Reflex recruitment of medullary gasping mechanisms in

Transcription

1 MS 2233, pp Journal of Physiology (1994), Reflex recruitment of medullary gasping mechanisms in eupnoea by pharyngeal stimulation in cats Man-Lung Fung, Walter M. St John and Zoltan Tomori Department of Physiology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, NH 3755, USA and Department of Pathophysiology, Faculty of Medicine, Safarik University, Trieda SNP1, 466 Kosice, Slovakia 1. Mechanical stimulation of the naso- and oropharynx causes the replacement of the eupnoeic ventilatory pattern by a brief, but large, burst of activity of the phrenic nerve. Our purpose was to define whether these changes in phrenic activity represent a switch to gasping. 2. In decerebrate, vagotomized, paralysed and ventilated cats, mechanical stimulation of the pharynx was performed during eupnoea, apneusis and gasping. The latter two ventilatory patterns were produced by ventilating the experimental animal with 1- % carbon monoxide in air or with 1 % nitrogen. Eupnoea could be re-established by a recommencement of ventilation with oxygen. 3. The rate of rise of phrenic activity and its peak height were much greater following mechanical stimulation of the pharynx than the phrenic bursts of eupnoea or apneusis. The durations of phrenic burst and the period between these were much less following pharyngeal stimulation. In contrast, these variables of phrenic activity were the same during pharyngeal stimulation and in gasping. 4. Previous studies had established that activity within a region of the lateral tegmental field of medulla is critical for the manifestation of gasping. Hence, electrical stimulation of this region during gasping elicits premature gasps whereas its ablation irreversibly eliminates gasping. 5. We positioned a multibarrelled pipette in the critical medullary region for gasping. Its location was verified, once gasping was established in hypoxia or anoxia, by the elicitation of premature gasps following electrical stimulation. Neurons in this region were destroyed by microinjections of the neurotoxin kainic acid; in a few experiments the region was destroyed by electrolytic lesions. 6. Following destruction of the region of the lateral tegmental field, gasping could no longer be provoked in anoxia. In contrast, the eupnoeic pattern of phrenic activity continued. However, mechanical stimulation of the pharynx no longer caused any changes in the on-going pattern of phrenic activity. 7. We conclude that mechanical stimulation of the pharynx elicits a powerful reflex by which eupnoea is suppressed and gasping is elicited. Stated differently, the changes in phrenic activity during this pharyngeal stimulation in fact represent gasps. 8. Gasps are dependent upon activity within a region of the lateral tegmental field of the medulla. This region plays no role in the neurogenesis of eupnoea. Hence, our results provide additional support for the concept that there are multiple sites for ventilatory neurogenesis in the mammalian brainstem. A number of previous studies have led us to conclude that medulla; this region appears to play no role in the the mechanisms underlying the neurogenesis of the gasp are neurogenesis of eupnoea. Thus, during gasping, electrical fundamentally different from those responsible for stimulation of this region produces premature gasps generating eupnoeic ventilatory activity (St John, 199). whereas its ablation irreversibly eliminates gasping. In Most obviously, gasping is critically dependent upon animals having an intact pons and medulla, neither activity within a region of the lateral tegmental field of the stimulation nor ablation of this medullary region alters the

2 52 J. Physiol M. -L. Fung, W M. St John and Z. Tomori eupnoeic ventilatory rhythm (St John, Bledsoe & Sokol, 1984; St John, Bledsoe & Tenney, 1985). Different mechanisms, located in different regions of the brainstem, may therefore underlie the neurogenesis of eupnoea and gasping. Inherent to these concepts concerning eupnoea and gasping is the assumption that neural elements responsible for their neurogenesis must interact. Specifically, medullary mechanisms generating the gasp must be suppressed during eupnoea. These gasping mechanisms can be released by removal of influences from the pons. Removal of these pontile influences is achieved irreversibly by brainstem transections at the pontomedullary junction or reversibly by exposure to severe hypoxia (St John, 199). Interestingly, during recovery from hypoxia, the interactions between the mechanisms for eupnoea and gasping may be manifested. Thus, eupnoeic inspirations become interposed between gasps and some bursts of phrenic activity, which commence as those of eupnoea, are suddenly transformed into gasps (Richardson, 1986). In addition to recovery from anoxia, there were indications that the medullary mechanisms generating the gasp might be released from inhibition during eupnoea by the 'aspiration reflex'. This reflex is elicited by mechanical stimulation of the pharyngeal mucosa (Tomori, 1979). Upon stimulation, a premature inspiration is elicited which, like the gasp, has a rate