Effects of postural changes and vestibular lesions on diaphragm and rectus abdominis activity in awake cats

J Appl Physiol 91: 137 144, 2001. Effects of postural changes and vestibular lesions on diaphragm and rectus abdominis activity in awake cats L. A. COTTER, 1 H. E. ARENDT, 1 J. G. JASKO, 1 C. SPRANDO, 1 S. P. CASS, 2 AND B. J. YATES 1 1 Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and 2 Department of Otolaryngology, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received 19 December 2000; accepted in final form 13 February 2001 Cotter, L. A., H. E. Arendt, J. G. Jasko, C. Sprando, S. P. Cass, and B. J. Yates. Effects of postural changes and vestibular lesions on diaphragm and rectus abdominis activity in awake cats. J Appl Physiol 91: 137 144, 2001. Changes in posture can affect the resting length of the diaphragm, requiring alterations in the activity of both the abdominal muscles and the diaphragm to maintain stable ventilation. To determine the role of the vestibular system in regulating respiratory muscle discharges during postural changes, spontaneous diaphragm and rectus abdominis activity and modulation of the firing of these muscles during nose-up and ear-down tilt were compared before and after removal of labyrinthine inputs in awake cats. In vestibularintact animals, nose-up and ear-down tilts from the prone position altered rectus abdominis firing, whereas the effects of body rotation on diaphragm activity were not statistically significant. After peripheral vestibular lesions, spontaneous diaphragm and rectus abdominis discharges increased significantly (by 170%), and augmentation of rectus abdominis activity during nose-up body rotation was diminished. However, spontaneous muscle activity and responses to tilt began to recover after a few days after the lesions, presumably because of plasticity in the central vestibular system. These data suggest that the vestibular system provides tonic inhibitory influences on rectus abdominis and the diaphragm and in addition contributes to eliciting increases in abdominal muscle activity during some changes in body orientation. respiration; abdominal muscle; plasticity CHANGES IN POSTURE CAN AFFECT the resting length of respiratory muscles, requiring alterations in the activity of these muscles if ventilation is to be unaffected. For example, it is well established that nose-up tilt of quadrupeds or standing in humans from a supine position can produce diaphragm shortening (11, 14, 16, 18, 25). Compensation for the effects of gravity on diaphragm length during supine to head-up body tilts includes both an increase in diaphragm activity and a cocontraction of the abdominal muscles (5, 6, 8, 10, 13, 18, 25). Experiments in anesthetized dogs have suggested that vagal afferents play an important role in eliciting changes in respiratory muscle activity during postural alterations (7, 10). However, recent studies in decerebrate, unanesthetized animals showed that the vestibular system also contributes to altering respiratory muscle activity during movement and changes in posture (22, 23, 27, 29). Furthermore, anatomic studies have demonstrated that many bulbospinal neurons in the medial medullary reticular formation provide inputs to both phrenic and rectus abdominis motoneurons (2); because this region of the reticular formation receives substantial vestibular and other movementrelated inputs (19, 20, 26), it seems likely that reticulospinal neurons could adjust the activity of both diaphragm and abdominal motoneurons during postural alterations. The present study had several objectives. One goal was to compare changes in diaphragm and abdominal muscle activity during whole body tilts in awake cats, to reassess the relative activation of these muscles during movement. Unlike previous studies in anesthetized dogs (8, 10, 18, 25), body tilts were performed in prone animals, because postural alterations from the supine position (which were examined in the prior studies) are infrequent in quadrupeds. Abdominal muscle recordings were made from rectus abdominis, because this is the only expiratory muscle thus far demonstrated to be coactivated with the diaphragm by the medial medullary reticular formation (1, 2). Although previous studies have indicated that rectus abdominis displays less expiratory-related activity than deeper abdominal muscles such as transversus abdominis (12), rectus abdominis is well suited to tonically restrain the abdominal contents during postural changes that allow gravity to displace the viscera (9). In addition, rectus abdominis has been shown to be activated during expiratory loading in cats and plays a significant role in increasing abdominal cavity pressure during some behaviors such as coughing (3). A second goal was to determine whether tonic firing of rectus abdominis and the diaphragm or alterations in the activity of these muscles during postural changes is influenced by the vestibular system. For this purpose, recordings were made from the muscles before and after removal of vestibular inputs by means of a com- Address for reprint requests and other correspondence: B. Yates, Dept. of Otolaryngology, Univ. of Pittsburgh, Eye and Ear Institute, Rm. 106, 203 Lothrop St., Pittsburgh, PA 15213 (E-mail: byates@pitt.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.jap.org 8750-7587/01 $5.00 Copyright 2001 the American Physiological Society 137

