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J Appl Physiol 88: 1996-2007, 2000;
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Vol. 88, Issue 6, 1996-2007, June 2000

CO2/H+ chemoreception in the cat pre-Bötzinger complex in vivo

Irene C. Solomon, Norman H. Edelman, and Marvin H. O'Neal III

Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effects of focal tissue acidosis in the pre-Bötzinger complex (pre-BötC; the proposed locus of respiratory rhythm generation) on phrenic nerve discharge in chloralose-anesthetized, vagotomized, paralyzed, mechanically ventilated cats. Focal tissue acidosis was produced by unilateral microinjection of 10-20 nl of the carbonic anhydrase inhibitors acetazolamide (AZ; 50 µM) or methazolamide (MZ; 50 µM). Microinjection of AZ and MZ into 14 sites in the pre-BötC reversibly increased the peak amplitude of integrated phrenic nerve discharge and, in some sites, produced augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst). Microinjection of AZ and MZ into this region also reversibly increased the frequency of eupneic phrenic bursts in seven sites and produced premature bursts (i.e., doublets) in five sites. Phrenic nerve discharge increased within 5-15 min of microinjection of either agent; however, the time to the peak increase and the time to recovery were less with AZ than with MZ, consistent with the different pharmacological properties of AZ and MZ. In contrast to other CO2/H+ brain stem respiratory chemosensitive sites demonstrated in vivo, which have only shown increases in amplitude of integrated phrenic nerve activity, focal tissue acidosis in the pre-BötC increases frequency of phrenic bursts and produces premature (i.e., doublet) bursts. These data indicate that the pre-BötC has the potential to play a role in the modulation of respiratory rhythm and pattern elicited by increased CO2/H+ and lend additional support to the concept that the proposed locus for respiratory rhythm generation has intrinsic chemosensitivity.

central respiratory chemoreceptors; control of breathing


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MULTIPLE FOCI IN THE BRAIN STEM have been shown to be CO2/H+ chemosensitive and have the potential to play a modulatory role in respiratory control. Work by Nattie and colleagues (3, 7, 8, 24) has established that multiple central chemosensitive regions modulating respiration may be identified in vivo by producing focal tissue acidosis with small-volume microinjections of the carbonic anhydrase inhibitor acetazolamide (AZ). These injections produce a circumscribed region of reduced tissue pH that leads to an increase in phrenic nerve discharge. As systemic PCO2 is maintained constant, the evoked increases in respiratory output can be ascribed to a central chemoreceptor response. The technique has revealed central CO2/H+ chemosensitivity modulating respiration in the rostral, caudal, and intermediate chemosensitive areas of the ventral medullary surface and the nucleus tractus solitarii, locus coeruleus, medullary raphe, and rostral ventral respiratory group (RVRG).

Results obtained from in vitro studies have recently suggested that chemosensitive neurons within the ventrolateral medulla may play a key role in the generation and modulation of respiratory rhythm (13-16, 42). The pre-Bötzinger complex (pre-BötC) located in the rostral ventrolateral medulla (RVLM) has been postulated to be essential for respiratory rhythm generation in neonatal mammals (for a recent review, see Ref. 28) and has been demonstrated to play a role in the generation and modulation of respiratory rhythm in adult mammals (1, 27, 37).

Recent work from our laboratory has also shown that the respiratory rhythm generator located in the pre-BötC is hypoxia chemosensitive (38). Focal hypoxia, produced by microinjection of small volumes of sodium cyanide, in the pre-BötC elicited modulation of phrenic burst pattern, increases in respiratory burst frequency, and tonic inspiratory discharge. Preliminary data involving GABAergic disinhibition suggest that the respiratory rhythm generator located in the pre-BötC can function as an O2 sensor in the brain over the physiological range (when disinhibited), acting as a medullary analog to chemoreceptors of the carotid bodies. Because peripheral chemoreceptors exhibit CO2/H+ chemosensitivity as well as hypoxic chemosensitivity and because other respiratory-related neurons in the brain stem manifest CO2/H+ sensitivity, we examined the effects of focal tissue acidosis in the pre-BötC on phrenic nerve discharge and arterial blood pressure.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Experiments were conducted in 26 adult cats weighing 3.4-5.4 kg. The cats were anesthetized initially by placing them inside a sealed, plastic induction chamber into which a gaseous mixture of halothane (5%) and O2 was introduced. After the cats were anesthetized, they were removed from the chamber, and anesthesia was maintained by delivering halothane (1.5-4%) and O2 through a face mask placed over the cat's nose and mouth. The right brachial vein was cannulated, and alpha -chloralose (35 mg/kg iv) was administered. The gaseous anesthetic was gradually reduced over a 30-min period as the alpha -chloralose took effect. Supplemental alpha -chloralose (3-5 mg/kg iv) was given as needed. The adequacy of anesthesia was regularly verified by firmly pinching a toe. If an increase in blood pressure was evoked or, during the absence of paralysis (see below), if the cat withdrew its limb, additional anesthetic was given. Dexamethasone (2 mg iv) was administered to minimize brain swelling. Both brachial arteries were cannulated. The left brachial cannula was connected to a Statham transducer (P23XL) for measurement of arterial blood pressure; the right brachial cannula was used for sampling arterial blood.