of rise of activity far in excess of that of eupnoea. Moreover, pressures generated against an occluded airway were very similar during the aspiration reflex and the gasp; these pressures were much in excess of those of the eupnoeic inspiration (Tomori, Donic & Kurpas, 1993). Other specific characteristics of ventilatory activity during the aspiration reflex also more closely resemble gasping than eupnoea. Underlying the phrenic burst of eupnoea is the discharge of two populations of phrenic motoneurons, which commence their discharge 'early' and 'late' in neural inspiration. In gasping, the time of onset of the late phrenic motoneuronal activity is switched to the beginning of the burst (St John & Bartlett, 1981). Similarly, all phrenic motoneurons commence activity at the same time during the aspiration reflex (Jodkowski, Guthrie & Cameron, 1989) and, as in gasping, expiratory-modulated activities are greatly diminished or eliminated following pharyngeal stimulation (Batsel & Lines, 1973; Tomori, 1979; St John, Zhou & Fregosi, 1989; Zhou, Wasicko, Hu & St John, 1991). A final similarity between the aspiration reflex and gasping is that this reflex burst of inspiratory activity can be obtained during both eupnoea, as noted above, and after eupnoeic activity has been eliminated in severe hypoxia or following brainstem transections at the pontomedullary junction (Tomori, 1979; Jakus, Tomori, Boselova, Nagyova & Kubinec, 1987; Tomori, Benacka, Donic & Tkacova, 1991). Hence, the 'gasp-like' inspirations characteristic of the aspiration reflex can be obtained during medullary gasping. The above similarities of ventilatory activity during the aspiration reflex and gasping have led us to hypothesize that a common mechanism underlies both. Results obtained support this hypothesis in that specific patterns of neural activities during this reflex and gasping, recorded in the same experimental animal, were very similar. Moreover, the medullary region which is critical for the neurogenesis of the gasp is also required for manifestation of the aspiration reflex. METHODS General experimental preparation Sixteen adult cats of either sex were used. The surgical preparation has been described in detail previously (St John, 1979; St John et al. 1984, 1985, 1989). Animals were initially anaesthetized with 4 % halothane in oxygen. The concentration of halothane was reduced to 2- % in oxygen following cannulation of the trachea. Catheters were placed in the femoral arteries and femoral veins. The vagi were sectioned bilaterally at a midcervical level. The animals were mounted in a stereotaxic apparatus and the brainstem was transected at midcollicular level. All neural tissue rostral to the transection was removed (Kirsten & St John, 1978). A partial cerebellectomy was performed by aspiration in order to expose the medullary obex and the floor of the lvth ventricle. Halothane anaesthesia was then discontinued. The animals were paralysed with gallamine triethiodide (5 mg kg1 initially and -2'5 mg kg' approximately every 3 min thereafter). Artificial ventilation with a hyperoxic gas mixture was administered. End-tidal fractional concentrations of CO2 and 2 were continuously monitored and the animals were maintained in normocapnic hyperoxia or normoxia. Normocapnia was considered as an end-tidal fractional concentration of CO2 of approximately '5. Rectal temperature was maintained at 'C. Arterial blood pressure was a minimum of 8 mmhg and intravenous infusions of a metaraminol-dextran solution were delivered, if required. C5 or C6 rootlets of the phrenic nerve were exposed and sectioned. Efferent activity was recorded from the cut central end. This activity was integrated by a R-C (resistance-capacitance) circuit (i.e. 'leaky integrator', time constants, -2 s 'on' and -4 s'off'). Elicitation of the aspiration reflex As described previously (Tomori, 1979), a ventrolateral pharyngostomy was performed to allow visualization of the pharynx. This region was mechanically stimulated by touching the mucosa with a nylon fibre. Alterations of the pattern of neural respiration The pattern of activity of the phrenic nerve can be reversibly altered from eupnoea to apneusis and then gasping by ventilating the experimental animal with a gas mixture containing 1% carbon monoxide (CO) in air (Zhou et al. 1991). Approximately 1-15 min are required for gasping to be produced. Replacement of the CO mixture by oxygen results in the reverse pattern of changes with gasping being replaced by apneusis and then eupnoea. This sequence can be repeated several times. Arterial pressure was allowed to decline to hypotensive levels during the exposure to CO. Eight animals underwent this exposure to CO. A similar sequence of changes in phrenic activity and arterial blood pressure is produced if the animal is ventilated with 1 % nitrogen (St John, 199). Under these conditions of anoxia, the changes from eupnoea to gasping occur within 2-3 min. Again, eupnoea can be restored by ventilation with oxygen. Recordings were obtained from seven animals under these conditions; recordings had also been obtained from two of these upon exposure to carbon monoxide.

