INTRODUCTION

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The pre-Bötzinger complex (pre-BötC) is hypothesized to be the primary locus of respiratory rhythm generation in mammals (Rekling and Feldman 1998; Smith et al. 1991, 2000). Selective destruction of neurokinin-1 receptor-expressing neurons in this region has recently been demonstrated to abolish the normal "eupneic" pattern of breathing in vivo (Gray et al. 2001), while activation of the pre-BötC, both in vivo and in vitro, increases the frequency of eupneic inspiratory motor activity (Chitravanshi and Sapru 1999; Gray et al. 1999; McCrimmon et al. 2000; Solomon et al. 1999). Recent studies have suggested that this region plays a role not only in the generation and modulation of eupneic breathing but also in the production and/or expression of different patterns of inspiratory motor activity, including gasping (Lieske et al. 2000; Solomon et al. 1999). Work from our laboratory, for example, has demonstrated that focal chemical stimulation of neurons located in the pre-BötC of anesthetized adult cats increases the frequency of phasic phrenic bursts and, in some cases, shifts the eupneic motor pattern to one of augmented or gasp-like discharge (Solomon et al. 1999). Further, focal hypoxia in the pre-BötC similarly elicits frequency modulation (FM) of eupneic phrenic motor discharge as well as these modified patterns of phrenic motor activity in vivo, suggesting intrinsic hypoxic chemosensitivity of this region (Solomon et al. 2000). Whether this region plays a role in FM of modified inspiratory motor patterns remains unclear. Therefore we examined the effects of chemical stimulation of neurons using DL-homocysteic acid (DLH) and focal hypoxia using sodium cyanide (NaCN) in the pre-BötC during gasping induced by severe brain hypoxia. We hypothesized that stimulation of neurons located in the pre-BötC during hypoxia-induced gasping would increase the frequency of gasp-like bursts and that similar FM would be elicited by focal hypoxia in this region.

	METHODS

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General methods

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 (Solomon et al. 1999).

In brief, anesthesia was induced in adult cats (3.4-4.4 kg; n = 12) with halothane (5%) in oxygen and maintained with intravenous -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. 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, 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.

Experimental protocol

We examined the effects of chemical stimulation and focal hypoxia in the pre-BötC on the patterning, timing, and frequency of phrenic nerve discharge during hypoxia-induced gasping. Sites in the pre-BötC were functionally identified using microinjection of DLH under hyperoxic conditions (Fig. 1A) as previously described (Solomon et al. 1999). In all experiments, gasping was produced by lowering the fraction of O₂ in the inspired gas mixture to 2-6% O₂ in a balance of N₂. A minimum of two gasps were recorded, and then 10-20 nl of 10 mM DLH (n = 7) or 1 mM NaCN (n = 3) was microinjected. In four cats, a second hypoxic challenge was performed without microinjection as a (time) control. In two additional cats, DLH microinjection (20 nl) was made into the adjacent (within 400 µm) rostral ventral respiratory group (rVRG) as a control for spread. No more than two hypoxic challenges were performed in a cat, and only one response to microinjection into the pre-BötC during hypoxia-induced gasping was studied. Prior to the hypoxic challenge and at the onset of gasping, whenever possible, an arterial blood sample was obtained for measurement of P_O2, P_CO2, pH, Sa_O2, and Ca_O2 (Radiometer ABL-500/OSM3). 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 (Fig. 1B) as previously described (Solomon et al. 1999, 2000).

View larger version (33K): [in this window] [in a new window]   Fig. 1. A: examples demonstrating functional identification of pre-Bötzinger complex (pre-BötC) sites. Microinjection of DL-homocysteic acid (DLH) into the pre-BötC during hyperoxia produced a rapid series of high-amplitude, short-duration inspiratory bursts (Aa), a tonic excitation of phrenic nerve discharge (Ab), or augmented bursts in conjunction with a respiratory rhythmic tonic discharge (Ac). B: schematic drawing of coronal sections of the medulla showing location of pre-BötC sites that received microinjection of DLH () and sodium cyanide (NaCN, ) during hypoxia-induced gasping. 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 level indicated ±0.2 mm (rostrally and caudally). C: characteristic pattern of hypoxia-induced gasping (without microinjection). Severe brain hypoxia produced abrupt onset, high-amplitude, short-duration phrenic bursts, separated by prolonged TE. Note: this trace corresponds to control experiment for NaCN microinjection presented in Fig. 3. 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.

