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J Appl Physiol 91: 777-788, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 2, 777-788, August 2001

Multiple rostral medullary nuclei can influence breathing in awake goats

J. M. Wenninger, L. G. Pan, P. Martino, L. Geiger, M. Hodges, A. Serra, T. R. Feroah, and H. V. Forster

Departments of Physiology and Pediatrics, Medical College of Wisconsin, Milwaukee 53226; Program in Physical Therapy, Marquette University, Milwaukee 53295; and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine the effect on breathing of neuronal dysfunction in the retrotrapezoid (RTN), facial (FN), gigantocellularis reticularis (RGN), or vestibular (VN) nuclei of adult awake goats. Microtubules were chronically implanted to induce neuronal dysfunction by microinjection of an excitatory amino acid (EAA) receptor antagonist or a neurotoxin. The EAA receptor antagonist had minimal effect on eupneic breathing, but 8-10 days after injection of the neurotoxin, 7 of 10 goats hypoventilated (arterial PCO2 increased 3.2 ± 0.7 Torr). Overall there were no significant (P > 0.10) effects of the EAA receptor antagonist on CO2 sensitivity. However, for all nuclei, >= 66% of the antagonist injections altered CO2 sensitivity by more than the normal 12.7 ± 1.6% day-to-day variation. These changes were not uniform, inasmuch as the antagonist increased (RTN, n = 2; FN, n = 7; RGN, n = 6; VN, n = 1) or decreased (RTN, n = 2; RGN, n = 3; VN, n = 2) CO2 sensitivity. Ten days after injection of the neurotoxin into the FN (n = 3) or RGN (n = 5), CO2 sensitivity was also reduced. Neuronal dysfunction also did not have a uniform effect on the exercise arterial PCO2 response, and there was no correlation between effects on CO2 sensitivity and the exercise hyperpnea. We conclude that there is a heterogeneous population of neurons in these rostral medullary nuclei (or adjacent tissue) that can affect breathing in the awake state, possibly through chemoreception or chemoreceptor-related mechanisms.

rostral medulla; excitatory amino acid receptors; carbon dioxide sensitivity; exercise


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CURRENT THEORIES ON THE ROLE of medullary nuclei in the control of breathing are based largely on data obtained in reduced preparations (14, 29). The relevance of these theories to physiological wakefulness and sleep remains speculative. We, therefore, chronically implanted thermodes on the rostral ventrolateral medulla (RVLM) of goats to induce reversible neuronal dysfunction in anesthetized, awake, and sleep states (16, 33). Cooling the RVLM in the anesthetized state caused sustained apnea, but cooling in awake and sleep states caused only a mild reduction in breathing. Cooling in the awake state attenuated CO2 sensitivity more than the hypoxic and exercise responses, but in no case was the attenuation >60% of normal. These data suggest that in the awake state the RVLM facilitates breathing through a chemoreceptor-dependent and chemoreceptor-independent mechanism (16).

Subsequently, we implanted microtubules bilaterally on the RVLM surface at the site where cooling under anesthesia resulted in apnea (17). Ejections of selective excitatory amino acid (EAA) receptor antagonist did not alter awake eupneic breathing or CO2 sensitivity. However, multiple ejections of a nonselective EAA receptor antagonist caused a modest hypoventilation during room air breathing and attenuated CO2 sensitivity by an average of 60%. Microejection of the neurotoxin kainic acid (KA) in the awake state increased breathing by 50-200% for 10 min to 5 h, and KA also increased heart rate (HR), blood pressure, salivation, swallowing, and mastication. A few hours after the ejections, all functions were normalized except CO2 sensitivity, which was reduced by ~60%. These findings contrast to the terminal apnea often found after KA injection in the anesthetized state on or in the RVLM (30, 32). Accordingly, the facilitation of breathing by the RVLM is critical to sustain breathing in the anesthetized state, but in the awake state, other components of the control system appear capable of partially or completely compensating for attenuated RVLM facilitation.

The RVLM site we studied includes the retrotrapezoid nucleus (RTN) (10), but with surface cooling and surface microejections, neuronal dysfunction may have extended to the facial nucleus (FN). Accordingly, the major objective of the present study was to determine the effect on breathing in the awake state of neuronal dysfunction localized primarily to the RTN or the FN. Neuronal dysfunction was created by microinjection of EAA receptor antagonists or a neurotoxin into microtubules chronically implanted into these nuclei from the dorsal medullary surface. Precise placement of the microtubules proved to be difficult, inasmuch as we found in postmortem histology of the medulla that microtubules in some initial goats were placed such that microinjection had actually been made into the gigantocellularis reticularis (RGN) or the vestibular nuclei (VN). Because some injections into these nuclei affected breathing, we studied these nuclei on a more systematic basis. On the basis of our laboratory's previous findings (16, 17), we initially hypothesized that breathing at rest and during exercise would not be altered by neuronal dysfunction at any site but that CO2 sensitivity would be attenuated by RTN neuronal dysfunction. We further hypothesized that, over days, there would be recovery of CO2 sensitivity after the acutely attenuated response caused by neurotoxic neuronal dysfunction.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All aspects of the study were reviewed and approved by the Medical College of Wisconsin Animal Care Committee before the studies were initiated.

