INTRODUCTION

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IT WAS DEMONSTRATED BY Doba and Reis (8) that in the cat the vestibular system participates in neural regulation of the circulation. They showed that arterial pressure and vascular resistance were unable to be maintained during tilt when the vestibular nerve (VIII cranial nerve) was transected. Stimulation of the vestibular nerve in animals alters sympathetic activity to a number of vascular beds (6, 13, 21, 42). More importantly, natural vestibular stimulation (i.e., nose-up tilt in the cat) has been demonstrated to increase sympathetic nerve activity (53). It was observed that, as the magnitude of the nose-up tilt increased, splanchnic nerve activity increased proportionally.

In humans, substantial evidence exists for vestibular regulation of sympathetic activity. Using head-down rotation (HDR) in the prone posture to activate the otolith organs, we have observed prompt increases in muscle sympathetic nerve activity (MSNA) (12, 26-28, 36). In these studies, potential nonlabyrinthine mechanisms that could have elicited the increases in MSNA with HDR were excluded. These mechanisms were change in visual inputs (36), unloading of the cardiopulmonary and arterial baroreflexes (36), activation of neck muscle afferents (26), engagement of central command (26-28), and input from nonspecific receptors in the head that could be activated by increases in cerebral pressure (12). In addition, because the subject's body is stationary during the HDR maneuver, activation of possible extravestibular gravitational receptors has been eliminated (22, 52). Other supporting evidence for the vestibular-mediated sympathetic outflow during HDR is that MSNA increases are graded to the degree of HDR and that MSNA fails to increase when head-down neck extension is performed in the supine posture (12). Head-down neck extension in the supine posture would stimulate the otolith organs in the opposite manner compared with HDR in the prone position. Finally, natural stimulation of the horizontal semicircular canals in humans does not activate MSNA (28). This finding is in agreement with animal studies that indicate that the semicircular canals do not participate in the vestibulosympathetic reflex (53). Therefore, the cumulative evidence demonstrates that HDR in humans activates MSNA via the vestibulosympathetic reflex.

Extracellular recordings have indicated that brain regions involved in cardiovascular regulation (i.e., caudal and rostral ventrolateral medulla) receive both vestibular and skeletal muscle afferent input (3, 4, 39, 50). Additionally, studies utilizing c-fos immunocytochemistry demonstrated that static muscle contractions in the cat resulted in increased c-fos labeling of neurons in the vestibular nuclei (18, 49). Similarly, Iwamoto et al. (15) found increased c-fos labeling in the vestibular nuclei during running in rats compared with nonexercising controls. These studies indicate that skeletal muscle afferents have inputs to the vestibular nuclei during muscle contractions. Therefore, it appears from these studies that neural integration could exist between the vestibulosympathetic and skeletal muscle reflexes. Understanding of this interaction is important because locomotory exercise engages both vestibular and skeletal muscle afferents.

With this background, the purpose of the current study was to investigate the neural interaction between the vestibulosympathetic and skeletal muscle reflexes in humans. MSNA, arterial pressure, and heart rate responses were examined in healthy volunteers during 1) HDR to engage the vestibulosympathetic reflex, 2) isometric handgrip (IHG) to activate the skeletal muscle reflexes, and 3) the simultaneous performance of both IHG and HDR.

	METHODS

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Subjects. Seventeen healthy volunteers (4 men and 13 women; age 27 ± 1 yr., height, 168 ± 2 cm, weight 69 ± 2 kg) who were normotensive, nonsmokers, not on medication, and free of any known vestibular disorder were studied. Ten of the 13 women were taking oral contraceptives. After a medical history and physical examination by a physician and verbal explanation of the testing procedures, written, informed consent was obtained from all of the subjects. The study and experimental protocols were approved by the Institutional Review Board at The Pennsylvania State University College of Medicine.

Experimental design. To determine the nature of the interaction between the vestibulosympathetic and skeletal muscle reflexes, subjects performed three experimental trials in the prone position. One trial examined responses to HDR (i.e., vestibular activation of otolith organs) for 2 min. The second trial examined responses to IHG at 30% of maximum voluntary contraction (i.e., skeletal muscle reflex) for 2 min. The third trial examined responses during simultaneous performance of HDR and IHG for 2 min. The order of the three trials was randomized with 15-min rest periods interspersed between trials.

Measurements. Multifiber recordings of MSNA were made by inserting a tungsten microelectrode into a peripheral nerve located in the popliteal region behind the right knee (43). A reference electrode was positioned subcutaneously 2-3 cm away from the recording electrode. To ensure that an adequate recording site for MSNA was obtained, the following criteria were met: 1) weak electrical stimulation through the electrode elicited involuntary muscle contractions of the appropriate muscles but not paresthesia, 2) tapping of muscles or tendons innervated by the impaled nerve fascicle evoked afferent mechanoreceptor discharges, but stroking the skin did not elicit afferent activity, 3) sympathetic impulses occurred as spontaneous bursts within the cardiac rhythm, 4) held expiration (apnea) resulted in increased sympathetic nerve activity, and 5) a sudden arousal stimulus (yell) did not elicit an increase in sympathetic nerve activity. The nerve signal was amplified (20,000-50,000 times) and filtered with a bandwidth of 700-2,000 Hz. The filtered signal was rectified and integrated (time constant 0.1 s) to obtain a mean voltage display of the nerve activity. Sympathetic recordings that indicated possible electrode site shifts or EMG artifact during the experimental interventions were excluded.