Data analysis

Peak amplitude of integrated phrenic nerve discharge, inspiratory duration (T_I), expiratory duration (T_E), rate of rise, and frequency of phrenic bursts were determined in response to microinjection of DLH and NaCN into the pre-BötC during hypoxia-induced gasping. Preinjection baseline values were calculated by averaging the values obtained for all hypoxia-induced gasps preceding microinjection (typically 2-5 gasps/cat) and peak values were calculated by averaging the values obtained for the first five phrenic bursts immediately following microinjection. Amplitude is reported as a percent change from preinjection baseline levels of discharge that were set at 100% in each cat. Rate of rise was determined over the linear phase of activity and is reported as a percentage of the maximal rate of rise recorded in each cat.

Data are reported as means ± SE. Student's paired t-tests or the paired nonparametric Wilcoxon signed-rank test, as appropriate, were used to determine statistical significance for which the criterion level was set at P < 0.05.

	RESULTS

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Characteristics of hypoxia-induced gasping

Hypoxia-induced gasping was characterized by abrupt-onset, high-amplitude, short-duration bursts of phrenic nerve activity, separated by prolonged periods of phrenic silence (T_E; Figs. 1-3). This pattern was typical for both the gasps produced in trials without microinjection (Fig. 1C) as well as those preceding microinjection of DLH or NaCN (i.e., preinjection baseline; Figs. 2, A-C, and 3). The mean frequency of hypoxia-induced gasps under these circumstances was 2.5 ± 0.3 gasps/min (range: 1-5), and arterial P_O2, P_CO2, pH, Sa_O2, and Ca_O2 at the onset of gasping (n = 11) were 21 ± 3 mmHg, 39 ± 2 mmHg, 7.40 ± 0.01, 14.7 ± 4.4%, and 2.5 ± 0.8 vol%, respectively. Prior to the hypoxic challenge, arterial P_O2, P_CO2, pH, Sa_O2, and Ca_O2 were 188 ± 4 mmHg, 40 ± 1 mmHg, 7.35 ± 0.01, 98.3 ± 0.1%, and 17.1 ± 0.6 vol%, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 2. FM evoked by DLH-induced activation of pre-BötC during hypoxia-induced gasping. Microinjection of DLH into the pre-BötC during hypoxia-induced gasping produced either a rapid series of phrenic bursts (A) or a more moderate increase in phrenic burst frequency (B). C: summary data showing the effects of DLH-induced activation of the pre-BötC during hypoxia-induced gasping on the timing and patterning characteristics of phrenic nerve discharge. Microinjection of DLH during hypoxia-induced gasping significantly increased the phrenic burst frequency and reduced TE. No significant changes were observed in TI, amplitude, or rate of rise. *, a significant difference (P < 0.05) from preinjection baseline gasps.

View larger version (11K): [in this window] [in a new window]   Fig. 3. FM evoked by focal hypoxia in the pre-BötC during hypoxia-induced gasping. Microinjection of NaCN into the pre-BötC during hypoxia-induced gasping produced a rapid series of phrenic bursts by reducing TE. In this example, there is also a progressive decrease in amplitude of integrated phrenic nerve discharge following microinjection of NaCN.

Effects of DLH-induced activation and focal hypoxia in the pre-BötC during hypoxia-induced gasping

