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J Appl Physiol 94: 2151-2157, 2003. First published February 28, 2003; doi:10.1152/japplphysiol.01192.2002
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Vol. 94, Issue 6, 2151-2157, June 2003

Focal CO2/H+ alters phrenic motor output response to chemical stimulation of cat pre-Bötzinger complex in vivo

Irene C. Solomon

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

Microinjection of DL-homocysteic acid (DLH), a glutamate analog, into the pre-Bötzinger complex (pre-BötC) can produce tonic excitation of phrenic nerve discharge. Although this DLH-induced tonic excitation can be modified by systemic hypercapnia, the role of focal increases in pre-BötC CO2/H+ in this modulation of the DLH-induced response remains to be determined. Therefore, we examined the effects of unilateral microinjection of DLH (10 mM; 10-20 nl) into the pre-BötC before and during increased focal pre-BötC CO2/H+ (i.e., focal tissue acidosis) in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Focal tissue acidosis was produced by blockade of carbonic anhydrase with either focal acetazolamide (AZ) or methazolamide (MZ) microinjection. For these experiments, sites were selected in which unilateral microinjection of DLH into the pre-BötC produced a nonphasic tonic excitation of phrenic nerve discharge (n = 10). Microinjection of 10-20 nl AZ (50 µM) or MZ (50 µM) into these 10 sites in the pre-BötC increased the amplitude and/or frequency of eupneic phrenic bursts, as previously reported. Subsequent microinjection of DLH produced excitation in which phasic respiratory bursts were superimposed on tonic discharge. These DLH-induced phasic respiratory bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.05). These findings demonstrate that modulation of phrenic motor activity evoked by DLH-induced activation of the pre-BötC is influenced by focal CO2/H+ chemosensitivity in this region. Furthermore, these findings suggest that focal increases in pre-BötC CO2/H+ may have contributed to the modulation of the DLH-induced responses previously observed during systemic hypercapnia.

respiratory rhythm generation; neural control of breathing; central CO2/H+ chemosensitivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CHEMICAL STIMULATION IN THE pre-Bötzinger complex (pre-BötC) in vivo has been shown to increase the frequency of phrenic bursts (2, 11, 18, 20, 23) as well as to produce nonphasic tonic excitation of phrenic nerve discharge (18, 20). The precise mechanism(s) by which chemical stimulation of this region elicits phasic vs. tonic excitation of phrenic motor output remains to be resolved. Recently, I demonstrated, in the anesthetized cat, that increased respiratory network drive produced by systemic hypercapnia can modify the nonphasic tonic excitation of phrenic nerve discharge evoked by DL-homocysteic acid (DLH)-induced activation of the pre-BötC to include increased-frequency phasic phrenic bursts (19). These findings suggest that hypercapnia, which increases neuronal excitability within the brain stem respiratory network, including presumptive pre-BötC rhythmogenic neurons (5, 7, 14, 16, 17), is capable of altering pre-BötC-mediated DLH-induced modulation of phrenic nerve discharge.

Although previous studies have suggested that hypercapnia produces a generalized increase in neuronal excitability within the brain stem respiratory network, recent studies have demonstrated that the pre-BötC also exhibits intrinsic CO2/H+ chemosensitivity (6, 21). Increasing H+ (at constant CO2) of the superfusate bathing the neonatal rat medullary slice elicits an increase in burst frequency of synaptically isolated pre-BötC pacemaker neurons (6) and focally increasing CO2/H+ in the pre-BötC in anesthetized cat elicits an increase in phrenic nerve output (21), suggesting the presence of CO2/H+ chemosensitivity within this region.