The trachea was cannulated low in the neck, and the lungs were mechanically ventilated with room air enriched with O2. The cats were then paralyzed with gallamine triethiodide (2-4 mg/kg iv) or vecuronium bromide (0.2-0.4 mg/kg iv), with supplemental doses administered as needed. The chest was opened bilaterally through the sixth or seventh intercostal spaces, and the expiratory outlet of the ventilator was placed under 2-3 cmH2O to prevent collapse of the lungs during expiration. End-tidal CO2 was monitored continuously through a side port in the tracheal cannula and was maintained constant (±0.2%) between 4.0 and 6.0% by adjusting the tidal volume and frequency of the ventilator. Arterial PO2, PCO2, and pH were measured at hourly intervals (Radiometer ABL-500), and, when necessary, blood-gas values were corrected by either adjusting the ventilator or intravenous infusion of sodium bicarbonate (8.5%). No microinjections were made within 30 min of bicarbonate infusion. At the start of the experimental protocol (see below), arterial PO2, PCO2, and pH averaged 181 ± 5 (SE) Torr, 39 ± 1 Torr, and 7.34 ± 0.01, respectively. Body temperature was maintained at 36-38°C with the use of a heating pad and a heat lamp.

Both cervical vagus nerves were exposed and cut bilaterally. In 12 cats, the carotid sinus nerves were also exposed and cut bilaterally. The cat's head was then placed in a stereotaxic instrument, and the dorsal surface of the medulla was exposed by separating the nuchal musculature along the midline, removing the basioccipital bone, and opening the atlantooccipital membrane. The dorsal surface of the brain stem was covered with saline or mineral oil to prevent drying.

The C5 rootlet of one or both phrenic nerves was isolated in the neck via a lateral approach, cut, and desheathed, and the central end was placed on a bipolar platinum-rod electrode. The nerve was then covered with a mixture of mineral oil and petroleum jelly. Phrenic nerve discharge was amplified (×1,000-10,000) and filtered (10-10,000 Hz), and a moving average was obtained by using a third-order Paynter filter with a 100-ms time constant. Both the raw and averaged nerve outputs were recorded on digital tape (model 4000A, A. R. Vetter) and on a chart recorder throughout the experimental protocol.

Experimental protocol. We examined the effects of focal tissue acidosis in the pre-BötC on phrenic nerve discharge and arterial blood pressure. Focal tissue acidosis was produced by unilateral microinjection of AZ or methazolamide (MZ), both of which inhibit carbonic anhydrase. Responses to unilateral microinjection of AZ and MZ from a total of 14 sites in the pre-BötC were recorded. Only one pre-BötC site was examined per animal. In six sites, we also recorded responses to unilateral microinjection of vehicle (see below) into the pre-BötC. To control for spread of injectate, we also recorded responses to unilateral microinjection of AZ from an additional 10 sites adjacent (within 300-500 µm) to the pre-BötC, including the RVRG. Only one microinjection of AZ or MZ was made into a single site, and no more than two microinjections were made in a single animal. All sites in the pre-BötC were initially localized by using predetermined stereotaxic coordinates relative to the calamus scriptorius (3.4-4.2 mm rostral), midline (3.8-4.1 mm lateral), and dorsal surface (4.2-4.5 mm ventral). The range in values for the coordinates used to find the pre-BötC reflects the variability in the dimensions of the brain stems in cats of different sizes. Sites were then functionally identified as pre-BötC by using DL-homocysteic acid (DLH), as previously described (37), and were histologically confirmed after completion of the experiments (see Location of injection sites). The functional criteria for identification of the pre-BötC were production of either a series of high-amplitude, short-inspiratory-duration bursts, tonic excitation, or augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst) of phrenic nerve discharge.

Microinjections into the pre-BötC were made with the use of a triple-barreled glass pipette (12- to 20-µm tip diameter) attached to a pressure injection device (General Valve Picospritzer II). One barrel of the pipette contained 10 mM DLH. The second barrel contained 50 µM AZ or 50 µM MZ. The third barrel contained Fast Green dye (2%), which was used to mark the injection sites (<= 100 nl). AZ and MZ were initially dissolved in DMSO (0.6 µl/ml) and brought to their final concentration by adding distilled water or saline. A vehicle solution using the same concentration of DMSO was used in six additional experiments instead of AZ or MZ as a control for nonspecific effects. DLH and Fast Green dye were dissolved in a saline solution. The pH of all microinjected chemicals was adjusted to 7.29-7.37. Microinjection volumes of DLH, AZ, and MZ were 10-20 nl, and microinjection typically required 1-2 s to complete. Microinjection of vehicle was 20 nl, and microinjection similarly required 1-2 s to complete. The volume of each injection was monitored by observing the displacement of the fluid meniscus by using a microscope equipped with an eyepiece reticule.

Histology. At the conclusion of the experiment, the cat was killed under deep anesthesia by an injection of saturated KCl solution. The brain stem was removed and placed in 4% Formalin for at least 48 h. The brain stem was then frozen, sectioned coronally (40 µm), mounted on slides, and stained for cell bodies by using 1% Neutral Red dye. With the use of a microprojector, we made drawings of tissue sections containing sites marked with Fast Green dye.

