimal depressor response was evoked by aortic nerve A-fibers. The response, however, was markedly augmented on activation of C-fibers, even though A-fibers were already maximally excited. As was demonstrated by experiments in which activation of aortic nerve A-fibers of the rabbit was suppressed by anodal block [24], a possible contribution of A-fibers to the reflex response was expected to be small over the range of stimulus frequency below 20 Hz. It is most likely that the depressor response to a stimulus train of 20 Hz here predominantly reflected the reflex action of aortic nerve C-fibers. The effect of stimulus frequency on the depressor response was summarized in the lower panel of Fig. 6. When the A-fibers were excited near maximally without activating C-fibers, the depressor response was elicited at stimulus frequencies above 10 Hz. The response was increased in magnitude as the stimulus frequency was raised and reached a maximum at 150 Hz. On simultaneously activation of A- and C-fibers, a depressor response was elicited at frequencies as low as 2 Hz and the maximal response was attained at 100 Hz. Considering a minimal contribution of aortic nerve A-fibers on the baroreceptor reflex over a stimulus frequency range below 20 Hz (see above), the reflex depressor response of C-fibers of the rat, like those of rabbits [9,24] and cats [10,19], appear to operate over a stimulus frequency range lower than that of A-fibers.
Reflex response to stimulation of the superior laryngeal nerve The rat's superior laryngeal nerve carries part of the afferent barosensory signal from the aortic arch region [2]. That nerve may even constitute a significant afferent pathway for the aortic baroreceptor reflex, since the reflex cardiovascular changes are greatly attenuated by its transection [11]. Furthermore, by a suitable choice of stimulus parameters, stimulation of the superior laryngeal nerve produces a pattern of hemodynamic responses identical to that of the arterial baroreceptor r e f l e x [ l l ]. We therefore examined in 5 rats whether or not stimulation of the superior laryngeal nerve elicited a reflex sympathetic response identical to that following aortic nerve stimulation. Throughout these experiments, the intensity and duration of the stimulating pulse was fixed at 15 V and 0.5 ms, respectively. When the superior laryngeal nerve was stimulated over a range of stimulus frequencies between 1 and 100 Hz, the reflex reponse of the renal and cardiac nerves was typically a mixture of inhibitory and excitatory components (Fig. 7). Although
75 !
a 5 Hz
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5 0 Hz
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e~ 1 /~.~ ] 2 / , v 0.5 s e c Fig. 7. Effect of electrical stimulation of the superior laryngeal nerve on the cardiac nerve activity, when the stimulus frequency was fixed at 6 levels between 1 and 100 Hz as shown at each specimen record. Stimulus consisted of a 1 s train of square wave pulses of 15 V intensity and 0.5 ms duration. The cardiac nerve activity was averaged over 32 successive sweeps produced every 20 s. Stimulation is applied at the time indicated by small triangles or horizontal bars at the top of each tracing.
the sequence of excitatory and inhibitory components depended on individuals, s, the stimulus parameters employed and depth of anesthesia, the excitatory component, which consisted wither of multiple spikes or a single deflection, usually appeared prior to or in the latter part of the inhibitory component. At frequencies below 10 Hz, the inhibitory component tended to be greater than the excitatory component. In conclusion, the reflex sympathetic responses to stimulation of the aortic and superior laryngeal nerves were not identical.
Discussion The present paper correlated, for the first time, the renal and cardiac sympathetic discharges to activation of aortic nerve A- and C-fibers in the rat. Our findings are summarized as follows. Electrical stimulation of aortic nerve A- a n d / o r C-fibers of the rat reflexly evoked inhibition of renal and cardiac nerve activities. In contrast with stimulation of the carotid sinus or aortic nerve of cats [6] and rabbits [24], the excitatory component preceding the inhibitory component was never elicited. With respect to the regional non-uniformity of the reflex response, cardiac and renal nerves responded in a similar manner. As observed in another animal species, the sympatho-inhibitory action of aortic nerve C-fibers was more powerful and longerlasting thatn that of A-fibers. Furthermore, the C-fiber reflex could be elicited at lower stimulus frequencies. On the other hand, the reflex sympathetic response to stimulation of the superior laryngeal nerve was typically a mixture of excitatory and inhibitory components.