Data analysis. MSNA was expressed as bursts per minute and total MSNA. Sympathetic bursts were identified from inspection of the mean voltage neurogram, and the sum of the area of those bursts for each minute was measured by a computer program (Peaks, ADInstruments) and reported as total MSNA, expressed in arbitrary units.

	RESULTS

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Absolute values for MSNA (burst frequency and total activity) during the three experimental trials are presented in Fig. 1. MSNA increased during both the first and second minute of HDR. During IHG, MSNA did not significantly increase until the second minute of exercise. When HDR and IHG were done together, MSNA was significantly increased during both minutes. The change in MSNA during the three trials is shown in Fig. 2. MSNA was increased by 5 ± 1 bursts/min and 48 ± 16 units during HDR. IHG elicited a 10 ± 1 bursts/min and 80 ± 25-unit increase in MSNA. During simultaneous HDR and IHG, MSNA increased by 14 ± 2 bursts/min and 142 ± 38 units. These changes during the combination trial were not different from the algebraic sum of the HDR and IHG performed individually (14 ± 2 bursts/min and 130 ± 41 units). Figure 3 shows an original recording of the effect that HDR, IHG, and HDR plus IHG had on MSNA.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Muscle sympathetic nerve activity (MSNA) responses (burst frequency and total activity) during the 3 experimental trials. Open symbols are significantly different from baseline (BL) (P < 0.05). MSNA significantly increased during head-down rotation (HDR), isometric handgrip (IHG), and HDR plus IHG. Rec, recovery.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Change () in MSNA from baseline during HDR and IHG performed alone, the algebraic sum of HDR and IHG values, and change when HDR and IHG were performed together. The algebraic sum of the individual trials and the combination trial (HDR + IHG) were not different from each other. These findings suggest that an additive interaction exists between the vestibulosympathetic reflex and exercise pressor reflex. *Response is significantly different from baseline, P < 0.05; NS, not significant.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Recordings of MSNA from 1 subject during the 3 experimental trials.

MAP and heart rate during the three experimental trials are presented in Fig. 4. MAP was significantly increased during the IHG and the combination trials. HDR did not significantly increase MAP. The change in MAP for the three trials and the algebraic sum of the HDR and IHG trials are shown in Fig. 5. MAP increased by 22 ± 2 mmHg for the combination trial. This was not significantly different from the algebraic sum of the HDR and IHG trials (21 ± 2 mmHg). Heart rate was significantly increased during all experimental trials (Fig. 4). The increase in heart rate was 2 ± 1, 9 ± 1, and 10 ± 1 beats/min for the HDR, IHG, and combination trials, respectively (Fig. 5). The increase in heart rate during the combination trial was similar to the algebraic sum of the HDR and IHG trials (11 ± 2 beats/min).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Heart rate and mean arterial pressure (MAP) responses during the 3 experimental trials. Open symbols are significantly different from BL (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Change in heart rate and MAP from baseline during HDR and IHG performed alone, the algebraic sum of HDR and IHG values, and change when HDR and IHG were performed together. The algebraic sum and the HDR and IHG combination trial were not different from each other for both heart rate and MAP. These findings suggest that an additive interaction exists between the vestibulosympathetic reflex and exercise pressor reflex. *Response is significantly different from baseline, P < 0.05.

	DISCUSSION

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The major finding from this study is that the neural interaction between the vestibulosympathetic and skeletal muscle reflexes for sympathetic and cardiovascular parameters is additive. This finding suggests that no central modulation exists between these two reflexes and that MSNA output from both reflexes are independent of each other in humans.

Exercise-induced increases in MSNA are primarily mediated by the activation of skeletal muscle afferents (19, 20, 29, 45). However, during exercise, a number of neural reflexes are engaged in addition to the afferent feedback from the contracting muscle. The presence of these neural reflexes can modulate muscle-related increases in MSNA. For example, the arterial baroreflex has been shown to alter exercise-induced MSNA. Phenylephrine-induced increases in arterial pressure reduced MSNA during IHG, whereas lowering arterial pressure with nitroprusside increases MSNA during exercise (35). Because the vestibular system and skeletal muscles play important roles in locomotion and both can regulate sympathetic outflow, it is reasonable to suspect that the vestibulosympathetic reflex may modulate sympathetic outflow during activation of the skeletal muscle afferents.