Our laboratory's recent report (19) did not assess the effects of focal increases in CO2/H+ (i.e., focal tissue acidosis) in the pre-BötC on the DLH-induced modulation of phrenic nerve discharge; therefore, the contribution of intrinsic pre-BötC CO2/H+ chemosensitivity to modulation of the DLH-induced responses observed is unknown. We propose that intrinsic chemosensitivity plays a role, at least in part, in this modulation. If this prediction is correct, increases in focal pre-BötC CO2/H+ should modify the response elicited by chemical activation of this region. Thus the purpose of this study was to examine specifically whether increases in focal pre-BötC CO2/H+ modify the nonphasic tonic excitation of phrenic nerve discharge evoked by DLH-induced activation of the pre-BötC. Accordingly, we hypothesized that, during focal increases in pre-BötC CO2/H+, activation of the pre-BötC would elicit frequency modulation of phasic phrenic bursts. In other words, the nonphasic tonic excitation of phrenic nerve discharge evoked by DLH-induced activation of the pre-BötC would be modified during increases in focal pre-BötC CO2/H+ to include increased frequency phasic phrenic bursts. To test this hypothesis, we examined the effects of repeated microinjection of DLH into the same site in the pre-BötC on phrenic nerve discharge before and during increases in focal pre-BötC CO2/H+.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at the State University of New York at Stony Brook in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals. A detailed description of the general methods has been published previously (20).

In brief, anesthesia was induced in adult cats (3.4-4.9 kg; n = 10) with halothane (5%) in O2 and maintained with intravenous alpha -chloralose (initial 35-50 mg/kg; supplemental 3-5 mg/kg). The adequacy of anesthesia was regularly verified by absence of a withdrawal reflex (in the unparalyzed state) or blood pressure response (during muscular paralysis) to a noxious paw pinch. If the cat withdrew its limb during the absence of paralysis or if an increase in blood pressure was evoked, additional anesthesia was given. The right brachial vein and both brachial arteries were cannulated for administration of drugs, measurement of arterial blood pressure (Statham transducer, P23XL), and sampling of arterial blood. The trachea was cannulated, the cat was vagotomized bilaterally (to eliminate the influence of cardiopulmonary afferent input to the central respiratory network), and the lungs were mechanically ventilated with 40% O2 in a balance of N2. The cat was then paralyzed with vecuronium bromide (0.2-0.4 mg/kg iv), supplemented as needed. The dorsal surface of the brain stem was exposed, and the C5 rootlet of one or both phrenic nerves was isolated for recording. Raw phrenic nerve discharge was amplified (×103) and filtered (0.1-10 kHz); the filtered signal was rectified, and a moving average was obtained by using a third-order Paynter filter with a 100-ms time constant.

Experimental protocol. We examined the effects of DLH-induced activation of neurons located in the pre-BötC on the patterning, timing, and frequency of phrenic nerve discharge before and during focal tissue acidosis in this region. Focal tissue acidosis was produced by unilateral microinjection of acetazolamide (AZ) (3) or methazolamide (MZ), both of which inhibit carbonic anhydrase (8, 9). Responses from 10 sites in the pre-BötC were recorded during focal tissue acidosis; 3 of these sites were also examined after recovery from focal tissue acidosis. Only one pre-BötC site was examined per animal because of the long-lasting effects (up to 3 h) of AZ and MZ. For these experiments, only functionally identified pre-BötC sites in which unilateral microinjection of DLH (10 mM; <= 20 nl) under control conditions (i.e., hyperoxic normocapnia) produced nonphasic tonic excitation of phrenic nerve discharge were selected. After functional identification, AZ (50 µm; 10-20 nl) or MZ (50 µm; 10-20 nl) was microinjected into the pre-BötC. At least 30 min were allowed for AZ or MZ to exert an effect, and then the DLH microinjection was repeated with the same volume used for functional identification. This time frame was chosen on the basis of our previous study using these agents (21). All microinjections into the pre-BötC were made with a triple-barreled glass pipette (~20 µm tip diameter) attached to a pressure injection device (General Valve Picospritzer II), and the volume of injectate was measured by observing the displacement of the fluid meniscus with use of a microscope equipped with an eyepiece reticule. In all experiments, arterial PO2, PCO2, and pH were measured (Radiometer ABL-500) immediately preceding microinjection of DLH. At the end of each experiment, fast green dye (2%; <=  120 nl) was microinjected to mark the site, the brain stem was removed, and the tissue was processed for histological analysis and verification of the location of the injection site, as previously described (20).