Data were obtained on 18 female and 11 castrated male adult goats weighing 35-58 kg. The goats were housed in an environmental chamber with the ambient temperature and photoperiod adjusted seasonally. They had free access to hay and water, except for periods of the study. The goats were trained to relax in the sternal position in a stanchion and to walk on a treadmill.

Surgical Procedures

An initial surgery was performed to elevate the carotid arteries. Anesthesia was induced with ketamine (15 mg/kg iv) and xylazine (0.01 mg/kg iv). After intubation for mechanical ventilation, anesthesia was maintained with 1-1.5% halothane in oxygen. Under sterile conditions, a 5-cm segment of each carotid artery was elevated to just below the skin, and electrodes were placed in the diaphragm and upper airway muscles for purposes not related to the present objectives. Ceftiofur sodium (2 mg/kg) was administered daily as an antibiotic for >= 1 wk after surgery.

After >= 3 wk, surgery was performed for chronic implantation of microtubules. An occipital craniotomy was performed to expose the cerebellum and dorsal medullary surface. The dura was removed for visualization of the obex. The obex, the midline, and the dorsal surface were reference points for rostral-caudal, medial-lateral, and dorsal-ventral directions, respectively. The distance in each direction for implants was obtained from our goat anatomic atlas (10). In goats, the cerebellum extends within 2 mm of the obex; thus microtubules were implanted through the cerebellum and then advanced into the medulla. Microtubules were implanted bilaterally into the same (n = 6) or different nuclei.

The microtubules were PE-50 tubing drawn to a 0.8-mm (OD) tip. A 25-gauge stainless steel tube was inserted into the tubing during manual advancement into the tissue with the use of a micromanipulator. After the tubing was in place, it was anchored to the bone by means of metal screws and dental acrylic. About 6 mm of tubing remained exterior to the skull through which injections were made. A 31-gauge stylet was placed in the microtubule, except during periods of study.

In 27 goats, microtubules were implanted into the medulla. In one other goat the medulla was exposed but the microtubules were not implanted, and in another goat the microtubules penetrated the cerebellum but not the medulla. These latter two goats served to aid in discerning the cause of changes in physiological function after brain surgery per se. In 6 of the 27 goats, a chronic tracheostomy was created for purposes other than those reported here.

At least for the initial 24 h after microtubule placement, the goats were continuously monitored by laboratory personnel. Some goats were unable to maintain normal sternal recumbent posture for 3-6 h after surgery, and some were unable to stand for up to 4 days after the implant. Food and water intake were decreased over this time period. To minimize brain edema, goats were medicated three times per day with dexamethasone (0.4 mg · kg-1 · day-1 for 2 days, then decreasing by 0.05 mg · kg-1 · day-1). To minimize infection, chloramphenicol (20 mg/kg) was administered three times per day for 3 days, and thereafter ceftiofur sodium (2 mg/kg) and gentamyacin (3 mg/kg) were administered three times per day. Buprenorphine was administered 3 and 12 h after implantation to minimize pain.

Procedures and Protocols

For most studies on airway-intact goats, a fitted mask was taped to the snout and a breathing valve was attached to the mask. For the tracheostomized goats, the breathing valve was attached to a cuffed endotracheal tube inserted into the trachea. The inspired port of the valve was connected to a pneumotachograph connected to a Grass recorder, which was connected to a Citus 436 computer. The expired port of the valve was connected to a Tissot spirometer for collection of expired air analyzed for O2 and CO2 concentration. The elevated carotid artery was chronically catheterized to monitor arterial blood pressure (ABP) and to obtain blood samples for pH, arterial PCO2 (PaCO2), and arterial PO2 determination (model 278, Ciba-Corning). ABP was monitored because medullary sites are also important in its control. Rectal temperature (Tre) was measured after each blood sample was taken.

Studies before and for 2 wk after microtubule placement. For >= 4 days before and nearly every day for 2 wk after brain surgery, the goats were studied to establish the effects of the brain surgery per se. Breathing, ABP, and Tre were monitored for 15 min during room air breathing and then for 15 min when inspired CO2 was increased in 2.5% increments each 5 min for assessment of CO2 sensitivity. Arterial blood (2-ml aliquots) was withdrawn over the last 2 min during room air breathing and at each level of elevated inspired CO2. Even though the goats appeared to be in good physical health within a few days after brain surgery, these studies indicated that breathing, ABP, and Tre were not stable and at control levels until 10-14 days after surgery (see RESULTS). Therefore, we did not begin microinjections until ~15 days after surgery.

Studies after injection of EAA receptor agonists. The initial day of microinjection was for determining whether neurons with EAA receptors were located at the injection site. While the goats were breathing room air, breathing and ABP were continuously monitored, arterial blood was periodically sampled, and Tre was periodically recorded. After a control period of >= 15 min, a 31-gauge stainless steel tube was loaded with mock cerebrospinal fluid (CSF) with a composition (in mM) of 124 NaCl, 2.0 KCl, 2.0 MgCl2, 1.3 NaH2PO4, 2 CaCl2, 26 NaHCO3, and 11 glucose. The solution was equilibrated (at 39°C) in a tonometer with 45 Torr PCO2-100 Torr PO2-balance N2. The injection tube was inserted into one of the microtubules for the exact (known) length of the tubule, and the solution (100 nl) was manually microinjected. After 30 min, mock CSF was similarly injected into the contralateral microtubule. After 30 and usually 60 min later, 100 nl of 100 mM N-methyl-D-aspartic acid (NMDA; mixed with mock CSF) were injected into each of the microtubules. Our laboratory previously showed that this concentration is sufficient to elicit a response in awake goats (17).