138 VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS Table 1. Period of data recording and total number of experimental trials for each animal Prevestibular Lesions Postvestibular Lesions Animal No. Muscles Recorded Recording period, days Total no. of tilts performed Recording period, days Total no. of tilts performed 1 Diaphragm RA 79 1,038 22 401 2 Diaphragm RA 41 1,134 40 707 3 Diaphragm RA 109 998 34 632 4 Diaphragm RA 106 703 33 417 5 Diaphragm RA 146 798 30 363 6 Diaphragm Only 106 424 20 355 7 RA only 79 549 25 302 RA, rectus abdominis. bined bilateral labyrinthectomy and transection of the eighth cranial nerves. However, rapid plastic changes occur in the central vestibular system after removal of labyrinthine inputs (21, 24, 28), in part because the vestibular nuclei receive substantial nonlabyrinthine inputs signaling body position in space (28). Thus effects of vestibular lesions on respiratory muscle activity were monitored for 1 mo to determine whether long-term compensation for deficits in respiratory regulation produced by the lesions would occur. METHODS All of the procedures used in this study conformed to the American Physiological Society s Guiding Principles in the Care and Use of Animals and were approved by the University of Pittsburgh s Institutional Animal Care and Use Committee. Overview of data collection procedures. Data were collected from seven female adult cats that had been spayed before the onset of data collection. The general procedures for producing body tilts were similar to those in a previous study that considered the effects of vestibular lesions on orthostatic tolerance in awake cats (15). Animals were trained to remain sedentary in the prone position on a tilt table during nose-up or ear-down whole body rotations of 20, 40, or 60 amplitude. A restraint bag with attached Velcro straps was placed around the animal s body; the Velcro straps were secured to the sides of the tilt table to prevent the animal s position from shifting during testing. The animal s head was immobilized by inserting a screw into a bolt mounted on the skull. Activity of rectus abdominis and the costal diaphragm was typically recorded using pairs of Teflon-insulated stainless steel wire (Cooner Wire, Chatsworth, CA) stripped of insulation for 5 mm and sutured to the muscle epimysium together with an insulating patch of Silastic sheeting. In one animal, however, wire pairs with uninsulated tips of 1 mm length were inserted directly into the muscles and secured using sutures. The insulated portions of the wires were led subcutaneously and soldered to a connector mounted on the animal s head. Data were collected during recording sessions with a duration of 30 min; one to three recording sessions were conducted per day. During each recording session, only one direction of tilt was performed (either nose up or left or right ear down), although table rotations of 20, 40, and 60 amplitude were randomly disbursed so that animals could not predict the amplitude of tilt at the onset of each rotation. Tilts persisted for 40 60 s and were separated by at least 1 min. Only data recorded during trials in which animals were observed to remain sedentary were analyzed. The tilt table was rotated manually and was secured in the tilted position using a spring device that permitted movement to one of three predetermined amplitudes. Rotations from the Earthhorizontal to the nose-up or ear-down position were performed rapidly, at a velocity of 60 /s at all three amplitudes. Respiratory muscle responses to tilt were recorded in animals with eighth cranial nerves intact over a period of 41 146 days (Table 1). Average activity of the diaphragm and rectus abdominis was determined for the period during which the animal was tilted maximally and compared with activity averaged over an equivalent time period immediately before the tilt, as indicated in Fig. 1. Subsequently, vestibular inputs were removed bilaterally, and respiratory muscle activity before and during tilts was measured in the same manner as before the lesion. The postlesion recordings Fig. 1. Example of respiratory muscle activity recorded during whole body tilt, illustrating the method used to quantify data in this study. Trace A shows rectus abdominis activity, trace B shows diaphragm activity, and trace C is a recording of table position. In this example, the animal was tilted from the prone position to 60 nose up. Average respiratory muscle activity was determined for the period in which the animal was tilted maximally as well as an equivalent time interval immediately before each tilt.

VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS 139 began the day after the surgery and continued for 20 34 days (see Table 1). Data recording was performed daily for the first week after the lesions and subsequently at least every 3 days. Training procedures. A training period of 2 3 mo was required for animals to learn to remain sedentary during experimental sessions. The training was performed in two phases. As a first step, animals learned to tolerate body restraint in a vinyl bag. The restraint period initially lasted for only a few minutes, but it was gradually increased until animals remained sedentary for at least 30 min. Food was provided at the end of the testing period as a reward, and the experimental session was terminated promptly if the animal attempted to move from the restraint bag or showed any signs of distress (e.g., vocalization). The second phase of training involved teaching the animal to tolerate head fixation. Initially, the head was fixed for only a few minutes, but this interval was increased over time until the animal remained relaxed throughout a head-fixation period of 30 min. No data were collected until training was complete and the animal could be restrained throughout the testing period without vocalization or any indication of discomfort. Surgical procedures. Two recovery surgeries were required for each animal. Both surgeries were performed using sterile procedures in a dedicated operating suite. The first surgical procedure was performed to secure a bolt to the skull to permit head fixation and to implant electromyogram (EMG) recording electrodes. The second surgery to produce peripheral vestibular lesions was performed after initial data collection was complete. For each surgery, animals were initially anesthetized using an intramuscular injection of ketamine (15 mg/kg) and acepromazine (0.2 mg/kg). Subsequently, an endotracheal tube and intravenous catheter were inserted. Anesthesia was supplemented as necessary using 1 1.5% isoflurane vaporized in O 2 to maintain areflexia and stable heart rate. Ringer lactate solution was infused intravenously to replace fluid loss during the surgery, and a heating pad was used to maintain rectal temperature near 38 C. To place EMG electrodes for recording diaphragm activity, a small incision was made through linea alba, and the liver and adjacent viscera were retracted to provide access to the costal diaphragm on one side. After implantation of diaphragm electrodes, the abdominal musculature was closed with the use of sutures, and EMG electrodes were attached to a portion of rectus abdominis near the border with the external oblique. Subsequently, the animal s head was secured in a stereotaxic frame, and a head fixation bolt was mounted with the use of procedures described in a previous publication (15). Wires from the EMG recording electrodes were attached to a connector that was mounted on the skull behind the fixation plate. To eliminate vestibular inputs, the tympanic bulla on each side was exposed using a ventrolateral approach and opened to expose the cochlea. A drill was used to remove temporal bone near the base of the cochlea, thereby producing a labyrinthectomy that rendered the vestibular apparatus dysfunctional. This procedure also provided access to the internal auditory canal. The eighth cranial nerve was then transected under microscopic observation within the internal auditory canal. Thus two independent lesions affecting the vestibular system were made on both sides to ensure that vestibular inputs were removed. In no case did nystagmus or deviation in eye position occur after the surgery, suggesting that the peripheral lesions were complete bilaterally. Furthermore, our laboratory has previously demonstrated that this procedure is effective in eliminating vestibular input (15). To ensure that animals received proper hydration and nutrition during the postsurgical period, an intravenous injection port remained in place for 2 days after surgery, and 50 ml of 5% dextrose solution were administered intravenously each day. In addition, feeding was done by hand until the animal s spontaneous consumption of food and water returned to prelesion levels, which required 3 5 days. Data recording procedures. During recording sessions, a cable was attached to the head-mounted connector to allow EMG signals to be fed to an alternating-current amplifier (model 1700, A-M Systems, Carlsberg, WA); activity was amplified by a factor of 10 4, filtered with a band pass of 10 10,000 Hz, and full-wave rectified with a time constant of 1 ms. Subsequently, signals were recorded digitally using a 1401-plus data collection system (Cambridge Electronic Design, Cambridge, UK) interfaced with a Macintosh G3 computer (sampling rate of 1,000 Hz). A potentiometer mounted on the tilt table provided a recording of table position; the voltage from this potentiometer was digitized and sampled at 100 Hz. The Spike-2 software package (Cambridge Electronic Design) was used for data analysis. Average EMG activity was determined for the time period an animal was tilted from the prone position as well as over an