Data acquisition and analysis. Both raw and averaged phrenic nerve discharge were recorded on tape (A. R. Vetter, model 4000A) and on a chart recorder (Astro-Med, model MT95K2) throughout the experimental protocol. Appropriate segments of data were then transferred to a Macintosh PowerBook 3400c computer for off-line analyses (PowerLab, chart 3.6.1, AD Instruments). Peak amplitude of integrated phrenic nerve discharge, inspiratory duration (TI), expiratory duration (TE), and frequency of phrenic bursts were determined in response to unilateral microinjection of DLH into the pre-BötC before and during focal tissue acidosis. In some cases, these variables were also determined in response to unilateral microinjection of DLH after recovery from focal tissue acidosis. Preinjection baseline values were determined by averaging the values obtained for five consecutive breaths immediately preceding DLH microinjection. Response values were determined as the peak change from preinjection baseline values for a tonic excitatory pattern of phrenic nerve discharge or by averaging the values obtained for five consecutive breathing cycles displaying the greatest change from preinjection baseline values for phasic phrenic nerve discharge responses. For the DLH-induced nonphasic tonic component of phrenic nerve discharge, TI represents the duration of tonic firing, and TE was not determined. Amplitude of integrated phrenic nerve discharge and frequency of phasic phrenic bursts are reported as a percent change from preinjection baseline levels of discharge, which were set at 100% in each cat. The onset latency for DLH-induced responses was measured from the beginning of microinjection.

All values are reported as means ± SE. The effects of DLH-induced modulation on both the phasic and tonic components of phrenic nerve discharge were evaluated. For the phasic component of phrenic nerve discharge, comparisons were made between the preinjection baseline and peak response values evoked by DLH-induced activation of the pre-BötC. For the tonic component of phrenic nerve discharge, comparisons were made between the DLH-induced response before (pre-AZ/MZ) and during (post-AZ/MZ) focal pre-BötC tissue acidosis. All responses to DLH microinjection are presented as paired data. Student's paired t-tests or the paired nonparametric Wilcoxon's signed-rank tests, as appropriate, were used to determine statistical significance, for which the criterion level was set at P < 0.05. Statistical analyses were not conducted on recovery data because of the small sample size (n = 3).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of focal pre-BötC tissue acidosis on the DLH-induced response. In these experiments, before microinjection of AZ or MZ (i.e., control conditions), unilateral microinjection of DLH into the pre-BötC produced a nonphasic tonic excitation of phrenic nerve discharge in each of the sites examined. Unilateral microinjection of AZ or MZ into the pre-BötC (i.e., focal pre-BötC tissue acidosis) elicited an increase in the amplitude and, in some cases, frequency of phasic phrenic bursts, as previously reported (21). Subsequent microinjection of DLH into the same sites in the pre-BötC produced excitation of phrenic nerve discharge in which phasic bursts were superimposed on tonic activity (Fig. 1). The onset latency for DLH-induced excitation of phrenic nerve discharge was similar both before and during focal pre-BötC tissue acidosis (P > 0.05), with DLH-induced responses being observed within 1-2 s from the beginning of microinjection. Arterial blood gases and pH, which were measured immediately preceding unilateral microinjection of DLH into the pre-BötC, were maintained constant throughout the experimental protocol (Table 1).