Studies after injection of EAA receptor antagonists. For several subsequent days, we determined in the awake state the effects on breathing and ABP of injecting into the microtubules mock CSF or mock CSF mixed with 250 mM kynurenic acid (KynA), 5 mM 2-amino-5-phosphonovalerate (AP5), or 300 µM 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX). These antagonists are effective for several minutes; thus these injections provided a means of creating prolonged neuronal dysfunction. The concentration of the antagonists was as described in our laboratory's previous studies (17). These studies lasted ~150 min. During the initial 15 min (control period), breathing and ABP were continuously monitored while the animals breathed room air. Two 2-ml samples of arterial blood were withdrawn over the last 2 min of this period. Mock CSF with or without an EAA receptor antagonist was then injected over 3 s either unilaterally or bilaterally, as described above. Breathing and ABP were continuously monitored, and arterial blood was withdrawn 15, 30, and 60 min after the injection. Then a second injection identical to the first was made. After 15 min, CO2 sensitivity was assessed as described above. At least 2 h were allowed for recovery before a second and final protocol was completed on the same day. The order of antagonist injection was randomized. The unilateral injection was always completed before the bilateral injection.

In 10 goats, we subsequently evaluated the effect of the EAA antagonists on regulation of breathing during exercise. As in our past studies (6), we did not place the mask on the goats because of concerns that it affects the exercise hyperpnea. The exercise response was evaluated by establishing the temporal pattern of PaCO2 during exercise. The goats stood on a treadmill during a control period and for 15 min after unilateral or bilateral injection of mock CSF with or without an EAA receptor antagonist, and then the goat walked at 1.8 miles/h, 5% grade, for 4 min and then for 4 min at 15% grade. Samples of arterial blood were withdrawn over the last 2 min of the control period and the 2 min preceding the onset of exercise. During exercise, blood was withdrawn nearly continuously at 15- or 30-s intervals. ABP was monitored at rest before blood sampling and over the last 30 s at each workload. Tre was continuously monitored. The order of antagonist injection was randomized.

Studies after injection of a neurotoxin. In 11 goats, after completion of the above studies, 50 mM ibotenic acid (IA) were microinjected unilaterally. IA, rather than KA, was injected, because IA causes neuronal death only at the injection site (37). Breathing, HR, ABP, and metabolic rate were monitored continuously for 30 min before and for 5 h after injection, with arterial samples being drawn every 0.5 h. Behavioral characteristics were also noted throughout the study. For several days after injection of IA, eupneic PaCO2 and CO2 sensitivity were assessed daily, and in three goats, exercise studies were also completed. Eventually, in 10 of these goats, an identical IA injection was made on the contralateral side and the 5-h study was repeated (the contralateral tube in 1 goat had become inadvertently occluded). Again the goats were studied for several days after this injection before they were killed.

Histological studies. To gain insight into the rate of cell death and the rate of reabsorption of dead neurons after IA microinjection, four additional goats were killed 5-10 h after unilateral injection of IA. In addition, to gain insight into tissue damage caused simply by implantation of the microtubule, two goats were killed 5-10 days after surgery before any microinjections were made.

After the goats were killed (Beuthanasia), the brain was perfused with phosphate-buffered saline (pH 7.3) and 4% paraformaldehyde fixative and then removed. In the initial 11 goats, Alcian blue dye (100 nl) was microinjected just before induction of anesthesia. Frozen, transverse sections (40 µm) were cut, stained (0.5% neutral red), and examined microscopically. The blue dye was found in tissue within 1 mm ventral to the lesion physically created by the microtubule. The microtubule lesion and the location of the dye identified the site of the injection. However, this analysis did not indicate the extent of diffusion of microinjected test substances. Accordingly, in all the goats in which IA had been microinjected, the harvested medulla was sectioned (20 µm) and stained with hematoxylin and eosin for identification of dead or dying neurons (eosinophilic and lacking a nucleus). Living and dead neurons were counted in the cubic millimeter volume just ventral to the lesion (absent or disrupted tissue) created by the microtubule and in most goats in the corresponding volume on the contralateral side at the same coordinates, which served as a control. In some goats, both microtubules were at the same approximate site; thus a control site was not obtained for these goats. Neuronal counts were also made in the cubic millimeter volume medial, lateral, rostral, caudal, and ventral to the cubic millimeter at the end of the lesion. Furthermore, the extent of the lesion was estimated by measuring the distance from the center of the end of the microtubule lesion to the most distal dead neurons in all aforementioned directions.

Data Analysis

Signals from the recorder were stored in a Citus 486 computer for subsequent computerized minute-by-minute computation of pulmonary ventilation (VE), tidal volume (VT), breathing frequency (f), inspiratory time, expiratory time, mean inspiratory flow rate (VT/TI), mean ABP (MABP), and HR. VE and expired gas concentrations were used to calculate metabolic rate (O2 uptake and CO2 output).