equivalent time period immediately preceding each tilt (see Fig. 1). To determine whether baseline respiratory muscle firing was altered by removal of vestibular inputs in a particular animal, levels of pretilt activity ascertained for pre- and postlesion trials were averaged and compared statistically as described in Statistical analysis of data. To pool results from all animals, an average was made of the mean percent change in respiratory muscle activity produced by eighth cranial nerve transection in each animal. Because percent changes in activity were considered in this analysis, differences between animals in baselines and noise levels of EMG recordings were accounted for. Verification of recording electrode locations and vestibular lesions. At the conclusion of data recording, animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg) and perfused transcardially with phosphate-buffered saline followed by the paraformaldehydelysine-sodium metaperiodate fixative developed by McLean and Nakane (17). The diaphragm was then removed so that the placement of EMG recording electrodes could be determined. Rectus abdominis was also inspected to confirm electrode placement. In three animals, the head was removed and decalcified using a solution of EDTA and hydrochloric acid. The temporal bone on each side was subsequently removed, embedded in 12% celloidin, cut in the coronal plane (30- m thickness), and stained using hemotoxilyn. The temporal bone sections were then inspected histologically to determine the extent of damage to the eighth cranial nerves and vestibular labyrinth. In all cases, it was obvious that peripheral vestibular inputs were eliminated on both sides. Statistical analysis of data. Statistical analyses of data were performed with the use of the Prism 3 software package (GraphPad Software, San Diego, CA) running on a Macintosh G4 computer. A nonparametric repeated-measures ANOVA procedure (Friedman test) in combination with a post hoc test (Dunn s multiple-comparison test) was used to compare respiratory activity measured before the peripheral vestibular lesion, in the first week after the lesion, and at subsequent times. This procedure was also used to compare changes in respiratory muscle activity elicited by different rotation amplitudes and directions. A Mann-Whitney test was used to compare changes in diaphragm and rectus abdominis activity during specific postural alterations. Statistical significance was set at P 0.05. Pooled data are presented as means SE.

140 VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS RESULTS Successful recordings of EMG activity were made from both rectus abdominis and the diaphragm in five of the seven animals; in one additional animal, only rectus abdominis activity was reliably recorded throughout the experiment, and, in another case, only the diaphragm provided consistent data during the entire recording period. Thus a sample of six animals was used to determine the effects of tilt on either rectus abdominis or diaphragm firing; the consequences of bilateral removal of vestibular inputs on spontaneous respiratory muscle activity and on tilt-elicited changes in muscle discharges were examined in all of these cases. Effects of body tilt on rectus abdominis and diaphragm activity before vestibular lesions. An example of diaphragm and rectus abdominis EMG activity recorded before and during a 60 nose-up tilt in one animal is shown in Fig. 1. The effects of 20, 40, and 60 nose-up and ear-down tilt on rectus abdominis and diaphragm firing are shown in Fig. 2; the data points in this figure represent pooled responses from six animals. Nose-up tilt produced an increase in rectus abdominis activity whose magnitude was dependent on the amplitude of the rotation; the relationship between tilt amplitude and the increase in muscle activity was shown to be statistically significant using a nonparametric repeated-measures ANOVA procedure (Friedman test, P 0.002). Similarly, ipsilateral ear-down tilt (toward the muscle that was recorded from) produced a statistically significant (P 0.001) amplitudedependent increase in rectus abdominis activity. During contralateral ear-down tilt, the increase in rectus abdominis activity with respect to rotation amplitude approached statistical significance (P 0.052). In contrast, no significant relationship between tilt amplitude and changes in diaphragm activity could be ascertained (P 0.05) for any direction of rotation. Although the increase in rectus abdominis activity (23%) during 60 nose-up tilt appeared larger than that during either 60 ipsilateral (13%) or contralateral (15%) ear-down tilt, these differences did not reach statistical significance (P 0.05, Friedman test). Nonetheless, the increase in rectus abdominis activity elicited by 60 nose-up pitch was significantly (P 0.02, 2-tailed Mann-Whitney test) larger than the increase in diaphragm activity (4%) elicited by the same rotation. However, the changes in rectus abdominis and diaphragm activity produced by 60 ipsilateral and contralateral ear-down roll were not statistically distinguishable (P 0.05, 2-tailed Mann-Whitney test). Effects