Data analysis. We measured peak amplitude of integrated phrenic nerve activity, frequency of phrenic bursts, arterial blood pressure, and arterial blood gases before, at 5 min after, and every 15 min after microinjection of AZ or MZ into the pre-BötC until phrenic nerve output returned toward control levels. Amplitude of integrated phrenic nerve discharge and frequency of phrenic bursts were determined from the phrenic neurogram off-line. Baseline and response values for these variables were determined by averaging the values obtained from 1-min intervals at each time point. These data are reported as a percent change from preinjection baseline levels of discharge, which were set at 100%. For mean arterial pressure (MAP), baseline and response values were taken as the steady-state values reached at each time point.

All values are reported as means ± SE. Responses before and after stimulation were analyzed by a repeated-measures ANOVA to determine statistical significance, followed by Fisher's least significant difference post hoc test, for which the criterion level was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General effects of microinjection of AZ and MZ into the pre-BötC. Six sites in the pre-BötC received AZ, and eight sites in the pre-BötC received MZ. The effects on phrenic nerve discharge were bilaterally symmetrical (n = 12 animals with bilateral recordings). Unilateral microinjection of AZ and MZ (50 µM, 10-20 nl) into the pre-BötC reversibly increased phrenic nerve discharge in each of the sites examined within 5-15 min of microinjection of either agent. The magnitude of the increase in phrenic nerve discharge in response to AZ and MZ was similar with each agent; however, the overall time course for these responses was different. In addition, similar phrenic neurogram and blood pressure responses were observed in cats with carotid sinus nerves intact (AZ, n = 2; MZ, n = 5) or cut (AZ, n = 4; MZ, n = 3); therefore, these data will not be considered separately. The effects on amplitude of integrated phrenic activity and frequency of phrenic bursts are described below.

Effects on amplitude of integrated phrenic activity. Unilateral microinjection of AZ and MZ (50 µM, 10-20 nl) into the pre-BötC reversibly increased peak amplitude of phrenic nerve discharge in each of the sites examined. Figure 1 shows an example depicting these amplitude effects. In this example, unilateral microinjection of AZ (Fig. 1, A and C) and MZ (Fig. 1, B and D) increased peak amplitude of integrated phrenic nerve discharge with no change in the frequency of phrenic bursts. This increase in amplitude had an onset latency of ~15 min for AZ and 5 min for MZ. Both AZ and MZ elicited similar peak (maximal) increases in amplitude; however, this peak (maximal) response followed slightly different time courses (75 min for AZ; 105 min for MZ). In addition, the duration of this increase in amplitude was less for AZ (135 min) than for MZ (180 min).


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  		Fig. 1.   Example tracings of integrated phrenic nerve activity depicting amplitude effects evoked by unilateral microinjection of acetazolamide (AZ; A) and methazolamide (MZ; B) into pre-Bötzinger complex (pre-BötC). Time after microinjection (in min) and corresponding arterial PCO2 (PaCO2) are shown under each trace. C and D: line drawings showing the complete time course and magnitude of the changes in amplitude of integrated phrenic nerve discharge () and frequency of phrenic bursts (open circle ) evoked by a single microinjection of AZ (C) and a single microinjection of MZ (D), respectively. In these sites, unilateral microinjection of AZ and MZ into pre-BötC reversibly increased amplitude of integrated phrenic nerve discharge with little or no change in frequency of phrenic bursts. Note that magnitude of increase in amplitude was similar for both agents microinjected. Phrenic nerve discharge (C and D) represents average values obtained from multiple respiratory cycles over a 1-min interval at each time point and is expressed as a percentage of preinjection baseline, with baseline levels of discharge set at 100%.

Averaged data for amplitude of integrated eupneic phrenic nerve discharge for preinjection baseline, onset, peak response, and recovery evoked by microinjection of AZ (n = 6) and MZ (n = 8) are shown in Fig. 2. Unilateral microinjection of AZ and MZ increased peak amplitude of integrated eupneic phrenic nerve discharge by 118.3 ± 40.6% (P < 0.01) and 110.0 ± 32.4% (P < 0.01), respectively, above preinjection baseline. The onset latency for this increase in amplitude averaged ~10 min for both agents; however, the time to the peak increase in amplitude and the time to recovery were on average shorter with AZ than with MZ. For AZ, the peak increase in amplitude typically occurred at 45 or 60 min (mean ± SE = 47.5 ± 7.2 min), after which the amplitude began to return toward baseline values. Recovery was usually noted within 120 min (mean ± SE = 97.5 ± 8.4 min). In contrast, for MZ, the peak increase in amplitude typically occurred within 60-105 min (mean ± SE = 72.9 ± 8.9 min), after which the amplitude began to return toward baseline values. Recovery was usually noted within 180 min (mean ± SE = 135.0 ± 19.6 min), although in two sites recovery occurred at 105 min.


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  		Fig. 2.   Summary data showing effects of unilateral microinjection of AZ and MZ into pre-BötC on amplitude of integrated phrenic nerve discharge. Data are expressed as a percentage of preinjection baseline. Baseline levels of discharge were set at 100%. A: unilateral microinjection of AZ into 6 sites in pre-BötC reversibly increased amplitude of integrated phrenic nerve discharge. B: unilateral microinjection of MZ into 8 sites in pre-BötC reversibly increased amplitude of integrated phrenic nerve discharge. * Statistically significant difference (P < 0.01) between peak response and baseline, onset, and recovery responses. Latency (in min; mean ± SE) to onset, peak, and recovery responses is shown. Note that the onset latencies were similar; however, the latency to the peak increase in amplitude and time to recovery were generally shorter with AZ than with MZ.