For statistical testing and data presentation, individual goat data were grouped according to the primary (although not always exclusive) nuclei in which the injection was made (RTN, FN, RGN, and VN). Linear regression analysis was used to calculate ventilatory CO2 sensitivity (Delta VE/Delta PaCO2). One-way analysis of variance for repeated measures was used to establish whether surgery and any injections had an effect on the physiological variables. The Newman-Keuls post hoc test was used to establish specific differences when P < 0.05 (by ANOVA). Finally, a paired t-test was also used in statistical analysis of some data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Neurosurgery

Irrespective of microtubule implant site, ~2 wk were required after neurosurgery before physiological functions stabilized at control levels. The Tre of the goats was reduced from control by ~0.4°C for the first few days after surgery, and then the Tre increased (P < 0.001) to 0.5°C above control for ~1 wk before returning to control levels. VE, f, MABP, and HR were increased (P < 0.05) above control 25-50% for ~1 wk after surgery, and then each gradually returned to control levels. Metabolic rate was not significantly (P > 0.10) altered after surgery, but it was increased or decreased in some goats. In most goats, PaCO2 during eupnea and CO2 sensitivity were unstable for several days after surgery (Fig. 1). Overall, there was a significant (P < 0.05) hyperventilation and reduction in CO2 sensitivity for a few days after surgery, but by 10-14 days after surgery, both variables were at stable, normal levels (Fig. 2). In all goats, arterial pH (Fig. 2) was ~0.1 unit above control (P < 0.001) 3-5 days after surgery, after which it gradually returned to control. This pH change occurred irrespective of whether eupneic PaCO2 increased or decreased. Finally, in the two goats that were subjected to brain surgery, but not to medullary microtubule placement, Tre, arterial pH, VE, f, eupneic PaCO2, and CO2 sensitivity were altered and unstable, similar to the pattern described above.


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  		Fig. 1.   Arterial PCO2 (PaCO2) during room air breathing and CO2 sensitivity (Delta VE/Delta PaCO2, where VE is ventilation) in 2 awake goats (black-lozenge  and diamond ) for 4 days before and for several days after implantation of microtubules into the facial (FN) and vestibular (VN) nuclei or the FN and gigantocellularis reticularis nucleus (RGN). Note stable resting PaCO2 and CO2 sensitivity before microtubule implants and unstable and altered levels for several days after implants.



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  		Fig. 2.   Arterial pH and PaCO2 during room air breathing and Delta VE/Delta PaCO2 in airway-intact (n = 13, squares) and tracheostomized (n = 6, circles) awake goats over 4 days before and 14 days after implantation of microtubules in the rostral medulla. All these variables changed (P < 0.05) after the implant. Filled symbols represent values different (P < 0.05) from preimplant values.

Effect of EAA Receptor Agonist Microinjection

A significant (P < 0.05) hyperpnea was elicited within minutes after NMDA was injected into the RTN, FN, RGN, and VN. At most sites, the injection increased VE 15-25%, but injections into the FN and RTN increased VE ~50% (Fig. 3). Particularly with FN injection, swallowing was also increased (Fig. 3), and MABP and HR were also increased with some injections. Injection of mock CSF at each site had no effect on VE (P > 0.10) over the same time period during which NMDA elicited a hyperpnea.


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  		Fig. 3.   Inspiratory flow (VI) and integrated electromyogram (EMG) activity of the inferior constrictor (IPC), laryngeal constrictor (TYA) and dilator (PCA), and the diaphragm (DIA) muscles in 1 awake goat before and after microinjection of 100 nl of 100 mM N-methyl-D-aspartic acid (NMDA) unilaterally into the FN. NMDA transiently stimulated VI and DIA, tonically activated the PCA, and (as shown by the large airway muscle contractions) caused a progressive, prolonged increase in swallowing frequency.

Effect of EAA Receptor Antagonist During Room Air Breathing (Eupnea)

Neither mock CSF nor the EAA receptor antagonists microinjected into the RTN or the RGN significantly (P > 0.10) altered any measured physiological variable during eupnea.

Microinjection of mock CSF and AP5 into the VN resulted in significant (P < 0.05) 1.5-Torr hyperventilation 30 min after the injection. The injection of mock CSF or NBQX into the VN also increased (P < 0.05) VI, VT, and/or f.

Injection of mock CSF into the FN caused (P < 0.05) hyperventilation during eupnea, but the only other injection into the FN that caused significant changes was KynA, which increased (P < 0.05) VT and MABP and decreased VT/TI and HR. In addition, in four goats, KynA injections into the FN resulted in seizures 50 min to 4 h after the injection. In one of these goats, ~5 min after the injection, VE, f, VT/TI, HR, and MABP increased above control, reaching peak values during each seizure. This pattern was also evident in the other goats, except the onset of the physiological changes and seizures occurred 2-4 h after the injection. All these goats recovered in good health from the seizures.

Effect of EAA Receptor Antagonist on CO2 Sensitivity

Unilateral microinjections of the EAA receptor antagonists did not have the same effect at all sites within any single nucleus; thus, with ANOVA treatment of data, there were no statistically significant (P > 0.10) differences in CO2 sensitivity between values after mock CSF and the antagonist injections (Fig. 4). However, at some sites in each nucleus, at least two of three, or 66%, of the antagonists altered CO2 by >13% from the mock CSF value (Fig. 5). Indeed, CO2 sensitivity was 1) decreased (25-75%, n = 5) or increased (15-40%, n = 2) by RTN injections, 2) increased (15-85%, n = 7) or not altered (n = 6) by FN injections, 3) increased (13-33%, n = 6), decreased (18-43%, n = 3), or not altered by RGN injections, and 4) increased (27%, n = 1), decreased (24%, n = 2), or not altered (n = 4) by VN injections (Fig. 5). These changes are noteworthy, because in goats studied for several days before microtubule implantation and again without injections 25-35 days after implantation the coefficient of variation (×100) in CO2 sensitivity was 12.7 ± 1.6 and 10.0 ± 1.2%, respectively.