of bilateral eighth cranial nerve transection on spontaneous rectus abdominis and diaphragm activity. After bilateral removal of vestibular inputs, background activities of rectus abdominis and the diaphragm recorded when animals were in the prone, untilted position were compared with firing measured before the lesions. Figure 3 compares average EMG activity before and after the vestibular neurectomy in each animal. Removal of vestibular inputs produced a highly statistically significant (P 0.0001, Friedman test) increase in rectus abdominis and diaphragm activity in every animal. Respiratory muscle activity typically diminished after the first 3 days after removal of vestibular inputs, but it was still significantly higher than before the lesions in the majority of animals (see Fig. 3). In four cases for both rectus abdominis and diaphragm recordings, spontaneous activity did not recover to prelesion levels even after more than a week. To further consider the effects of eighth cranial nerve transection on baseline respiratory muscle activity, the percent changes in spontaneous EMG activity from prelesion values determined for all animals were pooled. The results of this analysis are shown in Figure 4. Because spontaneous rectus abdominis and diaphragm activity typically remained elevated for at least a week after peripheral vestibular lesions (see Fig. 3), all data collected in the first week after the removal of vestibular inputs were combined to form a single group. When all animals were considered together, it was determined that the postlesion increase in both rectus abdominis and diaphragm activity was statistically significant (P 0.006 for rectus abdominis and P 0.03 for the diaphragm, Friedman test). However, a post hoc test (Dunn s multiple-comparison test) indicated that this significant increase in muscle activity could only be demonstrated for the first week after removal of vestibular inputs. Rectus abdominis and diaphragm EMG activity recorded subsequently was not statistically different (P 0.05) from prelesion Fig. 2. Effects of nose-up and ear-down tilts of different amplitudes on electromyogram activity recorded from rectus abdominis and the diaphragm. Every data point represents pooled responses from 6 animals. Error bars indicate SE.

VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS 141 Fig. 3. Effects of removal of vestibular inputs on baseline rectus abdominis (A) and diaphragm (B) activity in each animal. Electromyogram activity was recorded when animals were in the prone position, both before and after peripheral vestibular lesions. The postlesion data were separated into 3 groups for this analysis: those recorded in the first 3 days after the lesion (D1 3), those recorded in the following 3 days (D4 6), and those recorded subsequently (Subs). Error bars indicate SE. *Postlesion average significantly different from that before removal of vestibular inputs, P 0.05. values, despite the fact that in many animals the baseline level of firing remained significantly elevated (see Fig. 3). Effects of bilateral eighth cranial nerve transection on changes in rectus abdominis and diaphragm activity during whole body tilts. The effects of removal of vestibular inputs on changes in rectus abdominis and diaphragm activity during 60 nose-up and ear-down tilt were also examined and are shown in Fig. 5. Figure 6 provides examples of rectus abdominis responses to 60 nose-up tilt before and 1 day after vestibular lesions in one animal (animal 5). Eighth cranial nerve transection resulted in a significant (P 0.006, Friedman test) decrement in the percent increase in rectus abdominis activity during 60 nose-up pitch. A post hoc test (Dunn s multiple-comparison test) indicated that this decrease in the muscle s response to tilt was only significant during the first week after the lesion. However, removal of vestibular inputs resulted in no reduction in the percent increase in rectus abdominis activity during either ipsilateral or contralateral ear-down tilt or in the modest increases in diaphragm activity during any direction of tilt. Because spontaneous respiratory muscle activity was elevated after the removal of vestibular inputs, one possibility is that a ceiling effect prevented further increases in firing levels during body tilts. However, in all animals, transient increases in muscle activity that were much higher than the mean baseline level were noted after the lesions, particularly during voluntary movements at the end of recording sessions. Figure 6 illustrates an example of a transient burst of Fig. 4. Percent increase in baseline respiratory muscle activity after removal of vestibular inputs for all animals pooled. To perform this analysis, prelesion rectus abdominis and diaphragm activity in each animal was standardized at 100%, and the percent change in muscle activity after the lesion was determined for each animal. Subsequently, the values from each animal were averaged. W 1, first week; Subs, subsequent to first week. Error bars indicate SE. *Postlesion average significantly different from that before removal of vestibular inputs, P 0.05.