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  		Fig. 1.   Examples demonstrating the effects of focal pre-Bötzinger complex (pre-BötC) tissue acidosis on DL-homocysteic acid (DLH)-induced activation of the pre-BötC. Before focal pre-BötC tissue acidosis (i.e., control conditions), microinjection of DLH into the pre-BötC evoked a nonphasic tonic excitation of phrenic nerve discharge. During focal pre-BötC tissue acidosis produced by microinjection of acetazolamide (AZ; A) or methazolamide (MZ; B), microinjection of DLH into the pre-BötC produced a tonic excitation of phrenic nerve discharge in which phasic respiratory bursts were superimposed. These DLH-induced phasic bursts had an increased frequency compared with the preinjection baseline frequency. It should be noted that focal tissue acidosis increased either the amplitude of phrenic nerve discharge (as shown in A) or both the amplitude and the frequency of phrenic bursts (as shown in B). Regardless of the pattern of the AZ- or MZ-induced response, subsequent microinjection of DLH into the pre-BötC elicited increased-frequency phasic bursts superimposed on tonic discharge. ipsi, Ipsilateral.


                              
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  		Table 1.   Arterial blood gases and pH immediately preceding microinjection of DLH into the pre-BötC

In all experiments, after microinjection of AZ or MZ into the pre-BötC, the DLH-induced response was modified, such that it included phasic phrenic bursts. This modulation of the DLH-induced response was observed in sites in which microinjection of AZ or MZ into the pre-BötC elicited only an increase in amplitude (Fig. 1A) as well as in sites in which microinjection of AZ or MZ elicited increases in both amplitude and frequency of phrenic bursts (Fig. 1B). These DLH-induced phasic bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.01), resulting from a reduction in both TI (P < 0.05) and TE (P < 0.01). Summary data describing these responses are provided in Fig. 2. In addition, the patterning of these DLH-induced phasic bursts was quite variable and included bursts with an increase in the rate of rise (Fig. 1A), as well as bursts with a high-amplitude, short-duration burst component (Fig. 1A), such as those associated with the augmented burst (i.e., sigh) pattern. In some cases, multiple bursts were also observed within an inspiratory phrenic cycle. No statistically significant effect on the peak amplitude of integrated phasic phrenic nerve activity was noted (P > 0.05). In fact, an increase in the peak amplitude of integrated phasic phrenic nerve activity was observed in five of the sites examined, whereas a decrease in the peak amplitude of integrated phasic phrenic nerve activity was observed in the remaining five sites examined (including the response shown in Fig. 1B).


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  		Fig. 2.   Summary data illustrating the effects of DLH-induced activation of the pre-BötC during focal tissue acidosis on the timing and patterning characteristics of phasic phrenic nerve discharge. During focal tissue acidosis, microinjection of DLH significantly increased the frequency of phasic phrenic bursts and reduced both TI and TE. No significant difference was observed in the DLH-induced increase in peak amplitude of integrated phrenic nerve discharge. * Significant difference (P < 0.05) from preinjection baseline values. TI, inspiratory duration; TE, expiratory duration.

Although microinjection of DLH during focal pre-BötC tissue acidosis produced phasic phrenic bursts, it should be noted that these DLH-induced phasic bursts were superimposed on an underlying tonic discharge in each of the experiments conducted. For these analyses, the DLH-induced tonic component of phrenic nerve discharge during focal pre-BötC tissue acidosis is compared with the DLH-induced tonic excitation observed before microinjection of AZ or MZ into the pre-BötC. The peak amplitude of integrated tonic phrenic nerve activity evoked by microinjection of DLH during focal pre-BötC tissue acidosis was similar to that evoked by microinjection of DLH before microinjection of AZ or MZ into the pre-BötC (P > 0.05). In addition, no statistically significant effect on the duration of the DLH-induced tonic excitation was noted before vs. during focal pre-BötC tissue acidosis (P > 0.05); however, shorter durations of tonic discharge were observed in response to microinjection of DLH during focal pre-BötC tissue acidosis in 6 of the 10 sites examined. Summary data illustrating these amplitude and timing characteristics of this DLH-induced tonic discharge are provided in Fig. 3.