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  		Fig. 4.   Group average Delta VE/Delta PaCO2 after injections of mock cerebrospinal fluid (CSF) or excitatory amino acid (EAA) receptor antagonists [2-amino-5-phosphonovalerate (AP5), 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), and kynurenic acid (KynA)] in the retrotrapezoid nucleus (RTN), FN, RGN, and VN. There were no statistically significant (P > 0.10) differences in CO2 sensitivity over the 4 conditions at any nucleus. Numbers above bars indicate number of EAA antagonist or mock CSF injections into each respective nucleus. Error bars, SE.



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  		Fig. 5.   Delta VE/Delta PaCO2 and change in PaCO2 between rest and moderate exercise (Delta PaCO2, R-SS2) in 4 different goats after injection of mock CSF or EAA receptor antagonists into RTN, FN, RGN, or VN. EAA receptor antagonists decreased (RTN), increased (FN and VN), or did not alter CO2 sensitivity, while the exercise response was minimally (RTN and FN) affected or attenuated (RGN and VN) by the antagonist. Qualitatively, there was no correlation between effects on CO2 sensitivity and exercise hyperpnea.

In goats where microtubules were placed in two different nuclei (1 on each side), the effect of EAA receptor antagonist was specific to one nucleus. Bilateral injection into two different nuclei usually had an effect on CO2 sensitivity similar to that of one of the unilateral injections. When microtubules were bilaterally placed in the same nuclei, the two unilateral injections of EAA receptor antagonists had similar effects on CO2 sensitivity. In addition, bilateral injections usually attenuated CO2 sensitivity more than the unilateral injections (Fig. 6).


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  		Fig. 6.   Delta VE/Delta PaCO2 in 2 goats (A and B) on several days after injection of mock CSF (circles), AP5 (squares), or KynA (diamonds) into the rostral medulla. Filled symbols, bilateral injections. In A, all injections were in the RTN; in B, injections were in the RGN (1st diamond) or RTN (2nd diamond). Most injections of EAA receptor antagonists attenuated CO2 sensitivity, which did not return to normal for several days thereafter.

The responses in four goats are noteworthy, because the EAA receptor antagonist reduced CO2 more than in most other goats and because often after antagonist injections, several days were required for full recovery of CO2 sensitivity (Fig. 6).

Effect of EAA Receptor Antagonist During Exercise

Similar to most nonhuman mammals (3), goats usually hyperventilate during exercise. After injections of mock CSF in the present study, hyperventilation was observed in 10 of 20 exercise studies. Injections of the EAA receptor antagonists into the RTN, FN, RGN, or VN did not have a statistically significant (P > 0.10) effect on this exercise response (Fig. 7). However, in some individual goats, the three antagonists consistently attenuated the exercise hyperventilation (Fig. 5). Bilateral injections into the same, or into two different, nuclei usually had an effect on the exercise PaCO2 response similar to the unilateral injections. The effect of the EAA antagonists on the exercise hyperpnea did not consistently correlate with the antagonist effects on CO2 sensitivity (Fig. 5).


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  		Fig. 7.   Group average difference in PaCO2 between rest and steady-state moderate exercise after microinjection of mock CSF or EAA receptor antagonist into RTN, RGN, FN, and VN. Numbers below bars indicate number of EAA antagonist or mock CSF injections in each respective nucleus. Error bars, SE. There were no statistically significant (P > 0.10) differences in responses to the different injections. Data are presented for RTN injections at only 2 sites; thus an SE is not included.

Effects for Five Hours After Injections of the Neurotoxin

Periods of increased f, VE, O2 uptake, MABP, and HR were evident in the 5-h period following a unilateral IA injection. In addition, periods of increased swallowing, salivation, and vocalization were observed. Periods of increased excitation were interspersed with periods of decreased HR, MABP, f, and VE below pre-IA injection levels. At times, the animals were very relaxed, their eyes were shut, and they appeared drowsy and sometimes in a trancelike condition. This pattern of response was consistent irrespective of the site of the IA microinjection.

Effects Over Days After Injections of the Neurotoxin

There were no significant changes in Tre, ABP, or metabolic rate over the several days after IA injection into any of the nuclei studied.

For the first 2 days after unilateral IA injection into the RTN, FN, or RGN, there was no statistically significant (P > 0.10) change in eupneic PaCO2 from preinjection values (Fig. 8A). The random changes that did occur were unrelated to whether an injection was the first or second unilateral injection. Seven of the 10 goats slightly hypoventilated (3.2 ± 0.7 Torr) 8-10 days after injection of the neurotoxin into the RTN, FN, or RGN.