142 VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS Fig. 5. Effects of removal of vestibular inputs on increases in rectus abdominis and diaphragm activity during 60 nose-up and ear-down tilt. Bars represent pooled data from 6 animals. Error bars indicate SE. *Postlesion average significantly different from that before removal of vestibular inputs, P 0.05. muscle activity that occurred during nose-up tilt after eighth cranial nerve transection. These observations indicate that a ceiling effect did not result in the postlesion decrement of rectus abdominis responses to 60 nose-up tilt. DISCUSSION Nose-up or ear-down tilt from the prone position in awake cats resulted in an amplitude-dependent increase in rectus abdominis activity, presumably to restrain the abdominal viscera during these movements. Although prior studies that considered the effects of postural changes on abdominal muscle activity (e.g., Refs. 8, 10) largely ignored rectus abdominis, mainly because this muscle is typically silent (12), the present data show that rectus abdominis exhibits posturally related discharges in awake cats. Furthermore, changes in rectus abdominis activity during 60

VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS 143 Fig. 6. Example of effects of removal of vestibular inputs on spontaneous activity of rectus abdominis and on increases in muscle activity during 60 nose-up tilt from the prone position. The postlesion activity was recorded 1 day after bilateral transection of the eighth cranial nerves. An arrow denotes a brief burst of activity during the postlesion recording. The occurrence of this burst of muscle firing indicates that activity was not maximal and that the lack of a response to nose-up tilt after the removal of vestibular inputs was not due to a ceiling effect. nose-up tilts were significantly larger than alterations in diaphragm activity during these rotations. It was previously observed in anesthetized dogs that prevention of diaphragm shortening during nose-up tilts from a supine position was more related to increases in abdominal muscle activity than diaphragm activity (13). The present data show that similar relative increases in diaphragm and rectus abdominis activity occur during nose-up rotations from a prone position in awake cats. The present study also demonstrated that the vestibular system tonically influences respiratory muscle activity in awake animals, because removal of vestibular inputs produced a significant increase in spontaneous firing of both rectus abdominis and the diaphragm. The simplest explanation for this observation is that the vestibular system provides tonic drive to spinally projecting neurons that inhibit phrenic and abdominal motoneurons. In addition, modulation of rectus abdominis activity during 60 nose-up tilts from a prone position was significantly reduced after removal of vestibular inputs, indicating that the vestibular system provides phasic, posturally related influences on activity of this abdominal muscle in addition to the tonic influences described above. It seems likely that posturally related increases in abdominal muscle activity that are elicited by vestibular inputs are produced through descending excitatory pathways. Thus two bulbospinal projections may relay vestibular signals to rectus abdominis and perhaps other abdominal motoneurons: an inhibitory pathway that receives tonic vestibular drive that is unrelated to body position in space and an excitatory pathway that is only activated when the animal assumes particular postures. At present, the descending pathways that relay vestibular signals to respiratory motoneurons are largely unknown. In decerebrate animals, large brain stem lesions that either destroyed the dorsal and ventral respiratory groups or removed the projections of respiratory group neurons to spinal motoneurons did not abolish vestibular influences on diaphragm or abdominal muscle activity (22, 23, 29). Thus other pathways must be largely responsible for mediating vestibulorespiratory responses. One possibility is bulbospinal projections from the medial medullary reticular formation, because neurons in this region have been demonstrated to make synaptic connections with diaphragm and abdominal motoneurons (1, 2, 30) and to receive vestibular inputs (4, 20). However, further experiments will be required to establish whether either the tonic inhibition or the posturally related excitation of respiratory motoneurons