In addition to the increase in amplitude of the integrated eupneic bursts, examination of the phrenic neurogram also revealed augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst) (Figs. 1A, 3, and 4A) in response to microinjection of AZ and MZ. Augmented bursts were evoked by microinjection of AZ into five of the six sites examined and by microinjection of MZ into four of the eight sites examined. An expanded view of these augmented bursts may be seen in Fig. 3. In this example, augmented bursts appear at the beginning and end of each trace and are separated by a series of eupneic breaths. In general, one augmented burst was interspersed between eupneic breaths; although, in some cases, a small series of two to five augmented bursts was seen (not shown). The appearance of augmented bursts followed a time course similar in onset and recovery to the amplitude effects in each of the nine sites.


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  		Fig. 3.   Example tracings depicting augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst) in phrenic neurogram evoked by microinjection of AZ (A) and MZ (B) into pre-BötC. In this example, augmented bursts are shown at beginning and end of each trace and are separated by a series of eupneic breaths. Traces from top to bottom: integrated phrenic nerve activity [ipsilateral (ipsi)], raw phrenic nerve activity (ipsilateral), integrated phrenic nerve activity [contralateral (contra)], and raw phrenic nerve activity (contralateral). Asterisks identify augmented bursts. Time after microinjection is shown under each trace.



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  		Fig. 4.   Example tracings of integrated phrenic nerve activity depicting both frequency and amplitude effects evoked by unilateral microinjection of AZ (A) and MZ (B) into pre-BötC. Time after microinjection (in min) and corresponding PaCO2 are shown under each trace. C and D: line drawings showing complete time course and magnitude of changes in frequency of phrenic bursts (open circle ) and amplitude of integrated phrenic nerve discharge () evoked by a single microinjection of AZ (C) and a single microinjection of MZ (D), respectively. In these sites, unilateral microinjection of AZ and MZ into pre-BötC reversibly increased frequency of phrenic bursts and amplitude of integrated phrenic nerve discharge. Note that magnitude of the increases in frequency and amplitude was similar for each agent microinjected, although the peak responses to AZ vs. MZ were different. Phrenic nerve discharge in C and D represents average values obtained from multiple respiratory cycles over a 1-min interval at each time point and is expressed as a percentage of preinjection baseline, with baseline levels of discharge set at 100%.

Effects on frequency of phrenic bursts. Unilateral microinjection of AZ and MZ (50 µM, 10-20 nl) into the pre-BötC reversibly increased the frequency of eupneic phrenic bursts in 7 of the 14 sites examined. In these seven sites, microinjection of AZ and MZ also produced an increase in amplitude of integrated phrenic nerve discharge (as described above). In five of these sites, microinjection of AZ and MZ also evoked augmented bursts. Figure 4 shows an example depicting these frequency effects. In this example, unilateral microinjection of AZ (Fig. 4, A and C) and MZ (Fig. 4, B and D) increased both amplitude of integrated phrenic nerve discharge and frequency of phrenic bursts. The increases in amplitude and frequency followed a similar time course and were similar in magnitude. Both agents elicited an increase in amplitude and frequency of ~10% within 5 min from the beginning of microinjection, and the peak (maximal) responses occurred at 60 and 75 min for AZ and MZ, respectively. Although the onset latency and time to peak for the increases in amplitude and frequency were similar, the duration of these increases was less for AZ (105 min) than for MZ (165 min).

Unilateral microinjection of AZ and MZ elicited an increase in the frequency of eupneic phrenic bursts in 50% of the sites examined; in the remaining sites, no change in the frequency of eupneic bursts was evoked by microinjection of AZ or MZ. Overall for the group as a whole (including both frequency responders and nonresponders; n = 14), however, there was an increase in the frequency of eupneic bursts, which averaged 26.0 ± 11.2% (P < 0.05). Averaged data for the frequency of eupneic phrenic bursts for preinjection baseline, onset, peak response, and recovery evoked by unilateral microinjection of AZ and MZ in those sites that showed a frequency response (n = 7) are shown in Fig. 5. Because there were no differences in the magnitude of the increases in frequency in response to AZ (range = 27-105% increase above preinjection baseline; n = 3) and MZ (range = 25-123% increase above preinjection baseline; n = 4), these data have been combined. In these sites, unilateral microinjection of AZ and MZ increased the frequency of phrenic bursts by 55.6 ± 15.7% (P < 0.01) above preinjection baseline.


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  		Fig. 5.   Summary data showing effects of unilateral microinjection of AZ and MZ into pre-BötC on frequency of eupneic phrenic bursts. Data are expressed as a percentage of preinjection baseline. Baseline levels of discharge were set at 100%. Unilateral microinjection of AZ and MZ into 7 sites in pre-BötC reversibly increased frequency of eupneic phrenic bursts. * Statistically significant difference (P < 0.01) between peak response and baseline, onset, and recovery responses.