There is strong evidence that skeletal muscle afferents activate the vestibular nuclei. Li et al. (18) and Iwamoto et al. (15) reported increased c-fos immunoreactivity in the vestibular nuclei during isometric and dynamic muscle contractions, respectively. Vissing et al. (46) reported that local glucose uptake in the rat vestibular nuclei increased 30% during dynamic exercise. Recently, Williams and co-workers (49) have shown in the cat that the increase in c-fos immunoreactivity during isometric muscle contraction is specifically mediated by the contracting muscle. They showed that the increase in c-fos immunoreactivity in the vestibular nuclei was not mediated by changes in baroreceptor activity. Both elevation and reduction of arterial pressure failed to increase c-fos immunoreactivity to the vestibular nuclei. These findings strongly indicate that afferent inputs from contracting skeletal muscle increase neuronal activity in the vestibular nuclei during exercise. In addition to the convergence of afferent input to the vestibular nuclei, both the vestibulosympathetic and skeletal muscle reflexes appear to activate a number of common brain regions associated with sympathetic regulation. Both vestibular and skeletal muscle afferents have been demonstrated to stimulate the caudal and rostral ventrolateral medulla (3, 4, 39, 50, 54). The nucleus tractus solitarius has also been shown to receive inputs from the vestibular and skeletal muscle afferents (41, 51). However, vestibular inputs to the nucleus tractus solitarius are believed not to be associated with sympathetic responses observed during vestibular-mediated activity (51). Additionally, the lateral tegmental field has been shown to be important in the vestibulosympathetic reflex; lesion in this brain area reduces sympathetic outflow in response to vestibular nerve stimulation (38). The lateral tegmental field has been shown to be responsive to muscle contraction (14). Despite all the evidence that vestibular and skeletal muscle afferents may interact centrally to modify sympathetic outflow, the results from the present study suggest that this does not occur in humans.

It has been reported that elevation of arterial pressure attenuated sympathetic outflow elicited by electrical stimulation of the feline vestibular nerve (16). This finding suggested that the arterial baroreflex buffered increases in sympathetic outflow mediated by the vestibular system. However, in the present study, increases in arterial pressure mediated by IHG did not appear to alter MSNA responses elicited by the vestibulosympathetic reflex (i.e., HDR). This conclusion is based on the finding that MSNA responses during HDR and IHG were additive when arterial pressure was significantly greater during the combination trial than during the HDR trial. It would have been expected that MSNA responses during the combination trial would have been less than the sum of the two individual trials (i.e., neural inhibition) if arterial pressure modulates the vestibulosympathetic reflex. It is possible that, unlike pharmacological manipulation of arterial pressure in the cat (16), exercise resets the arterial baroreflex to elicit a different effect on the vestibulosympathetic reflex.

The reflex interaction between the vestibular and skeletal muscle afferents may change importantly during spaceflight and aging. During both spaceflight and aging, both the otolith organs and skeletal muscle undergo morphological and functional changes. For example, muscle atrophy and weakness accompany spaceflight and aging (9, 44, 48). In humans, a significant portion of muscle fibers are denervated in the elderly (5, 10). Similarly, otolith hair cell loss is associated with both spaceflight (7, 32, 33) and aging (1, 2, 24). This change is associated with significant reductions in the sensitivity of the otolith organs to gravitational input (25, 31, 47). Moreover, both the vestibulospinal and vestibuloocular reflexes are diminished in the elderly (11, 17, 23, 24, 37). Thus, with these physiological changes, it is possible that the interaction between the vestibulosympathetic reflex and skeletal muscle afferents will be altered. These vestibular and muscle afferent changes may increase the susceptibility to orthostatic hypotension in astronauts after spaceflight (40) and in the elderly (34).

The results of the current study are limited to vestibular input from the otolith organs. Because the head was stationary during HDR, input from the semicircular canals is eliminated. Animal and human studies indicate that the vestibulosympathetic reflex is primarily mediated by the otolith organs (28, 53). In addition, the influence of central command during IHG could not be eliminated. It is possible that descending central input to the vestibular nuclei will modify its interaction with skeletal muscle afferents. However, the increase in MSNA during IHG is primarily the result of afferent input from the exercising muscle and not central command (20). Finally, the conclusions reached from this study are limited to the conditions that were tested. Only one experimental intervention was used to engage the vestibular and skeletal muscle afferents (i.e., 30% MVC and maximal HDR). It is possible that the interaction of these reflexes would change under other testing conditions. For example, using IHG at 5% MVC and HDR of only 50% of maximum may have elicited different results. Furthermore, whether afferent feedback from the otolith organs mediated by HDR is similar to that observed during locomotion is not clear. Future studies are needed to address this issue.

In summary, IHG and HDR performed together elicited MSNA and arterial pressure responses that were equal to the sum of each stimulus when performed individually. These results indicate that the vestibulosympathetic and skeletal muscle reflexes demonstrate an additive interaction for MSNA and arterial pressure under the conditions that were tested. Thus these findings suggest that no central modulation exists between these two reflexes in humans.