provided by the vestibular system is mediated by medial medullary reticulospinal neurons. After a few days after bilateral eighth cranial nerve transection, tonic activity of both the diaphragm and rectus abdominis and modulation of rectus abdominis activity during nose-up tilts tended to return to prelesion values, although in many animals complete recovery was not observed. Such compensation is not unexpected, however, because after removal of labyrinthine inputs vestibular nucleus neurons quickly regain spontaneous activity and even modulation of their firing during whole body rotations (21, 24, 28). This adaptive plasticity is undoubtedly related to the presence of substantial nonlabyrinthine inputs reflecting body position in space to the vestibular nuclei (28). Thus, even after elimination of inputs from the inner ear, the central vestibular system could potentially influence the regulation of respiratory muscle activity. It is therefore likely that bilateral vestibular nucleus lesions would have larger effects on tonic respiratory muscle activity and alterations in this activity during changes in posture than would bilateral eighth cranial nerve transection. This prospect remains to be explored experimentally. Three caveats must be considered when interpreting the results of this study. First, the peripheral lesions used to remove vestibular inputs undoubtedly also diminished auditory signals to the central nervous system. Thus the possibility exists that the effects of eighth cranial nerve transections on respiratory muscle activity were related more to the peripheral auditory lesion than to removal of vestibular inputs. However, this prospect seems remote, because auditory stimulation has not been demonstrated to affect respiration. Second, the only abdominal muscle that was studied was rectus abdominis, and it is possible that

144 VESTIBULORESPIRATORY INFLUENCES IN AWAKE ANIMALS activity of other abdominal muscles would not have been altered by removal of peripheral vestibular inputs. Nevertheless, the present findings indicate that the vestibular system tonically influences intra-abdominal pressure through effects on at least one abdominal muscle and in addition contributes to producing necessary alterations in intra-abdominal pressure during some postural changes. Third, our diaphragm recordings were limited to the costal portions of this muscle, although previous studies have shown that larger increases in activity occur in the crural than in the costal diaphragm during nose-up tilts from a supine position (25). Thus more pronounced effects of tilt on diaphragm firing might have been observed in the present experiments if the crural region were studied. Perspectives. The present data show that the vestibular system provides tonic inhibitory influences on the activity of rectus abdominis and the diaphragm and in addition contributes to eliciting increases in the activity of rectus abdominis during nose-up rotations from the prone position. These observations demonstrate that control of respiratory muscle activity in awake animals is complex, in that sensory inputs in addition to those traditionally considered to influence breathing (such as those from chemoreceptors and pulmonary receptors) participate in the regulatory process. Further studies will be required to elucidate how these multiple sensory inputs interact in the adjustment of respiratory muscle contractions. We thank Drs. Robert Schor and Joseph Furman for comments on a previous version of this manuscript. We are also grateful to Brian Jian, Michael Holmes, Andrew Etzel, David Eisenberg, Sarah Graff, and Aaron Bergsman for assistance with the completion of these experiments. This study was supported by National Institute on Deafness and Other Communication Disorders Grants R01 DC-00693, R01 DC- 03732, and P01 DC-03417. Electronics support was provided through National Eye Institute Core Grant EY-08098. REFERENCES 1. Billig I, Foris JM, Card JP, and Yates BJ. Transneuronal tracing of neural pathways controlling an abdominal muscle, rectus abdominis, in the ferret. Brain Res 820: 31 44, 1999. 2. Billig I, Foris JM, Enquist LW, Card JP, and Yates BJ. Definition of neuronal circuitry controlling the activity of phrenic and abdominal motoneurons in the ferret using recombinant strains of pseudorabies virus. J Neurosci 20: 7446 7454, 2000. 3. Bolser DC, Reier PJ, and Davenport PW. Responses of the anterolateral abdominal muscles during cough and expiratory threshold loading in the cat. J Appl Physiol 88: 1207 1214, 2000. 