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  		Fig. 3.   Summary data illustrating the effects of DLH-induced activation of the pre-BötC before, during, and after focal pre-BötC tissue acidosis on tonic excitation of phrenic nerve discharge. Before focal tissue acidosis (pre-AZ/MZ) and after the return of phrenic nerve activity to pre-AZ or MZ levels (recovery), microinjection of DLH into the pre-BötC evoked a nonphasic tonic excitation of phrenic nerve discharge. In contrast, during focal tissue acidosis (post-AZ/MZ), microinjection of DLH produced phasic phrenic bursts superimposed on tonic activity. The peak amplitude of the DLH-induced tonic component of the integrated phrenic nerve discharge and the duration of this tonic excitation, however, were similar before (pre-AZ/MZ) and during (post-AZ/MZ) focal pre-BötC tissue acidosis (P > 0.05; n = 10). In addition, the nonphasic tonic excitation of phrenic nerve discharge evoked during recovery was similar to that evoked during control (pre-AZ/MZ) conditions in the 3 sites examined.

In three of these sites, we also examined the effects of DLH-induced activation of the pre-BötC on phrenic nerve activity after recovery from focal tissue acidosis. For these trials, we waited until phrenic nerve discharge returned to control levels (i.e., pre-AZ or pre-MZ) and then repeated the microinjection of DLH. Although during focal tissue acidosis the DLH-induced response consisted of excitation of phrenic nerve discharge with phasic bursts superimposed on tonic activity, after recovery from focal tissue acidosis similar microinjection of DLH into the same sites in the pre-BötC produced only a nonphasic tonic excitation of phrenic nerve discharge. Summary data illustrating the amplitude and timing characteristics of this DLH-induced tonic discharge after recovery from focal pre-BötC tissue acidosis are also provided in Fig. 3.

Location of injection sites. The distribution of sites in which DLH was microinjected into the pre-BötC is shown in Fig. 4. As landmarks for identifying the rostrocaudal level of the pre-BötC, we identified the caudal pole of the retrofacial nucleus, the 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.


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  		Fig. 4.   Schematic drawing of coronal sections of the medulla showing location of pre-BötC sites that received microinjection of DLH during and/or after focal pre-BötC tissue acidosis. Different symbols represent sites in which focal pre-BötC tissue acidosis was produced using AZ (open circle ) and MZ (). All sites in the pre-BötC are identified with reference to the caudal pole of the retrofacial nucleus (0 mm). Each section is meant to encompass the level indicated ±0.2 mm (rostrally and caudally). RFN, retrofacial nucleus; NA, nucleus ambiguus; LRN, lateral reticular nucleus; 5SP, spinal nucleus of the trigeminal nerve; 5ST, spinal tract of the trigeminal nerve; ION, inferior olivary nuclei; P, pyramidal tract

The histological analysis revealed that all microinjection sites functionally identified as pre-BötC were located within the anatomical boundaries described for the pre-BötC in adult cat (4, 13, 15, 20). The microinjection sites were located within 320 µm (i.e., 0-320 µm caudal) of the caudal pole of the retrofacial nucleus in the rostrocaudal plane, 3.68-3.96 mm lateral to midline and 4.20-4.36 mm ventral to the dorsal surface of the medulla. No differences were detected in the location of injection sites for experiments using AZ vs. those using MZ (Fig. 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have demonstrated that focal pre-BötC tissue acidosis alters the phrenic motor output response elicited by DLH-induced activation of the pre-BötC in vivo. We have shown that although DLH-induced activation of the pre-BötC under baseline conditions can produce a tonic (nonphasic) excitation of phrenic nerve discharge, during focal pre-BötC tissue acidosis produced by microinjection of either AZ or MZ, chemical stimulation of this region (using DLH) elicits phasic phrenic bursts that are superimposed on an underlying tonic phrenic nerve discharge. Furthermore, these phasic phrenic bursts exhibit an increased burst frequency compared with the preinjection baseline burst frequency. An alternate interpretation of the present findings, however, must also be considered. It is possible that DLH-induced activation of the pre-BötC is affecting the phrenic nerve discharge response elicited by AZ/MZ-induced pre-BötC tissue acidosis. In other words, chemical stimulation of the pre-BötC during focal pre-BötC tissue acidosis modifies or enhances the CO2/H+-induced phrenic nerve discharge response of the pre-BötC. Although this possibility cannot be excluded, these experiments were designed to assess the effects of focal increases in pre-BötC CO2/H+ (i.e., focal tissue acidosis) on the DLH-induced modulation of phrenic nerve discharge. Therefore, we interpret the present findings to suggest that phrenic motor activity evoked by DLH-induced activation of the pre-BötC is influenced by intrinsic CO2/H+ chemosensitivity of the pre-BötC.