3 Reflex recruitment of gasping during eupnoea J. Physiol Stimulation and ablation of the medullary region for gasping In seven cats, a multibarrelled glass pipette, having a total diameter of 2-3 #sm, was inserted into the medulla. The initial position of this pipette was 2-3 mm rostral to the medullary obex, 2-3 mm lateral to the midline and 3-4 mm ventral to the surface. This position approximates the medullary region for neurogenesis of gasping which we have previously defined (St John et al. 1984,1985). Two of the barrels of the micropipette were filled with 3 M NaCl. These barrels were used for delivery of electrical stimuli. Stimuli consisted of a ms train of pulses of 5 ms duration which were delivered at a frequency of 25 Hz; the maximal current was 1 1sA. Two other barrels contained solutions of kainic acid, to destroy neurons, and Fast Green FCF, to localize the site of injection. Both the Fast Green (1 % solution) and the kainic acid (4-69 mm) were dissolved in mock cerebrospinal fluid (St John et al. 1984). The ph of the solution containing kainic acid was adjusted to and the osmolality was about 3 mosmol kg-1h2o. Barrels were pressurized in order to eject solutions. The amount that was ejected was defined by reference to calibrations which had been established for similar micropipettes. Hence, we determined the impedance of a number of micropipettes and then defined the amount of solution which was ejected at different pressures for different times. This amount was determined by direct observation of the tip of the pipette through a calibrated reticule of a microscope. In three additional animals, the above protocol of stimulation was followed but using a bipolar metal electrode. The medullary region for gasping neurogenesis was lesioned by passing direct current through this electrode. Experimental protocol Recordings were obtained at normocapnia during eupnoea. The 'aspiration reflex' was elicited numerous times during these control recordings. Ventilation with carbon monoxide in air or with nitrogen was then commenced. Mechanical stimulations of the pharynx were performed periodically, as were electrical stimulations of the medulla. When periodic gasping was obtained, the position of the micropipette or metal electrode was altered, if required, until premature gasps were obtained by the electrical stimulations. In some experiments, several alterations between ventilation with oxygen and with carbon monoxide or nitrogen were required before the critical medullary area was located. Kainic acid was then injected into the region or the region was lesioned by direct current. Before and after lesions, stimulations of the pharynx and medulla were repeated after ventilation with oxygen had recommenced. Such stimulations were also repeated after eupnoea had been re-established and the pattern of neural respiration was again altered during one or more exposures to carbon monoxide or nitrogen. At the end of experiments, the brainstem was removed for histological localization of the region of injection of kainic acid or the placement of electrolytic lesions. Sections were stained with Cresyl Violet. Measurement of variables For integrated activity of the phrenic nerve, the following were determined: duration of the burst from onset to rapid decline (neural inspiration, T,), period to the commencements of the next burst (neural expiration, TE), peak height and mean rate of rise of activity. The rate of rise was determined over the linear phase of activity, i.e. from time of onset to a level approximating 9% of the peak value. For arterial blood pressure, systolic and diastolic levels were defined. Under conditions of eupnoea, apneusis and gasping, the above variables were defined for a minimum of six respiratory cycles and the average obtained. The aspiration reflex was elicit during each of these three ventilatory patterns. Comparisons were made between each pattern and the aspiration reflex recorded concomitantly. Statistical evaluations of data were made using the paired Wilcoxon test, adjusted, if required, for multiple comparisons. Probabilities less than 5 were considered statistically significant. Eupnoeic inspiration mmhg 25 5 s 2 4s Figure 1. Elicitation of the aspiration reflex in eupnoea The left panel shows recordings of activity of the phrenic nerve (), its integral (f) and arterial blood pressure () during control recordings and during mechanical stimulations of the pharynx (filled bar). The right panel shows superimposed integrated phrenic records during eupnoeic inspiration and elicitation of aspiration reflex. Vertical line designates start of neural inspiration.