In addition to the increase in frequency of these eupneic phrenic bursts, in some cases, we saw the appearance of premature bursts (i.e., doublets) in response to microinjection of AZ and MZ. Premature bursts were evoked by unilateral microinjection of AZ into two of the six sites examined and by unilateral microinjection of MZ into three of the eight sites examined. Both premature bursts and an increase in the frequency of eupneic phrenic bursts were seen in two of these sites; no increase in the frequency of eupneic phrenic bursts was noted in the remaining three sites. An example of three different patterns of premature (doublet) bursts may be seen in Fig. 6. A second burst of phrenic activity occurred either during a phrenic burst or immediately after a phrenic burst with an absent or reduced expiratory pause (i.e., phrenic silence). In general, the appearance of these premature bursts followed a time course similar in onset and recovery to the amplitude effects, and, in some cases, there was a reversible increase in the number of premature (doublet) bursts produced (not shown). In all sites in which microinjection evoked premature (doublet) bursts, augmented bursts were also produced. In addition, the incidence of augmented bursts reversibly increased following a similar time course in four of the nine sites in which augmented bursts were produced (not shown).


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  		Fig. 6.   Example tracings depicting 3 different patterns of premature (i.e., doublet) bursts in phrenic neurogram evoked by unilateral microinjection of AZ and MZ into pre-BötC. In these examples, a second burst of phrenic activity occurs either during a phrenic burst (A and B) or immediately after a phrenic burst (C) with an absent or reduced expiratory pause (i.e., phrenic silence). Traces from top to bottom: integrated phrenic nerve activity (ipsilateral), raw phrenic nerve activity (ipsilateral), integrated phrenic nerve activity (contralateral), and raw phrenic nerve activity (contralateral). Arrows identify premature bursts.

Overall, we found a modulation of respiratory rhythm in 11 of the 14 sites examined. This modulation consisted of one or more of the following: 1) an increase in the frequency of eupneic phrenic bursts, 2) production of premature bursts, or 3) increased incidence of augmented bursts.

Effects on MAP. Unilateral microinjection of AZ and MZ (50 µM, 10-20 nl) into the pre-BötC reversibly increased arterial blood pressure in 6 of the 14 sites examined; no change in MAP was seen in the remaining 8 sites. There were no differences in the magnitude of the increases in MAP produced by microinjection of AZ (n = 3) and MZ (n = 3) into these sites; therefore, these data have been combined. In these sites, unilateral microinjection of AZ and MZ reversibly increased MAP from 134 ± 11 (baseline) to 152 ± 12 (peak response) to 136 ± 11 mmHg (recovery) (P < 0.01). The time course for the onset and peak blood pressure and phrenic neurogram responses was similar; however, MAP usually recovered 15-30 min before the recovery of phrenic nerve discharge.

Control injections. Unilateral microinjections of vehicle (DMSO control, 20 nl) were made into six sites in the pre-BötC to control for nonspecific effects. Vehicle microinjection was ineffective in producing an increase in peak amplitude of integrated phrenic nerve discharge or frequency of phrenic bursts in five of these sites. In the remaining site, there was a small increase in phrenic neurogram amplitude (<10%); however, arterial PCO2 also increased in this animal.

We also examined the effects of unilateral microinjection of AZ into 10 sites adjacent (within 300-500 µm) to the pre-BötC including the RVRG as a control for the spread of injectate. In these experiments, a site in the pre-BötC was functionally identified by using DLH, and then the pipette was moved dorsal, medial, or caudal in increments of 100-500 µm. Subsequent microinjection of DLH into sites 300-500 µm dorsal or medial was ineffective in altering phrenic nerve discharge. For RVRG sites, subsequent microinjection of DLH produced a small, transient increase in the amplitude of integrated phrenic nerve discharge. In each of the sites examined in the RVRG (n = 5), unilateral microinjection of AZ (50 µM, 20 nl) reversibly increased peak amplitude of integrated phrenic nerve discharge with no change in the frequency of phrenic bursts. In addition, unilateral microinjection of AZ into these sites was ineffective in producing either augmented bursts or premature (doublet) bursts in the phrenic neurogram. Figure 7 shows the averaged data for preinjection baseline, onset, peak response, and recovery evoked by unilateral microinjection of AZ into the RVRG. Unilateral microinjection of AZ into the RVRG increased the amplitude of integrated phrenic nerve discharge by 38.1 ± 9.2% (P < 0.05) above preinjection baseline. The onset latency for this increase in amplitude averaged ~15 min. The peak increase in amplitude typically occurred between 30 and 60 min (mean ± SE = 41.3 ± 7.2 min), after which the amplitude began to return toward baseline values. Recovery was usually noted within 75 min (mean ± SE = 71.3 ± 9.4 min). In the remaining five sites adjacent to the pre-BötC, unilateral microinjection of AZ was ineffective in producing an increase in peak amplitude of integrated phrenic nerve discharge or frequency of phrenic bursts.


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  		Fig. 7.   Summary data showing effects of unilateral microinjection of AZ into 5 sites in the rostral ventral respiratory group (RVRG) on amplitude of integrated phrenic nerve discharge. Data are expressed as a percentage of preinjection baseline. Baseline levels of discharge were set at 100%. Unilateral microinjection of AZ into these 5 sites in the RVRG reversibly increased amplitude of integrated phrenic nerve discharge. * Statistically significant difference (P < 0.05) between peak response and baseline and recovery responses. Latency (in min; mean ± SE) to onset, peak, and recovery responses is shown.

Location of injection sites. The distribution of sites in which AZ and MZ were microinjected into the pre-BötC is shown in Fig. 8A. As landmarks for identifying the rostrocaudal level of the pre-BötC, we identified the caudal pole of the retrofacial nucleus, nucleus ambiguus, the rostral pole of the lateral reticular nucleus, and the rostral pole of the hypoglossal nucleus. All sites in the pre-BötC were identified with reference to the caudal pole of the retrofacial nucleus, not the obex.