Modulation of DLH-induced phrenic nerve discharge: focal pre-BötC tissue acidosis vs. systemic hypercapnia. Although the present findings demonstrate that focal pre-BötC tissue acidosis modifies the phrenic motor output response to DLH-induced activation of the pre-BötC, the production of phasic activity was accompanied by an underlying tonic phrenic nerve discharge in each of the experiments conducted. Furthermore, this tonic discharge appeared to be unaffected by focal pre-BötC tissue acidosis. This finding is in contrast to our recent observations in which the DLH-induced tonic component of phrenic nerve discharge was substantially reduced during systemic hypercapnia (19). Even though the modulation of phrenic motor output elicited by DLH-induced activation of the pre-BötC during focal pre-BötC tissue acidosis was not identical to that seen during systemic hypercapnia, both of these perturbations were capable of modifying the nonphasic tonic excitation of phrenic nerve discharge to include increased frequency of phasic phrenic bursts (Ref. 19; present findings). Therefore, we further interpret the present findings to suggest that intrinsic pre-BötC CO2/H+ chemosensitivity could have contributed to the DLH-induced modulation of phrenic nerve discharge observed during systemic hypercapnia in our previous study (19).

The precise reason(s) for the differences observed in response to DLH-induced activation of the pre-BötC during focal pre-BötC tissue acidosis and systemic hypercapnia is unclear; however, a number of possibilities exist. For example, the level of acidification elicited by these stimuli may be quite different. In our laboratory's previous experiments (19), systemic hypercapnia increased arterial PCO2 by ~21 Torr (range = 15.7-40.4 Torr). In contrast, focal tissue acidosis produced by small-volume microinjections of AZ has been demonstrated to reduce tissue pH to a level comparable to increasing end-tidal PCO2 by ~40 Torr (range = 36-47 Torr) (3). Another possibility is related to differences in neuronal excitability within the pre-BötC produced by focal increases in pre-BötC CO2/H+ vs. those produced by systemic hypercapnia. Although we did not attempt to directly measure neuronal excitability within the pre-BötC under either of these conditions, we suggest that focal pre-BötC tissue acidosis increases neuronal excitability of a population of CO2/H+-chemosensitive pre-BötC neurons, which includes the presumptive rhythmogenic neurons (21), whereas systemic hypercapnia enhances neuronal excitability of both CO2/H+-chemosensitive and nonchemosensitive pre-BötC neurons (including presumptive rhythmogenic neurons). In other words, within the pre-BötC, the precise neuroanatomical substrate affected and the mechanism(s) of excitation by each of these stimuli are not identical. We do not believe that the present findings result from increased pre-BötC neuronal excitability mediated by increases in excitatory synaptic input because arterial blood gases and pH were maintained constant throughout the experimental protocol. Thus modulation of the DLH-induced response during focal pre-BötC tissue acidosis presumably resulted from the direct effects of intrinsic CO2/H+-induced changes in pre-BötC excitability, whereas those observed during systemic hypercapnia would have resulted from both direct (i.e., intrinsic CO2/H+ chemosensitivity) and indirect (i.e., increased excitatory synaptic input mediated by enhanced release of excitatory neurotransmitters and neuromodulators) mechanisms.