4. Bolton PS, Goto T, Schor RH, Wilson VJ, Yamagata Y, and Yates BJ. Response of pontomedullary reticulospinal neurons to vestibular stimuli in vertical planes. Role in vertical vestibulospinal reflexes of the decerebrate cat. J Neurophysiol 67: 639 647, 1992. 5. De Troyer A. Mechanical role of the abdominal muscles in relation to posture. Respir Physiol 53: 341 353, 1983. 6. De Troyer A, Estenne M, Ninane V, Van Gansbeke D, and Gorini M. Transversus abdominis muscle function in humans. J Appl Physiol 68: 1010 1016, 1990. 7. Farkas GA, Baer RE, Estenne M, and De Troyer A. Mechanical role of expiratory muscles during breathing in upright dogs. J Appl Physiol 64: 1060 1067, 1988. 8. Farkas GA, Estenne M, and De Troyer A. Expiratory muscle contribution to tidal volume in head-up dogs. J Appl Physiol 67: 1438 1442, 1989. 9. Farkas GA and Rochester DF. Characteristics and functional significance of canine abdominal muscles. J Appl Physiol 65: 2427 2433, 1988. 10. Farkas GA and Schroeder MA. Mechanical role of expiratory muscles during breathing in prone anesthetized dogs. J Appl Physiol 69: 2137 2142, 1990. 11. Finkler J and Iscoe S. Control of breathing at elevated lung volumes in anesthetized cats. J Appl Physiol 56: 839 844, 1984. 12. Gilmartin JJ, Ninane V, and De Troyer A. Abdominal muscle use during breathing in the anesthetized dog. Respir Physiol 70: 159 171, 1987. 13. Gorini M and Estenne M. Effect of head-up tilt on neural inspiratory drive in the anesthetized dog. Respir Physiol 85: 83 96, 1991. 14. Green M, Mead J, and Sears TA. Muscle activity during chest wall restriction and positive pressure breathing in man. Respir Physiol 35: 283 300, 1978. 15. Jian BJ, Cotter LA, Emanuel BA, Cass SP, and Yates BJ. Effects of bilateral vestibular lesions on orthostatic tolerance in awake cats. J Appl Physiol 86: 1552 1560, 1999. 16. Leevers AM and Road JD. Effect of lung inflation and upright posture on diaphragmatic shortening in dogs. Respir Physiol 85: 29 40, 1991. 17. McLean IW and Nakane PK. Periodate-lysine-paraformaldehyde for immunoelectron microscopy. J Histochem Cytochem 22: 1077 1083, 1974. 18. Newman SL, Road JD, and Grassino A. In vivo length and shortening of canine diaphragm with body postural change. J Appl Physiol 60: 661 669, 1986. 19. Peterson BW, Anderson ME, and Filion M. Responses of pontomedullary reticular neurons to cortical, tectal and cutaneous stimuli. Exp Brain Res 21: 19 44, 1974. 20. Peterson BW, Filion M, Felpel LP, and Abzug C. Responses of medial reticular neurons to stimulation of the vestibular nerve. Exp Brain Res 22: 335 350, 1975. 21. Ris L and Godaux E. Neuronal activity in the vestibular nuclei after contralateral or bilateral labyrinthectomy in the alert guinea pig. J Neurophysiol 80: 2352 2367, 1998. 22. Rossiter CD, Hayden NL, Stocker SD, and Yates BJ. Changes in outflow to respiratory pump muscles produced by natural vestibular stimulation. J Neurophysiol 76: 3274 3284, 1996. 23. Shiba K, Siniaia MS, and Miller AD. Role of ventral respiratory group bulbospinal expiratory neurons in vestibular-respiratory reflexes. J Neurophysiol 76: 2271 2279, 1996. 24. Smith PF and Darlington CL. Neurochemical mechanisms of recovery from peripheral vestibular lesions. Brain Res Rev 16: 117 133, 1991. 25. Van Lunteren E, Haxhiu MA, Cherniack NS, and Goldman MD. Differential costal and crural diaphragm compensation for posture changes. J Appl Physiol 58: 1895 1900, 1985. 26. Wilson VJ and Peterson BW. Vestibulospinal and reticulospinal systems. In: Handbook of Physiology. The Nervous System. Motor Control. Bethesda, MD: Am. Physiol. Soc., 1981, sect. 1, vol. II, pt. 1, chapt. 14, p. 667 702. 27. Yates BJ, Jakus J, and Miller AD. Vestibular effects on respiratory outflow in the decerebrate cat. Brain Res 629: 209 217, 1993. 28. Yates BJ, Jian BJ, Cotter LA, and Cass SP. Responses of vestibular nucleus neurons to tilt following chronic bilateral removal of vestibular inputs. Exp Brain Res 130: 151 158, 2000. 29. Yates BJ, Siniaia MS, and Miller AD. Descending pathways necessary for vestibular influences on sympathetic and inspiratory outflow. Am J Physiol Regulatory Integrative Comp Physiol 268: R1381 R1385, 1995. 30. Yates BJ, Smail JA, Stocker SD, and Card JP. Transneuronal tracing of neural pathways controlling activity of diaphragm motoneurons in the ferret. Neuroscience 90: 1501 1513, 1999.