4 M. -L. Fung, W. M. St John and Z. Tomori 522 J. Physiol RESULTS Elicitation of the aspiration reflex Eupnoea Mechanical stimulation of the pharynx caused eupnoea to be interrupted and replaced by a series of phrenic bursts of high amplitude. These bursts were accompanied by a marked rise in arterial blood pressure (Fig. 1). Typically, a single phrenic burst followed each stimulus. Thus, in all trials, repeated stimulation of the pharynx was performed. Most commonly, the phrenic bursts were characterized by an extremely rapid rise to maximal or near-maximal levels (Fig. 1). We have taken these phrenic bursts as those defining the 'aspiration reflex'. However, if the pharynx was stimulated during neural inspiration, the rapid rise of phrenic activity was superimposed upon the eupnoeic pattern (Fig. 1, initial burst during stimulation). In two animals, the patterns of changes in phrenic activity described above could not be obtained. We have no explanation for the absence of the reflex, although damage to the neural innervation of the pharynx is obviously a possibility. No further data concerning these two animals are presented herein. As is evident in Fig. 1, the rate of rise of phrenic activity during the aspiration reflex was significantly greater than that of the eupnoeic phrenic burst. In addition, the peak level of integrated phrenic activity was significantly higher and the durations of neural inspiration and expiration were significantly less during the reflex (Fig. 2). Apneusis Upon exposure to carbon monoxide, the pattern of ventilatory activity was altered to apneusis, with marked prolongations of the period of the phrenic burst and the period between bursts. These alterations are shown in Fig. 3 for the same experimental animal for which eupnoeic data were presented in Fig. 1. Upon mechanical stimulation of the pharynx, the same patterns of phrenic activity could be elicited as during eupnoea. Thus, this stimulation caused a replacement of the apneustic pattern by the pattern characteristic of the 'aspiration reflex'. As during eupnoea, the rate of rise and peak phrenic activity were significantly higher and the durations of neural inspiration and expiration were significantly lower during the aspiration reflex than the apneustic ventilatory cycle (Fig. 4). Gasping Continuing exposure to carbon monoxide resulted in an extended period without detectable phrenic activity. Mechanical stimulation of the pharynx immediately terminated this apnoeic phase and elicited the stereotypical pattern of phrenic bursts. Such stimulation also elicited premature bursts of activity once spontaneous gasping was established (Fig. 5). With the establishment of gasping, electrical stimulation of the lateral tegmental field of medulla elicited premature gasps (Fig. 5). In contrast to findings during eupnoea or apneusis, rates of rise of phrenic activity were very similar during the aspiration reflex and the spontaneous gasp (Fig. 5). Rates of rise during spontaneous gasps and those resulting from electrical stimulations of the medulla were also similar (Fig. 5). In summary, as reported in Fig. 6, there were no significant differences between any variables of phrenic activity measured during the aspiration reflex and spontaneous and stimulus-induced gasping. For the latter, TE represented time to the commencement of the next spontaneous phrenic burst. When carbon monoxide was replaced with oxygen, the changes in ventilatory pattern reversed. Hence, gasping was replaced by apneusis and then by eupnoea. At all times during this gradual restoration of eupnoea, the high- ~-Z -U1) 2 r 1-5 F TI * k-! 4 3 1i * 25 a) Q 15 a) 1 - C: U) r 15 P 1 [ T 5 a) 5 Er. 5 oo _I o _I o f-l I Figure 2. Comparison of variables of phrenic activity during eupnoeic ventilatory cycles and aspiration reflex Mean values (+ S.E.M.) are presented for the durations of neural inspiration (TI) and expiration (TE), peak integrated phrenic height (Peak ) and the rate of rise of phrenic activity (Rate of rise). Open bars are eupnoeic cycles; filled bars are aspiration reflex. * P< 5 compared to eupnoeic value.

5 Reflex recruitment of gasping during eupnoea J. Physiol I -i i i 6-- ii, f f mmhg[ f.lfln.s, 25 mmhg 5 s nnasamaasa~~~~~~~~~~~!limwilitifl111olfltaanaaa1h s Aspiration reflex Apneustic inspiration 1'WN 2 4s Figure 3. Elicitation of the aspiration reflex in apneusis Upper panels are recordings of activity of the phrenic nerve (), its integral (f) and arterial blood pressure () during control recordings (left panel) and during mechanical stimulations of the pharynx (right panel, filled bar). Lower traces are superimposed integrated phrenic records during apneustic inspiration and elicitation of aspiration reflex. Vertical line designates start of neural inspiration k i i15 Ca CL 15 a. 1~~~~~ 2 rr 5 5 * I Figure 4. Comparison of variables of phrenic activity during apneustic ventilatory cycles and aspiration reflex Mean values (+ S.E.M.) are presented for the durations of neural inspiration (TI) and expiration (TE), peak integrated phrenic height (Peak ) and the rate of rise of phrenic activity (Rate of rise). Values of the last two variables are expressed as the percentage of these values during eupnoea. Open bars are apneustic cycles; filled bars are aspiration reflex. * P< 5 compared to apneustic value.