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  		Fig. 8.   Change in individual goat's eupneic PaCO2 (Delta PaCO2) (A) and Delta VE/Delta PaCO2 (B) 1-2 and 8-10 days after injection of ibotenic acid (IA) into RTN, FN, and RGN. open circle  and , 1st and 2nd injection of the neurotoxin, respectively. Responses were independent of the order of the injection. At 8-10 days after injection into FN and RGN, most goats hypoventilated and CO2 sensitivity was reduced in all goats.

CO2 sensitivity was significantly (P < 0.05) or nearly significantly (P < 0.07) reduced from preinjection 1-2 and 8-10 days after unilateral neurotoxic injection onto the RGN (Fig. 8B). One to 2 days after injections into the FN and RTN, CO2 sensitivity tended to be increased or decreased, respectively. However, in all three goats studied 8-10 days after FN injections, CO2 sensitivity was reduced.

Injection of IA into the RGN of one goat and FN of another goat attenuated the exercise hyperpnea, whereas IA injection into the FN of a third goat accentuated the exercise hyperpnea. There was a partial time-dependent recovery toward a normal exercise response in only one of these goats.

Histological Findings

Usually, disrupted and presumably fibrotic tissue and dead neurons were observed up to 0.3-0.5 mm lateral and ventral to the edge of the microtubule. In goats with microtubules in which no injections were made (5 in 3 goats), tissue damage and dead neurons were usually confined to the area 0.5 mm surrounding the microtubule tract.

At the medullary sites where IA was injected, neuronal death was greatest within the 1 mm3 ventral to the microtubule lesion where 20-30% of the neurons were dead. The percentage of dead neurons decreased (P < 0.05) with rostral and caudal distance, such that as few as only 3-9% (rostral) and 12-15% (caudal) of the neurons were dead 1.5 mm rostral and caudal to the lesion created by the microtubule. The area of dead neurons approximated a sphere with an average radius of 1.5 mm. In most goats, neuronal death was confined to the nucleus in which it was microinjected; in others it extended slightly into the adjacent nuclei (Fig. 9). We found no evidence of a "distant cell death," as has been reported after KA injections (37). Virtually no dead neurons were found contralateral to the IA injection site. The living plus dead neuron sum on the injection side was 10-15% less than the control side living count presumably because of reabsorption of dead neurons. This difference was greater in goats killed 2-3 wk after IA injection than in those killed 5-10 h after the injection.


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  		Fig. 9.   Drawings of transverse medullary sections in which the site of the microtubule tip through which microinjections were made into the RTN (A), FN (B), RGN (C), or VN (D) is indicated. open circle , , and ×, Sites at which >= 2 of the 3 EAA receptor antagonists increased, decreased, or did not alter, respectively, CO2 sensitivity in individual goats. HP, hypoglossi praepositus nucleus; V, vestibular nucleus; R, raphe nucleus, 5SP, spinal trigeminal nucleus; CON, cochlear nucleus; RB, restiform body; P, pyramids; 5ST, spinal trigeminal tract. Number above each section denotes distance rostral to obex. Camera lucida drawings of the area of dead neurons are indicated by lines in A-C; injection site is indicated by the corresponding open circle  or . Lesioned area encompassed only 2 sections of some goats, but it extended over 4 sections in other goats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study were that, in awake goats, microinjection of an EAA receptor agonist, EAA receptor antagonists, and a neurotoxin indicated that 1) not only the RTN, but also the FN, RGN, VN, and/or tissue immediately adjacent to these nuclei, is part of the neural circuitry that can affect breathing and 2) there is a heterogeneous population of neurons in each of these rostral medullary nuclei.

Limitation of Study

Various techniques have been utilized to identify the site of neurons affected by microinjections into the brain. To our knowledge, there is no totally satisfactory technique for such identification in chronic studies such as those presented here (13). Because it is widely accepted that neurotoxins cause neuronal death (18, 37), we utilized the extent of neuronal death as an indication of neurons presumably affected by IA. We found cell death on the injection side but only random cell death on the control side. Because there was a decrease in dead neurons with distance from the injection site, it appears that IA diffused passively to cause cell death in an area that approximated a sphere with an average radius of 1.5 mm. Neurons outside this sphere may have contributed to the physiological effects of the neurotoxin, but it is highly probable that the major effects of not only the neurotoxin but also the EAA receptor agonist and antagonists were mediated by neurons within the sphere of dead neurons. This area is large relative to the size of lesions made in rats and cats (1, 30), but it is small considering the relatively larger goat medulla (10). The medulla of a 35-kg goat is 160% of the size of the medulla of a 2-kg cat (10). For most goats, this volume of dead neurons was confined to a portion of the nucleus implanted, but in some goats, it extended slightly to an adjacent nucleus; thus tissue immediately adjacent to the target nucleus may have contributed to the responses. We recognize that this method for estimating the area affected by microinjections has its limitations, but we are unaware of another more feasible method for chronic studies (1, 13).

It was difficult to place the microtubules consistently at exactly the target sites. This difficulty reflects the cumulative effect of several factors, including variation in goat anatomy and gradual evolution of the stereotaxic apparatus for goats. Others have also had difficulty in placing lesions at precise locations in rats in which the anatomy is more defined and the stereotaxic apparatus is more refined (24). Difficulty in placing and controlling the size of the lesion likely contributed to the variation in the physiological responses to the induced neuronal dysfunction. This variation ultimately affected the conclusions, which are, however, warranted.