Furthermore, our recent report (19) suggested that, during systemic hypercapnia, presumptive rhythmogenic pre-BötC neurons (5, 13) are closer to threshold for eliciting a rhythmogenic (i.e., frequency modulation) response to DLH-induced activation of the pre-BötC and that DLH-induced activation of this region elicits less of a contribution of the respiratory-modulated, nonrhythmogenic pre-BötC neurons (4, 15, 22) because these neurons are in a more excited state owing to increased synaptic drive (resulting from a generalized increase in neuronal excitability within the brain stem respiratory network in response to hypercapnia). Thus, during systemic hypercapnia, frequency modulation of phasic phrenic bursts and a reduction in the level of tonic phrenic nerve discharge in response to microinjection of DLH into the pre-BötC would be expected. If this explanation is correct, then, during focal pre-BötC tissue acidosis, presumptive rhythmogenic pre-BötC neurons would similarly be closer to threshold for eliciting a rhythmogenic (i.e., frequency modulation) response to DLH-induced activation of the pre-BötC; however, the contribution of the respiratory-modulated, nonrhythmogenic pre-BötC neurons in response to DLH-induced activation of this region would remain unaltered (because focal pre-BötC tissue acidosis would presumably lead to only increased neuronal excitability of the CO2/H+-chemosensitive pre-BötC neurons and not a generalized increase in synaptic drive within this region). Thus, during focal pre-BötC tissue acidosis, frequency modulation of phasic phrenic bursts with little or no change in the level of tonic phrenic nerve discharge in response to microinjection of DLH into the pre-BötC would be expected. The present experiments found that, during focal pre-BötC tissue acidosis, microinjection of DLH into this region added a phasic component, which included frequency modulation, to an unaltered level of tonic phrenic nerve discharge. Thus these findings suggest that the basic rhythm-generating circuitry located in the pre-BötC (14, 16, 17) is still responsive to activation during focal pre-BötC tissue acidosis and that the predominant response under these conditions consists of both phasic and tonic phrenic nerve discharge, including frequency modulation of the induced phasic phrenic bursts.

Limitations of the present study. Although the present findings demonstrate that focal pre-BötC tissue acidosis modifies the nonphasic tonic excitation of phrenic nerve discharge evoked by DLH-induced activation of the pre-BötC to include increased frequency of phasic phrenic bursts, three primary limitations associated with the present investigation should be mentioned. First, for the present experiments, we only selected sites in the pre-BötC in which microinjection of DLH produced a nonphasic tonic excitation of phrenic nerve discharge under baseline conditions. Although sites were often encountered in which DLH-induced activation of the pre-BötC evoked other patterns of excitation of phrenic nerve discharge (as previously reported; Ref. 20), these sites were avoided because the DLH-induced response already included modulation of phrenic burst frequency. The sites not included in the present investigation were those in which DLH-induced excitation of phrenic nerve activity included a high-amplitude, short-duration burst component. When this type of response was encountered, the microinjection pipette was moved 200-300 µm rostral because this response type is generally obtained from sites caudal to those in which tonic discharge is evoked (12). Even though these sites were intentionally avoided, in some cases DLH-induced activation of the pre-BötC during focal pre-BötC tissue acidosis elicited phrenic bursts that exhibited these patterning characteristics. Nonetheless, it remains to be determined whether focal pre-BötC tissue acidosis influences these other pre-BötC-mediated DLH-induced patterns of excitation of phrenic nerve discharge.