6 524 J. Physiol M.-L. Fung, W. M. St John and Z. Tomori J A P I&A-LL-i,.l - 11* r 11 1 T -1mv"www T iorrw r 'T'W ---7 V I!1 fj v-..-, mmhg Pl 25 5I s t 25 5 s Gasping Aspiration reflex t 2 4s 2 4 s Figure 5. Elicitation of the aspiration reflex in gasping Upper panels are recordings of activity of the phrenic nerve (), its integral (f) and arterial blood pressure () during control recordings (left panel) and during mechanical stimulations of the pharynx (right panel, filled bar). Arrows below traces in panels designate gasps which were elicited by electrical stimulation. Lower traces in left panel are superimposed integrated phrenic records during spontaneous gasping and elicitation of aspiration reflex. Lower tracings in right panel compare spontaneous and electrically induced gasps. Vertical line designates start of neural inspiration. 2- r 12 r *5 1- II 8 k [ 5 F I I - Figure 6. Comparison of variables of phrenic activity during gasping ventilatory cycles and aspiration reflex Mean values (+ S.E.M.) are presented for the durations of neural inspiration (TI) and expiration (TE), peak integrated phrenic height (Peak ) and the rate of rise of phrenic activity (Rate of rise). Values of the last two variables are expressed as the percentage of these values during eupnoea. Open bars are spontaneous gasping cycles; filled bars are aspiration reflex; hatched bars are electrically induced gasps.

7 J. PhysioL Reflex recruitment of gasping during eupnoea 525 amplitude bursts of phrenic activity were observed upon stimulation of the pharynx. Influence of the medullary region for gasping upon the aspiration reflex Alterations of phrenic activity As shown in Fig. 5, when spontaneous gasping had been established during ventilation with carbon monoxide, premature gasps followed stimulation in the lateral tegmental field of medulla. Figure 7 illustrates data from an experimental animal in which a single injection of 1 nl of kainic acid was made into this critical region of the medulla. Within 2 min of this injection, the eupnoeic pattern of phrenic activity became somewhat irregular with the duration of neural expiration declining. At this time, mechanical stimulation of the pharynx caused only a modest alteration of peak phrenic activity; an elevation of J. T I Eupnoea J WItN f r- Gasping i i i.. 'L i, 14.,h I IdL nqw 4 e.~.i.. - hn Al * A. ^ m rr- mmhg ' ' I ' 1 l l ' II-~ 15 3C)s O 2 min post-ka -L s Co E f 1 L u.1 kll u... sl.- L., _~~~~~~~ - _ -. I _ - -rsm "'B Ie-R TI1- "pq- r-- "-r ~ 1r T17 i~~~~~~o *.66.= i X v6 d ~~~~~~~~~~~,1. Trp 'jtj'yti f'lw'si rfwr-wiswiyrt --sr-w r r -- T,,, [-, r TTr- nw mmhg [ M L W 15 3 s I.- 15 'lhwmmmffawmh,%w#*mam, 3 s 1 h post-ka Co mmhg L f AJK~~ZkV~~4AA%AAMAAJtJI' Nj',1 1 l.' l i. illl.lfi.ul!@ t.'v A4JJL~Ct. -E4..,-... I 15 3 s 15 3 s Figure 7. Elimination of aspiration reflex and gasping by lesions of the lateral tegmental field of medulla Each panel contains recordings of activity of the phrenic nerve (), its integral (f) and arterial blood pressure (). Recording in upper panels were obtained during eupnoea and gasping, induced by ventilation with carbon monoxide (CO). Mechanical stimulations of the pharynx are indicated by filled bars. Middle panels are comparable records obtained 2 min after a single injection of kainic acid (KA) and a subsequent exposure to CO. Lower panels are records taken approximately 1 h thereafter. Arterial catheter was flushed during a portion of the recording in the lower right panel.