Effect of Surgery for Microtubule Placement

Our findings indicate that neurosurgery in goats results in transient, extensive changes that have a generalized effect on physiological functions. These effects were observed even in two goats in which microtubules were not placed in the medulla, and in those goats with medullary implants, the changes were independent of the site of implant and extent of tissue damage. Some effects (plasma pH, body temperature) were uniform over all goats, but other effects varied qualitatively or quantitatively between goats (especially CO2 sensitivity). We cannot identify the exact cause of the changes, and we cannot discount the possibility that the microtubule lesions per se contributed to the transient physiological changes. Whatever the cause of the changes, there was eventual recovery and/or compensation, inasmuch as all the measured physiological functions were stable and at control levels 2 wk after the surgery.

Effect of Neuronal Dysfunction During Room Air Breathing (Eupnea)

Most microinjections of EAA receptor antagonist had minimal effect on breathing and other physiological functions during eupnea. These findings are consistent with previous findings of minimal effects on awake eupneic breathing when surface RVLM cooling (16, 33) or surface RVLM microejections of EAA receptor antagonists (17) were used to create neuronal dysfunction. For the initial hours and the first 2 days after the IA injection, eupneic breathing was slightly stimulated or depressed. Our laboratory previously observed that another neurotoxin, KA, when microejected on the RVLM of awake goats, stimulated breathing for several minutes to several hours (17). Others have observed in anesthetized mammals that neurotoxins microinjected on or in the RTN area cause a momentary increase in breathing (30, 32). This short-term response is consistent with the presumed excitotoxic action of the neurotoxins.

We presently observed hypoventilation during eupnea in 7 of 10 goats studied >= 8 days after microinjection of IA into the RTN, FN, or RGN. This effect suggests that the neurotoxin attenuated an excitatory component of the ventilatory control system. Why did it take 8 days for IA to induce hypoventilation, and why did the EAA receptor antagonists not induce hypoventilation? Conceivably, in the awake state, the entire process of IA excitotoxic effects differs from that in the anesthetized state. Several days might be required for neuronal death sufficient to decrease excitatory drives for breathing to below normal. With the EAA receptor antagonist, some neurons may have recovered from dysfunction before the maximal effect occurred at other neurons. However, with cell death, the neurons remain dysfunctional. The total number of dysfunctional neurons thus might be greater with the neurotoxin than with the EAA receptor antagonist, which could be the cause of the greater effect on breathing of neurotoxin-mediated neuronal dysfunction.

Effect of EAA Receptor Antagonist on CO2 Sensitivity

Until recently, intracranial chemoreception was thought to be restricted to neurons near the rostral and caudal ventrolateral medullary (VLM) surface (22, 23, 26). Neurons between these two sites were postulated to integrate all chemoreceptor activity (36). However, recently, it has been shown in reduced preparations that chemosensitive neurons are located at widespread brain sites (3, 7, 9, 12, 19-21, 34, 44). Moreover, it has been shown in awake goats that cooling-induced dysfunction of neurons of nearly the entire VLM surface only attenuates CO2 sensitivity by 60% (16), indicating that chemoreceptors at other sites must be capable of stimulating breathing and that in the awake state a chemoreceptor integration site is not located near the RVLM surface.

The data in the present study indirectly support the concept of widespread distribution of chemoreceptors in the medulla. Neuronal dysfunction at some sites in the RTN, FN, RGN, and VN increased or decreased CO2 sensitivity. The effects tended to be less than our laboratory previously found with surface RVLM cooling and chemically induced neuronal dysfunction (16, 17). This difference likely reflects in part at least the considerably smaller area of neuronal dysfunction in the present study. For example, with surface cooling, we likely caused dysfunction of nearly all RTN neurons, whereas with the present lesions, <50% of the RTN neurons were dysfunctional. For the FN, RGN, and VN lesions, it is likely that only 10-20% of the neurons were dysfunctional. There was no consistent correlation between effects on CO2 sensitivity and effects during eupneic breathing and the exercise hyperpnea. In fact, reduced CO2 sensitivity was in some goats associated with hyperventilation after the neurotoxic lesion. Accordingly, it is likely that the EAA receptor antagonists and the neurotoxin affected chemoreceptor neurons, a network of respiratory neurons that included chemoreceptor neurons, or factors that influence stimulus level at the chemoreceptors (such as cerebral blood flow and/or glial H+ buffering). Irrespective of which was affected, it seems that each of the nuclei studied consists of a heterogeneous population of neurons, because the effects on CO2 sensitivity were not uniform throughout any of the nuclei. Accordingly, in determining the physiological effects, the size of the lesion and area of neuronal dysfunction probably were not as important as the site of dysfunction. Heterogeneous responses are not unique to in vivo conditions, inasmuch as chemoreceptor neurons have previously been found in in vitro preparations to increase or decrease neuronal firing frequency with increases in CO2 (38).

The purpose of intracranial chemoreceptors (11, 15, 29), particularly widespread chemoreceptors, is unclear. There is considerable evidence that, in the awake state, CO2 sensitivity normally contributes minimally to the control of breathing. Apnea does not occur in humans when PaCO2 is reduced to below its threshold for stimulating breathing (8, 39). In central congenital hypoventilation syndrome (38) and after carotid body denervation (CBD) (18), neurosurgery (Figs. 1 and 2), and small medullary lesions (17, 28) (Figs. 4-7 and 9), CO2 sensitivity is attenuated, yet some of these effects occur without a change in eupneic breathing or the exercise hyperpnea. It is noteworthy that the dissociation of eupneic breathing and CO2 sensitivity was recently documented in human stroke patients with lesions in the rostral dorsal lateral medulla near or at the sites in which we created neuronal dysfunction in goats (28).