76
In the present study, the fastest conduction velocity of the aortic nerve was calculated to be 18.0 + 3.8 m / s and 0.9 _+ 0.4 m / s for A- and C-fibers, respectively. Although the velocity of C-fibers is in agreement with the result of other studies [4,9,34], that of A-fibers calculated from the latency-to-onset or latency-to-peak is generally smaller than those observed in cats (3-40 m / s [7,8,26]) or rabbits (5-4(I m / s [8,9,21]). However, in normotensive and spontaneously hypertensive strains of Wistar rats, a result comparable to ours was reported by Brown et al. [4]. Namely, the conduction velocity of myelinated aortic baroreceptor fibers in these strains of rats ranged between 3 and 22.5 m / s . Thus, the conduction velocity of the rat's aortic nerve A-fibers appears to fall within the velocity range of the slow conducting myelinated fibers of the arterial baroreceptor nerve of the cat and rabbit. With respect to the sensory modality of fibers comprising the rat's aortic nerve. Sapru and Krieger [29] reported that the discharge of that nerve was not affected by an intra-arterial administration of stimulants of arterial chemoreceptors such as sodium cyanide or lobeline HC1. Since electrical stimulation of the aortic nerve elicited decreases in the arterial pressure, heart rate and phrenic nerve activity [28], they concluded that the rat's aortic nerve consisted of barosensory afferents with few, if any, functional chemosensory fibers [28]. In our study, stimulation of the aortic nerve resulted exclusively in inhibition of renal and cardiac sympathetic discharges. As mentioned earlier, this finding provides electrophysiological evidence for the lack of functionally active non-barosensory afferents in the rat's aortic nerve. As we have demonstrated previously [24], averaging the evoked sympathetic response enables us to detect even a tiny excitatory component that might have been overlooked in the presence of a dominating inhibitory component. We are aware that the present electrophysiological approach cannot detect the effect of aberrant vagal fibers innnervating cardiopulmonary stretch receptors other than aortic arch baroreceptors such as those found in the rabbit's aortic nerve [33]. Activation of such fibers, if contained in the rat's aortic nerve, might account for part of the inhibition of sympathetic discharges [3], although, to our knowledge, no physiological and morphological evidence positively supports this possibility. We were able to correlate activity of aortic nerve A- and C-fibers with reflex responses of renal and cardiac ne~e. In agreement to the aortic nerve reflex in rabbits [9,24] and cats [19,25], C-fibers exerted depressor and sympatho-inhibitory actions more powerful and longer-lasting than that of A-fibers. It should be mentioned in this connection that, judging from stimulus parameters employed, Sapru, Gonzalez and Krieger activated in most of their experiments both A- and C-fibers simultaneously [28]. A characteristic feature of the sympathetic nervous system is its regional non-univormity of the reflex reponse to various types of stimuli [14]. To mild arterial hypoxia [12,13] or to a stretch of the arterial wall [17], for example, the activity of renal and cardiac sympathetic nerves responds in the opposite direction. On electrical stimulation of the aortic nerve in our study, the activity of the renal and cardiac nerves was diminished in a similar manner, although the maximal response of the cardiac nerve tended to be greater than that of the renal nerve. The result may
77
correspond to the observation by Ninomiya et al. in the cat [23] that the gain of the sympathetic nerve response in the arterial baroreceptor reflex was slightly greater with the cardiac nerve than the renal nerve. It seems likely that the cardiac nerve of the rat can respond more to the arterial baroreceptor inputs than the renal nerve. We demonstrated that stimulation of the superior laryngeal nerve resulted in an evoked response on renal and cardiac nerves typically consisting of both excitatory and inhibitory components. This finding seems to be reasonable, since the superior laryngeal nerve contains myelinated as well as non-mylinated afferent fiber groups innervating the mucous membrane of the upper larynx [2,26]. Somatic and visceral afferents of different fiber groups or sensory modalities are expected to elicit either excitation or inhibition of the sympathetic nerve activity [18,30]. Thus, the superior laryngeal nerve may substitute the aortic nerve in so far as to be able to produce an identical pattern of hemodynamic responses on electrical stimulation with appropriate combinations of stimulus parameters [11,16]. The two nerves, however, are quite distinct with respect to their reflex effect on the sympathetic nerve activity. Our study, together with those by Sapru and co-workers [28,29], indicates that the rat's aortic nerve is the most useful nerve preparation known to date to selectively activate central neural structures associated with barosensory (presumably arterial baroreceptor) afferents and to study the neural mechanism of the arterial baroreceptot reflex.
Acknowledgements We thank Miss M. Yoshikubo and Miss T. Maeda for technical assistance and Mrs. K. Nagaoka for the help in preparing the manuscript. This work was supported by Grants from Ministries of Education, Science and Culture, and Health and Welfare of Japan.
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79 29 Sapru, H.N. and Krieger, A.J,, Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol.: Respirat. Environ. Exercise Phsiol., 42 (1977) 344-348. 30to, A. and Schmidt, R.F., Samatic reflexes: afferent fibers, central pathways, discharge characteristics, Physiol. Rev., 53 (1973) 916-947. 31 Son, S.L. and Waud, D.R., A vagolytic action of neuromuscular blocking agents at the pacemaker of the isolated guinea pig atrium, Anesthesiology, 48 (1978) 191-194. 32 Terui, N., Numao, Y., Kumada, M. and Reis, D.. Identification of the primary afferent fiber group and adequate stimulus initiating the trigeminal depressor response, J. auton. Nerv. Syst, 4 (1981) 1-16. 33 Thor6n, P. and Jones, V., Characteristics of aortic baroreceptor C-fibres in the rabbit, Acta physiol. scand., 99 (1977) 448-456. 34 Thor6n, P., Saum, W.R. and Brown, A.M., Characteristics of aortic baroreceptors with non-medullated afferents in the rat, Circular. Res., 40 (1977) 231-237.