8 526 M.-L. Fung, W M. St cjohn and Z. Tomori J. Physiol arterial blood pressure was still observed. These modest changes were obviously very different from the pattern observed before the injection of kainic acid (Fig. 7). Moreover, when this animal was re-exposed to carbon monoxide, only a rapid and irregular pattern of phrenic bursts was recorded; gasping was not observed. Pharyngeal stimulations during these exposures to CO did not elicit the changes in phrenic activity characteristic of the aspiration reflex, as described above. Approximately one hour after the injection of kainic acid, the pattern of phrenic activity resembled more closely that of eupnoea. Mechanical stimulation of the pharynx now caused only a slight shift in the baseline of phrenic activity, perhaps reflecting an electrical artifact. No rapid rises in phrenic activity could be induced nor was arterial blood pressure altered. Likewise, while the pattern of phrenic activity was altered, exposure to carbon monoxide never resulted in elicitation of gasping. Gasping was not observed even when exposure continued until irreversible elimination of phrenic activity was obtained (Fig. 7, right lower trace). Results similar to those of Fig. 7 were obtained in all seven animals following injections of kainic acid. Thus, following a single injection in three cats, no discernible phrenic activity was observed upon stimulation of the pharynx. This pharyngeal stimulation resulted in only a low-amplitude burst of phrenic activity for the other four cats following the unilateral injection. This residual stimulus-induced phrenic activity was completely eliminated after larger injections of kainic acid (e.g. 6 nl) into the same medullary region in two cats or, in the other two cats, after a single injection into the comparable region of the contralateral medulla. In the three cats in which single electrolytic lesions were placed in the medulla, no changes in phrenic activity followed pharyngeal stimulation. In summary, following unilateral injections of kainic acid into the medulla or unilateral electrolytic lesions of the region, eupnoeic patterns of phrenic activity were still recorded. Exposure to carbon monoxide did not result in any reappearance of gasping. Following elimination of gasping, the phrenic activity induced by mechanical stimulation of the pharynx was either severely altered or completely eliminated. / K (I.' '.1 Figure 8. Examples of sites of injection of kainic acid in the medulla Each of the brainstem sections is from a different animal. A circle surrounds the area of injection. Damage above site of injection was caused by insertion of the micropipette. Bar to the lower right of sections indicates 1 mm. Abbreviations: 1, inferior olive; XII, hypoglossal nucleus.

9 Reflex recruitment of gasping during eupnoea J. Physiol Histology Histological evaluation revealed that the region of injection of kainic acid was in the lateral tegmental field of medulla. Comparable sites were found for electrolytic lesions. Examples of this region of injection in four animals appears in Fig. 8. A composite of the site of injection in all animals and of the electrolytic lesion is shown in Fig. 9. The minimum region of injection approximated a sphere of 5 mm in diameter. This critical region of injection was between the nucleus tractus solitarii and nucleus ambiguus. These two nuclei, respectively comprising the dorsal and ventral medullary respiratory nuclei, contain high densities of respiratory-modulated neuronal activities (e.g. von Euler, 1986). DISCUSSION The major conclusion of this study is that mechanical stimulation of the naso- and oropharynx activates a potent reflex by which mechanisms generating eupnoeic ventilatory activity are suppressed and those for gasping are released. In addition, our results provide support for the concept that there are multiple sites for ventilatory neurogenesis in the mammalian brainstem. As noted in the Introduction, release of the medullary mechanisms for gasping has heretofore been equated only with a complete removal of descending influences from the pons (St John, 199). Indeed, we have previously demonstrated that gasping is not obtained so long as any anatomical connection remains between the pons and the medulla (St John & Knuth, 1981). Thus, it appears that these pontile influences must tonically suppress the medullary mechanisms underlying the neurogenesis of gasping. This concept of a powerful pontile suppression is further supported by the response, or lack thereof, to electrical stimulation of the medullary region for gasping. In eupnoea, such stimulations produce no change in ventilatory activity. Identical stimulations elicit premature gasps once this pattern has been established by brainstem transections or exposure to hypoxia or anoxia (St John et al. 1984,1985). We have previously maintained that the lack of oxygen during hypoxia and anoxia releases medullary mechanisms for gasping by dual actions. First, pontile neuronal activities are suppressed (Neubauer, Melton & Edelman, 199). Second, Figure 9. Localization of sites of injection of kainic acid and electrolytic lesions in the medulla Sites of injection () and lesions (U) have been projected on the nearest section to those illustrated. Although bilateral injections were made in two animals, only injections on one side are shown. Likewise, injections were made on the right or left side in some animals, but all are projected to the right side of the medulla. Bar to the lower right of sections indicates 1 mm. Abbreviations: 1, inferior olive; LRN, lateral reticular nucleus; LTF, lateral tegmental field; VSP, spinal tract of trigeminal nerve; XII, hypoglossal nucleus.