Another noteworthy point is that, in four goats in which the EAA receptor antagonist greatly reduced CO2 sensitivity, the response to CO2 remained attenuated for several days. The area of dead neurons (lesion size) was not exceptionally large in these goats. The cause of the large sustained reduction in CO2 sensitivity is unknown, but it seems to indicate that an EAA receptor antagonist has effects beyond transient inactivation of the receptor. Similarly unexplained and unexpected were the seizures induced by KynA in four goats. The area of dead neurons in these goats was relatively confined, indicating that a large or distant lesion was not the cause of the seizure. In total, these data suggest that in the awake state the EAA receptor antagonists can have effects not usually observed in reduced preparations.

Effect of EAA Receptor Antagonists on the Exercise Hyperpnea

Even though, overall, the EAA receptor antagonists did not have a consistent effect on the exercise hyperpnea, it is notable that in a few goats neuronal dysfunction either consistently attenuated or accentuated the hyperpnea. The magnitude of these responses does not suggest that the effect was through the primary stimulus for the exercise hyperpnea. It seems possible that in some goats the effects could have resulted from altered airway resistance or dead space or through some modulatory system. It seems clear, however, that these data provide additional evidence of independence of the exercise hyperpnea and CO2 sensitivity.

Recovery of Responses After Chronic Medullary Lesions

We found minimal recovery in eupneic breathing, CO2 sensitivity, and the exercise hyperpnea after acute alterations. Recovery was greatest after the first IA injection, but noteworthy was the slight hypoventilation in most goats studied 8-10 days after a second IA injection and the sustained reduction in CO2 sensitivity after the second injection. We did not study recovery over a longer period after IA injections, because reabsorption of dead neurons potentially could have compromised identification of the lesioned area.

The findings of minimal recovery may suggest that there is a limit to the magnitude of compensation or plasticity after medullary lesions. This finding agrees with previous studies documenting that CO2 sensitivity is below normal: 1) in humans with unilateral focal lesions in the dorsal rostrolateral medulla (28) and 2) in cats 3 mo after neurotoxic lesions in the nucleus tractus solitarius (2). In addition, others found that rats did not demonstrate any recovery in CO2 sensitivity over 3 wk after IA lesions in the RTN (1). Relevant is that 2 wk after CBD in goats, there is a major, but incomplete, recovery of eupneic breathing, the exercise hyperpnea, and CO2 sensitivity (18). Recovery is more complete after unilateral than after bilateral CBD, which also suggests a limit to plasticity (18). In dogs, 21 days after CBD, there is some recovery of eupneic breathing but no recovery of CO2 sensitivity (35). In ponies (5), months are required before there is full recovery of eupneic breathing after CBD, but in neonatal goats (18) and piglets (18), recovery of eupneic breathing and CO2 sensitivity occurs within 2 wk. Finally, in adult goats (18), ponies (5), dogs (35), cats (40), and rats (25), there is only partial recovery of peripheral chemosensitivity after CBD. Accordingly, considering all these studies on effects of chronic lesions of chemoreceptors or presumed chemoreceptor pathways, the emerging concept is that there is recovery/plasticity, but the plasticity is not uniform across species or over all the normal functions of the pathway.

Influence of Rostral Medullary Nuclei on Breathing

There is now convincing evidence that the RTN is a site of intracranial chemoreception, which, along with chemoreceptor-independent facilitation of breathing, is critical to sustain breathing in the anesthetized state but not in the awake and sleep states (1, 16, 17, 29-33). On the other hand, the VN, RGN, and FN have generally been considered to have a minimal role or no role in the control of breathing. However, our present findings coupled with other recent findings indicate that each of these nuclei is part of the neural circuitry that can affect breathing. For example, Xu et al. (42) found that the increased breathing elicited by electrical stimulation of the cerebellar fastigial nucleus was critically dependent on functional neurons in the RGN. Others have found in reduced preparations that breathing is altered by electrical stimulation of the vestibular nerve or within the VN (43). This latter effect also appears dependent on functional RGN neurons (27). Transneuronal tracing studies suggest that abdominal muscle premotor neurons are located in the RGN, and it was speculated that RGN neurons might be important in coordinating the respiratory and nonrespiratory functions of several muscles (4). To our knowledge, there are no published data suggesting that neurons in the FN participate in the control of breathing. However, some FN are important in physiological functions (i.e., mastication, salivation) that directly or indirectly (i.e., through swallowing) involve use of some of the same muscles (soft palate, pharyngeal wall) involved in regulation of airflow. Accordingly, the changes we observed in CO2 sensitivity after neuronal dysfunction in the FN, and possibly also in the RGN and VN, might be due to altered feedback to respiratory neurons from neurons whose primary function is not the control of breathing. Whatever the mechanism, there is considerable evidence that multiple rostral medullary nuclei can influence ventilatory control in the awake state.


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grant HL-25739 and by the Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: H. V. Forster, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: bforster{at}mcw.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.

Received 21 March 2000; accepted in final form 12 March 2001.


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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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