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  		Fig. 8.   A: schematic drawing of coronal sections of the medulla showing sites in pre-BötC in which microinjections of AZ (open circle ), MZ (), and vehicle (DMSO control; dashed circles) were made. B: schematic drawing of coronal sections of the medulla showing sites adjacent to pre-BötC (, dorsal and medial; , RVRG) in which microinjections of AZ were made. Also shown in B are corresponding pre-BötC sites (open circle  and ). Lines in B connect pre-BötC site to corresponding adjacent site. All sites in pre-BötC are identified with reference to caudal pole of retrofacial nucleus (0 mm). Each section is meant to encompass level indicated ± 0.2 mm (rostrally and caudally). RFN, retrofacial nucleus; NA, nucleus ambiguus; 5SP, spinal nucleus of the trigeminal nerve; 5ST, spinal tract of the trigeminal nerve; LRN, lateral reticular nucleus; ION, inferior olivary nucleus; and P, pyramidal tract.

Our histological analysis revealed that all microinjection sites functionally identified as pre-BötC were located within the anatomic boundaries described for the pre-BötC in adult cat (9, 27, 29, 37). Our microinjection sites extended in the rostrocaudal plane from 480 µm caudal to the retrofacial nucleus to the caudal pole of the retrofacial nucleus. In mediolateral and dorsoventral planes, our microinjection sites were located 3.8-4.1 mm lateral to midline and 4.2-4.5 mm ventral to the dorsal surface of the medulla. From these analyses, we could not differentiate sites in which microinjection of AZ or MZ produced an increase in the amplitude of integrated phrenic nerve discharge from those in which microinjection of AZ or MZ produced both an increase in amplitude of integrated phrenic nerve discharge and frequency of phrenic bursts, including premature bursts (i.e., doublets), based on their histological distribution. Furthermore, we could not distinguish sites in which augmented bursts were produced from those in which this pattern was not seen.

Our histological analysis also revealed that our control injections of AZ (n = 10) were all within 500 µm of functionally identified and histologically confirmed sites in the pre-BötC, as illustrated in Fig. 8B (lines connect pre-BötC site to corresponding adjacent site). Sites in the RVRG (n = 5) were located within 500 µm caudal to the pre-BötC, and the remaining sites were located 300-500 µm dorsal (n = 3) or medial (n = 2) to the pre-BötC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that blockade of carbonic anhydrase in the pre-BötC elicits a long-lasting but reversible increase in phrenic nerve discharge. As do others, we attribute the AZ- and MZ-induced responses to focal tissue acidosis. The increase in phrenic nerve discharge consisted of an increase in peak amplitude of integrated activity, production of augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst), and an increase in the frequency of bursts (including premature bursts). These responses did not require input from the peripheral chemoreceptors, as similar results were obtained from animals with the carotid sinus intact or cut. In addition, it is unlikely that these responses resulted from nonspecific effects of the injectate, because they were not produced by microinjection of vehicle. We, therefore, suggest that the pre-BötC has intrinsic chemosensitivity mediated by focal changes in CO2 and/or H+ in this region.

Mechanisms of central chemoreception. The exact chemical stimulus by which central chemoreceptors are activated to drive ventilation is not completely resolved, although molecular CO2, extracellular pH, and intracellular pH have all been implicated (for reviews see Refs. 19 and 23). Both in vivo and in vitro studies have proposed that CO2 and H+ exert independent and differential effects on central respiratory chemoreceptors (5, 11, 13, 17, 20, 32, 33, 42). For example, Borison et al. (5) reported that, in the decerebrate, peripherally denervated cat, the respiratory response to an increase in arterial H+ was greater with respiratory-induced acidosis than with metabolically induced acidosis. Similarly, in the anesthetized, peripherally denervated cat, Eldridge et al. (11) found that raising PCO2 increased phrenic nerve activity to a greater extent than did infusion of HCl for the same change in medullary extracellular pH.

Data obtained from in vitro studies further suggest that CO2 and H+ exert differential effects with respect to frequency and amplitude of inspiratory motor output (13, 39, 42). In the isolated neonatal rodent brain stem-spinal cord preparation, for example, alteration of superfusate pH at constant PCO2 elicits changes in both respiratory burst frequency and amplitude of phrenic nerve bursts, whereas alteration of superfusate PCO2 at constant pH produces a transient change in amplitude but not frequency of phrenic bursts (13). It is not clear whether these reflect differences in the effect of CO2/H+ at one or more receptors or differential access of CO2 and H+ to receptors in the brain stem.

AZ- and MZ-induced tissue acidosis. The exact mechanism by which microinjection of AZ and MZ produces focal tissue acidosis is not completely understood. It has been demonstrated, however, that inhibition of carbonic anhydrase by intravenous administration of AZ in both rabbits (4) and cats (41) produces an increase in brain PCO2 and medullary extracellular fluid H+ concentration. This increase in brain PCO2 has been suggested to result from interference with the hydration of CO2 and impairment of transport in brain capillaries and red blood cells (4, 31, 41), whereas the increase in extracellular fluid H+ concentration has been suggested to result from an accumulation of metabolically produced H+ (4, 31). Thus the extracellular fluid H+ concentration and brain PCO2 can increase independently in response to inhibition of brain carbonic anhydrase (4). Because both of these mechanisms contribute to the production of focal tissue acidosis, and PCO2 and H+ may independently stimulate central chemoreceptors (5, 11, 13, 20, 32, 33, 40), it is unclear whether our responses were evoked by focal increases in PCO2 or changes in H+ concentration.