10 528 M. -L. Fung, W M. St John and Z. Tomori J. Physiol medullary mechanisms for gasping are activated directly. Such a hypoxia-induced activation has been demonstrated for medullary sympathetic premotor neurons (e.g. Sun, Jeske & Reis, 1992). A similar activation of medullary mechanisms for gasping is supported by our finding that the frequency of gasping increases in hypoxia in vagotomized animals having sectioned carotid sinus nerves (St John & Knuth, 1981). We hypothesize that mechanical stimulation of the pharynx releases medullary mechanisms for gasping by a similar dual process. Pharyngeal stimulation during eupnoea, apneusis or gasping was equally effective in eliciting a series of gasps. This elicitation during eupnoea or apneusis might simply be equated with a depression of pontile influences. However, premature gasps following such pharyngeal stimulation during anoxia-induced apnoea or gasping per se demonstrate an activation of the mechanisms for gasping neurogenesis. The neurophysiological processes by which pharyngeal stimulation produces a suppression of pontile and activation of medullary neuronal activities are undefined. Indeed, the neuroanatomical pathways underlying the aspiration reflex are not definitively established. The region of the naso- and oropharynx from which the aspiration reflex can be elicited is innervated by the glossopharyngeal, trigeminal and the intermediate branch of the facial nerve (Tomori, 1979). Given the overlapping pattern of innervation and the distortion resulting from mechanical stimulation, it is possible that afferent fibres in more than one nerve were activated. Yet Nail, Sterling & Widdicombe (1972) have reported that changes in phrenic activity which are characteristic of the aspiration reflex can be induced by electrical stimulation of the glossopharyngeal nerve alone. Again, how such glossopharyngeal stimulation would suppress the pattern generator for eupnoea and activate the medullary mechanisms for gasping is undefined. However, results from other studies involving electrical stimulation provide insights into such inhibition and excitation. Berger & Mitchell (1976) reported a three-phase response to stimulation of the glossopharngeal nerve. The first component is a short-latency activation of phrenic activity. This component reflects the paucisynaptic pathway from the glossopharyngeal nerve to the nucleus tractus solitarii and, thence, to the phrenic motoneuronal pool (e.g. Cottle, 1964; Biscoe & Sampson, 1974; von Euler, 1986). The second and third components represent an inhibition and delayed, but powerful, activation of phrenic discharge (Berger & Mitchell, 1976). We believe that this latter activation reflects the activation of the medullary centre for gasping. The earlier depression of phrenic activity is thought to reflect a depression of pontile activities and mechanisms underlying the neurogenesis of eupnoea. This concept of multiple changes following activation of the glossopharyngeal nerve and elicitation of the reflex changes in phrenic activity is also evident in the concomitant changes in cardiovascular activity. As noted in Results, mechanical stimulation of the pharynx resulted in a marked elevation in arterial blood pressure. This elevation reached a peak value long after the augmentation of phrenic activity. Since the experimental animals were paralysed, the changes in cardiovascular activity did not reflect mechanical changes resulting from the increased ventilation. Moreover, as described previously (Tomori & Widdicombe, 1969), such changes in arterial blood pressure reflect, as least in part, changes in sympathetic activity. Hence, mechanical stimulation of the pharynx elicits a complicated series of reflex changes in respiratory and cardiovascular activities within the brainstem. From our data, it does not appear that the changes in these two systems are inextricably linked. Within 5 min of injection of kainic acid into the lateral tegmental field of medulla, gasps could no longer be elicited by either mechanical stimulation of the pharynx or exposure to anoxia. However, the rise in blood pressure following pharyngeal stimulation was still evident. This cardiovascular response did ultimately disappear. We believe that these results are consistent with the conclusion that activition of medullary mechanisms for gasping is not essential for the cardiovascular changes following pharyngeal stimulation to be manifested. However, it does appear that these cardiovascular changes do require activities of neurons in a proximal region. As noted above, not only do the cardiovascular changes ultimately disappear following injections of kainic acid in the lateral tegmental field, but arterial blood pressure also typically increases following electrical stimulation in this region. No such blood pressure