Second, we believe that the modulation of phrenic nerve activity observed in the present experiments was specific to DLH-induced activation of the pre-BötC during focal tissue acidosis of this region and did not result from spread of injectate to the adjacent rostral ventral respiratory group (rVRG). Although we cannot exclude the possibility of spread, this is unlikely. Our laboratory's previous experiments have shown that focal pre-BötC tissue acidosis markedly increases phrenic burst amplitude (by ~110% above baseline amplitude) and, in most cases, frequency, whereas focal rVRG tissue acidosis elicits only a moderate increase in phrenic burst amplitude (by ~40% above baseline amplitude) (21). The effects of microinjection of AZ and MZ in the present experiments were consistent with these previous observations, and regardless of whether focal pre-BötC tissue acidosis elicited an increase in amplitude or both amplitude and frequency of phrenic bursts, subsequent microinjection of DLH into this region produced phasic phrenic bursts superimposed on an underlying tonic discharge. Additionally, in our laboratory's previous experiments, nonphasic tonic excitation of phrenic nerve discharge and modulation of phrenic burst frequency were only observed in response to chemical stimulation of the pre-BötC (20). This is in contrast to the effects of similar activation of the rVRG, which has been shown to elicit changes in only amplitude of phrenic nerve discharge (1, 2, 10, 11, 20). From our experiments, however, we cannot exclude the possibility that microinjection of AZ, MZ, or DLH had an effect on dendrites whose cell bodies were distant from the site of injection.

Finally, the effects of focal pre-BötC tissue acidosis on the phrenic nerve discharge responses evoked by DLH-induced activation of the pre-BötC were examined in chloralose-anesthetized adult cats. Because anesthesia has been previously demonstrated to depress respiratory network activity due to enhanced inhibitory synaptic interactions, the patterns of evoked phrenic nerve activity observed may have been influenced by the effects of anesthesia. Thus it remains to be determined whether alterations in pre-BötC excitability produced by focal tissue acidosis would similarly modify DLH-induced pre-BötC-mediated phrenic nerve discharge responses in the unanesthetized (awake and/or decerebrate) state.

In summary, these findings demonstrate that the phrenic motor output response evoked by chemical stimulation of the pre-BötC in vivo is strongly influenced by intrinsic CO2/H+ chemosensitivity (i.e., focal tissue acidosis) within this region. In the present experiments, DLH-induced activation of a single site in the pre-BötC during focal pre-BötC tissue acidosis elicited modulation of phasic phrenic burst frequency, which was not observed in response to similar activation during baseline conditions; thus modulation of CO2/H+ within the pre-BötC appears to be one mechanism capable of influencing the response type evoked by repeated activation of a single site in the pre-BötC. We suggest that focal pre-BötC tissue acidosis increases neuronal excitability of a population of CO2/H+-chemosensitive pre-BötC neurons, which includes the presumptive rhythmogenic neurons, resulting in modulation of the pre-BötC-mediated DLH-induced response. These findings further indicate that intrinsic pre-BötC CO2/H+ chemosensitivity has the potential to play a role, at least in part, in frequency modulation of phrenic motor output during increased respiratory network drive (e.g., systemic hypercapnia) because chemical stimulation of this region during focal pre-BötC tissue acidosis elicited an increase in frequency of phasic phrenic bursts. Thus this study provides additional in vivo evidence for a role of this region in modulation of phasic respiratory activity.


    ACKNOWLEDGEMENTS

The author thanks T. J. Halat for technical assistance.

This work was supported by National Heart, Lung, and Blood Institute Grant HL-63175.


    FOOTNOTES

Address for reprint requests and other correspondence: I. C. Solomon, Dept. of Physiology and Biophysics, Basic Science Tower T6 Rm. 140, State Univ. of New York at Stony Brook, Stony Brook, NY 11794-8661 (E-mail: icsolomon{at}physiology.pnb.sunysb.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.

First published February 28, 2003;10.1152/japplphysiol.01192.2002

Received 26 December 2002; accepted in final form 6 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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