In our experiments, focal tissue acidosis was produced pharmacologically by unilateral microinjection of AZ and MZ. Although both AZ and MZ are cell-permeable carbonic anhydrase inhibitors, MZ differs from AZ because of its slightly higher solubility, lower plasma protein binding, and longer activity (21, 22). In our experiments, we found that both agents were effective in eliciting a similar increase in peak amplitude of integrated phrenic nerve activity and frequency of phrenic bursts; however, the time course for these responses was dependent on the agent microinjected. In general, the onset latency was slightly shorter for MZ than for AZ, although this difference was not statistically significant. Furthermore, the time to the peak increase and the time to recovery were generally shorter with AZ than with MZ. These findings appear to be consistent with the pharmacological properties of AZ vs. MZ discussed above.

Widespread sites of central CO2/H+ chemosensitivity using AZ. Previous studies have used injection of AZ to demonstrate central CO2/H+ chemosensitivity for respiratory control in widespread medullary sites (3, 7, 8, 24). In these studies, AZ injection produced a region of sustained focal tissue acidosis that was sufficient to induce an increase in phrenic nerve discharge. The acidosis produced at the center of the injection site, as measured by pH electrodes, was reported to be similar to that evoked by increasing end-tidal PCO2 by ~40 Torr (range = 36-47 Torr), with no detectable pH change noted at distances 300-400 µm from the injection center (8, 24). In our experiments, we did not measure the medullary tissue pH; however, we believe that the changes in phrenic nerve activity obtained in our experiments are likely to be specific to focal tissue acidosis within the pre-BötC and not simply spread to other chemosensitive sites, although some effects from spread cannot be excluded. We do note that the spread of injectate, and presumably tissue acidosis, in our experiments most likely exceeded that reported in some other studies (3, 8, 24), as our injection volumes were larger. However, microinjection of AZ into sites located 300-500 µm dorsal or medial to the pre-BötC was ineffective in increasing phrenic nerve activity, and microinjection of AZ into sites in the RVRG (within 500 µm of the pre-BötC) produced only small increases in the amplitude of integrated phrenic nerve discharge, consistent with the increases in magnitude previously reported in other studies (24). Furthermore, examination of the data in the studies by Coates et al. (8) and Nattie and Li (24), in which focal tissue acidosis was produced in the rostral and caudal chemosensitive areas, the intermediate area of the ventral medullary surface, and the RVRG, (i.e., regions that are adjacent to the pre-BötC), indicates that there was an increase in the amplitude of integrated phrenic nerve activity, with little or no modulation of respiratory rhythm. In our experiments, microinjection of AZ and MZ into the pre-BötC dramatically increased the frequency of eupneic phrenic bursts in one-half of the sites examined and produced premature bursts (i.e., phase resetting of the respiratory rhythm), consistent with stimulation of the respiratory rhythm generator located in the pre-BötC.

It should be noted that, in our experiments, microinjection of AZ and MZ into the pre-BötC increased the amplitude of integrated phrenic nerve discharge in all of the sites examined. In contrast, previous studies have found that ~40% of the AZ microinjections are ineffective in eliciting an increase in phrenic nerve discharge (3, 8, 24). One possible explanation for our findings is related to the concentration and volume of AZ and MZ used in our experiments. Because our concentration of AZ was higher and our volume of AZ and MZ was larger, it is possible that microinjection of AZ and MZ produced a greater intensity and spread of tissue acidosis in our experiments. If so, then our microinjections had the potential to produce a more intense stimulation of CO2/H+ chemosensitive neurons in the region under investigation as well as to affect a greater number of neurons involved in respiratory control. It should be noted, however, when Coates et al. (7) microinjected just beneath the medullary surface even higher concentrations and larger volumes than those used in our study, only 79% of the sites examined showed an increase in phrenic nerve activity. Another possibility for the difference between these previous studies and our present findings results from the methods employed to select injection sites. In the studies by Bernard et al. (3) and Coates et al. (7), no attempt was made to functionally identify respiratory regions. In contrast, all sites used in our study were selected on the basis that microinjection of DLH modified respiratory rhythm and pattern. Thus sites in which AZ injections are ineffective may reflect sites within the medulla that do not contain sufficient numbers of neurons involved in respiratory control. In support of this idea, Nattie and Li (24) found in a recent study that, when sites in the RVRG were functionally identified by using glutamate, microinjection of AZ increased phrenic nerve activity in all sites examined, whereas, in functionally unidentified sites in the RVRG, microinjection of AZ elicited an increase in phrenic nerve activity in ~64% of the sites studied. These studies raise the idea that CO2/H+ chemosensitivity may be a widely distributed property of premotor respiratory neurons.