elevations followed the spontaneous gasps. Concerning the latter, results of the present study confirm our earlier reports that medullary mechanisms which are critical for the neurogenesis of gasping are separate from brainstem mechanisms responsible for generating eupnoeic ventilation activity (see St John, 199 for review). In summary, the results of this investigation establish that a region in the lateral tegmental field of medulla is necessary both for gasping to be elicited and for the 'aspiration reflex' to be manifested. It will be of interest to define whether other potent respiratory reflexes, such as cough or sighs, are also dependent upon this medullary region for gasping. REFERENCES BATSEL, H. L. & LINES, A. J. (1973). Bulbar respiratory neurons participating in the sniff reflex in the cat. Experimental Neurology 39, BERGER, A. J. & MITCHELL, R. A. (1976). Lateralized phrenic nerve responses to stimulating respiratory afferents in the cat. American Journal of Physiology 23, BISCOE, T. J. & SAMPSON, S. R. (1974). Field potentials evoked in the brain stem of the cat by stimulation of the carotid sinus, glossopharyngeal, aortic and superior laryngeal nerves. Journal of Physiology 24, COTTLE, M. K. (1964). Degeneration studies in primary afferents of the IXth and Xth cranial nerves in the cat. Journal of Comparative Neurology 122,

11 J. PhysioL Reflex recruitment of gasping during eupnoea 529 JAKUS, J., ToMORI, Z., BOSELOVA, L., NAGYOVA, B. & KUBINEC, V. (1987). Respiration and airway reflexes after transversal brain stem lesions in cats. Physiologia Bohemoslovaca 36, JODKOWSKI, J. S., GUTHRIE, R. D. & CAMERON, W. E. (1989). The activity pattern of phrenic motoneurons during the aspiration reflex: an intracellular study. Brain Research 55, KIRSTEN, E. B. & ST JOHN, W. M. (1978). A feline decerebration technique with low mortality and long term homeostasis. Journal of Pharmacological Methods 1, NAIL, B. S., STERLING, G. M. & WIDDICOMBE, J. G. (1972). Patterns of spontaneous and reflexly-induced activity in phrenic and intercostal motoneurons. Experimental Brain Research 15, NEUBAUER, J. A., MELTON, J. E. & EDELMAN, N. H. (199). Modulation of respiration during brain hypoxia. Journal of Applied Physiology 68, RICHARDSON, C. A. (1986). Unique spectral peak in phrenic nerve activity characterizes gasps in decerebrate cats. Journal of Applied Physiology 6, ST JOHN, W. M. (1979). Differential alteration by hypercapnia and hypoxia of the apneustic respiratory pattern in decerebrate cats. Journal of Physiology 287, ST JOHN, W. M. (199). Neurogenesis, control and functional significance of gasping. Journal of Applied Physiology 68, ST JOHN, W. M. & BARTLETT, D. (1981). Comparison of phrenic motoneuron activity in eupnoea and gasping. Journal of Applied Physiology 5, ST JoHN, W. M., BLEDSOE, T. A. & SOKOL, H. W. (1984). Identification of medullary loci critical for neurogenesis of gasping. Journal of Applied Physiology 56, ST JOHN, W. M., BLEDSOE, T. A. & TENNEY, S. M. (1985). Characterization by stimulation of medullary mechanisms underlying gasping neurogenesis. Journal of Applied Physiology 58, ST JOHN, W. M. & KNUTH, K. V. (1981). A characterization of the respiratory pattern of gasping. Journal of Applied Physiology 5, ST JOHN, W. M., ZHou, D. & FREGOsI, R. F. (1989). Expiratory neural activities in gasping. Journal of Applied Physiology 66, SUN, M.-K., JESKE, I. T. & REIS, D. J. (1992). Cyanide excites medullary sympathoexcitatory neurons in rats. American Journal of Physiology 262, R ToMORI, S. (1979). The sniff-like aspiration reflex. In Progress in Respiration Research, Cough and Other Respiratory Reflexes, ed. HERZOG, H., pp Karger, Basel, Switzerland. ToMORI, Z., BENACKA, R., DONIC, V. & TKACOVA, R. (1991). Reversal of apnoea by aspiration reflex in anaesthetized cats. European Respiratory Journal 4, ToMORI, Z., DONIC, V. & KURPAS, M. (1993). Comparison of inspiratory effort in sniff-like aspiration reflex, gasping and normal breathing in cats. European Respiratory Journal 6, ToMoRI, Z. & WIDDICOMBE, J. G. (1969). Muscular, bronchomotor and cardiovascular reflexes elicited by mechanical stimulation of the respiratory tract. Journal of Physiology, VON EULER, C. (1986). Brain stem mechanisms for generation and control of breathing pattern. In Handbook of Physiology, section II, The Respiratory System, vol. 2, ed. CHERNIACK, N. S. & WIDDICOMBE, J. G., pp American Physiology Society, Bethesda, MD, USA. ZHOU, D., WASICKO, M. S., HU, J.-M. & ST JOHN, W. M. (1991). Differing activities of medullary respiratory neurons in eupnoea and gasping. Journal of Applied Physiology 7, Acknowledgements These studies were supported by research grant HL2691 from the National Heart, Lung and Blood Institute, National Institutes of Health (USA). Received 24 March 1993; accepted 23 August 1993.