Unilateral microinjection of DLH or NaCN into the pre-BötC during hypoxia-induced gasping increased phrenic burst frequency in each of the sites examined (Figs. 2 and 3). Microinjection of DLH increased the frequency of phrenic bursts by ~630% over preinjection baseline gasp frequency (P < 0.01; Fig. 2C), resulting predominantly from a reduction in T_E (P < 0.01; Fig. 2C). Similarly, microinjection of NaCN increased phrenic burst frequency by 600% over preinjection baseline gasp frequency (range: 600-2,200%) in each of the sites examined. This DLH- and NaCN-induced FM was quite variable and consisted of either a rapid series of phrenic bursts (DLH: n = 3, Fig. 2A; NaCN: n = 2, Fig. 3) or a more moderate increase in phrenic burst frequency (DLH: n = 4, Fig. 2B; NaCN: n = 1). The precise pattern of FM appeared to be independent of the initial (hyperoxic) DLH-induced response or the histological location of the injection site; however, the rapid series of phrenic bursts was generally seen in cases with the more severe hypoxic blood gas values. In some cases, a small increase in basal (tonic) discharge was also observed following microinjection of DLH or NaCN. No significant changes in T_I, peak amplitude of integrated phrenic nerve discharge, or rate of rise between hypoxia-induced gasps and the DLH-induced bursts were observed (Fig. 2C); however, NaCN-induced bursts typically exhibited a small reduction in peak amplitude of integrated phrenic nerve discharge compared with the hypoxia-induced gasps (although this was not evaluated for statistical significance; n = 3). It should also be noted that although the early DLH- and NaCN-induced phrenic bursts have similar timing and patterning characteristics compared with the hypoxia-induced gasps, the later bursts exhibited more variability. Whether this increased variability resulted from chemical activation and focal hypoxia in the pre-BötC, severe brain hypoxia, or a combination of these effects is unclear.

Control experiments

In trials without microinjection (Fig. 1C; time control), no FM was observed. Further, unilateral microinjection of DLH into the adjacent rVRG during hypoxia-induced gasping was ineffective in increasing the frequency of phrenic bursts in either of the sites examined (not shown). In these experiments, the gasping pattern of phrenic bursts (i.e., T_I, peak amplitude of integrated phrenic nerve discharge, rate of rise) also remained unchanged in response to DLH microinjection.

	DISCUSSION

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We have demonstrated that both chemical stimulation of neurons and focal hypoxia in the pre-BötC during gasping induced by severe brain hypoxia increase the frequency of "gasp-like" phrenic bursts. Previous studies have shown FM of eupneic inspiratory motor activity in response to chemical stimulation of this region (Chitravanshi and Sapru 1999; Gray et al. 1999; McCrimmon et al. 2000; Solomon et al. 1999); however, these studies did not consider the effects of pre-BötC activation in FM of other patterns of inspiratory motor output, such as gasping. Our current findings suggest that the basic rhythm-generating circuitry located in the pre-BötC (Rekling and Feldman 1998; Smith et al. 1991, 2000) is still responsive to activation during hypoxia-induced gasping as evidenced by the FM. In these experiments, we did not observe changes in the patterning of "gasp-like" phrenic bursts in response to pre-BötC activation, although in some cases, a small tonic increase in basal discharge was detected. It has been reported that during brain hypoxia, most neurons with respiratory-modulated activities are depressed (Richter et al. 1991); thus we suggest that the necessary elements for pattern formation changes were most likely silent and/or unresponsive to pre-BötC input under the hypoxic conditions of our experiments.

Although brain hypoxia produces a generalized depression of respiratory-modulated neuronal activity, we have previously demonstrated that focal hypoxia in the pre-BötC produces excitation of phrenic motor output during hyperoxia, suggesting intrinsic hypoxic chemosensitivity of this region (Solomon et al. 2000). Our current data provide additional evidence in support of this concept. In these experiments, focal hypoxia in the pre-BötC during hypoxia-induced gasping modified the frequency of gasp-like phrenic bursts similar to the effects observed with DLH. Although our findings provide no insight into which population of pre-BötC neurons are hypoxia chemosensitive, we suggest that the most likely candidates mediating this FM are the proposed rhythm-generating pacemaker neurons (Rekling and Feldman 1998; Smith et al. 1991, 2000). In support of this proposal, it has recently been demonstrated in the in vitro transverse medullary slice from neonatal mice that some pre-BötC pacemaker neurons continue to burst rhythmically during anoxia (Thoby-Brisson and Ramirez 2000).

In summary, our results indicate that in addition to the proposed role of the pre-BötC in generation and modulation of eupneic inspiratory motor output, this region has the potential to play a role in FM of gasping during severe brain hypoxia. Our findings further suggest that during brain hypoxia when respiratory output is reduced, the pre-BötC rhythm-generating network can be excited by focal activation and focal hypoxia, yielding an increase in phrenic burst frequency. Thus this study provides additional in vivo evidence consistent with the proposed role of this region as the primary locus for respiratory rhythm generation.