CO2/H+ chemosensitivity and respiratory rhythm generation. It is well accepted that central chemosensitivity provides a major source of tonic input to the neurons that make up the respiratory rhythm generator, thus mediating basal respiration as well as changes in breathing in response to changes in CO2 and H+. Recent in vitro studies, moreover, suggest that chemosensitive neurons within the ventrolateral medulla play a key role in the generation and modulation of respiratory rhythm (13-16, 39, 42). For example, perfusing the isolated neonatal rat brain stem-spinal cord with a Krebs solution of high pH (addition of NaOH) markedly depresses the rate of respiratory rhythmic oscillations recorded from the C4 ventral root, whereas perfusion with low pH (addition of HCl) markedly enhances activity (39). Furthermore, CO2-induced reductions in pH of the superfusate bathing the isolated neonatal rat brain stem-spinal cord elicits a substantial increase in the frequency of respiration-related spinal (C2) or phrenic (C4 or C5) nerve bursts (16, 42). In addition, elevating the H+ in the superfusate bathing the neonatal rat medullary slice containing the pre-BötC increases hypoglossal burst frequency (14).

In our experiments, we found that focal tissue acidosis in the pre-BötC, the primary locus of respiratory rhythm generation (34), modifies both respiratory rhythm and pattern in addition to increasing the amplitude of eupneic bursts. We interpret the phase resetting of respiratory rhythm in our study, demonstrated by increases in the frequency of phrenic bursts and the production of premature bursts, as an indication of intrinsic chemosensitivity of the rhythm generating "kernel." This interpretation is supported by recent work from Johnson et al. (15) that demonstrated an increase in burst frequency of synaptically isolated pre-BötC pacemaker neurons in response to increasing H+ (at a constant CO2) of the superfusate bathing the neonatal rat medullary slice.

These pacemaker neurons in the pre-BötC exhibit conditional bursting properties and are thought to provide rhythmic drive to the rest of the respiratory network during the inspiratory phase of network activity (6, 12, 18, 28, 34, 35). In the hybrid pacemaker-network model for respiratory rhythm generation, synaptic interactions synchronize and modify the basic rhythm generator by modulating the intrinsic membrane conductances of the conditional bursting (i.e., voltage-dependent) pacemaker neurons. Although Johnson et al. (15) have demonstrated that pre-BötC pacemaker neurons are intrinsically chemosensitive, it is unclear from their study or our present findings whether other populations of neurons in this region are also CO2/H+ chemosensitive. For example, in the adult cat, the pre-BötC has been shown to contain a high concentration of preinspiratory (pre-I) (9, 29) or inspiratory-driver (I-driver) (30) neurons, which are proposed to be key elements in respiratory phase transition (35, 36). These pre-I or I-driver neurons are also known to provide excitatory drive to inspiratory neurons that exhibit augmenting and decrementing patterns of discharge in the respiratory network (30). It is possible that stimulation of these neurons elicited the modulations in respiratory rhythm reported in our study.

In our experiments, we found that, in addition to the modulation of respiratory rhythm, there was also an increase in the amplitude of integrated phrenic nerve discharge and the production of augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst). As the conditional bursting pacemaker neurons located in the pre-BötC are known to provide excitatory drive to other inspiratory neurons in the respiratory network, the patterning changes evoked by focal tissue acidosis in the pre-BötC could have resulted from CO2/H+-mediated excitation of these chemosensitive neurons. However, as noted above, chemosensitivity of other than pacemaker neurons (i.e., pre-I or I-driver neurons) can also explain the patterning (i.e., amplitude and augmented bursts) changes observed.

CO2/H+ chemosensitivity and arterial blood pressure. In addition to the increase in phrenic nerve discharge, in six sites we also evoked a reversible increase in MAP. Although stimulation of central chemoreceptors by increasing systemic CO2 is known to increase sympathetic nerve activity and arterial blood pressure (26), the increase in blood pressure evoked in our study most likely resulted from respiratory modulation of sympathetic activity (2, 10) mediated by the effects of focal tissue acidosis of the respiratory rhythm generator located in the pre-BötC. Previous neuroanatomic studies with the use of intracellular biocytin injections have demonstrated that inspiratory neurons originating in the RVRG (some in the vicinity of the pre-BötC) project directly onto tyrosine hydroxylase immunoreactive neurons located in the RVLM (25), suggesting that these respiratory neurons may provide direct synaptic input to RVLM sympathoexcitatory neurons.

Conclusion. We have demonstrated that focal tissue acidosis in the pre-BötC increases peak amplitude of integrated phrenic nerve activity, produces augmented bursts, and, in some sites, increases frequency of phrenic bursts (including production of premature bursts). The increase in frequency is in contrast to other CO2/H+ brain stem chemosensitive sites demonstrated in vivo, which have only showed increases in amplitude, but is consistent with the in vitro response to focal tissue H+ in the pre-BötC. Our findings suggest that the pre-BötC has the potential to play a role in the modulation of respiratory rhythm and pattern elicited by increased CO2/H+ and lends additional support to the concept that the locus for respiratory rhythm generation has intrinsic chemosensitivity.


    ACKNOWLEDGEMENTS

We thank Dr. Eugene Nattie for suggestions and thoughtful review of the manuscript and Tami Halat for excellent technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-16022 and American Lung Association (ALA) Grant RG-008-N. I. C. Solomon is an Edward Livingston Trudeau Scholar from the ALA.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. C. Solomon, Dept. of Physiology and Biophysics, Health Sciences Center, Basic Science Tower, Level 6, Rm. 140, State Univ. of New York at Stony Brook, Stony Brook, NY 11794-8661 (E-mail: ICSolomon{at}physiology.pnb.sunysb.edu).

Received 27 December 1999; accepted in final form 31 January 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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