The influence of descending inputs on breathing pattern formation in the isolated bullfrog brainstem-spinal cord

Transcription

1 Respiration Physiology 120 (2000) The influence of descending inputs on breathing pattern formation in the isolated bullfrog brainstem-spinal cord Stephen G. Reid a, *, Janice T. Meier b, William K. Milsom b a Department of Physiology, College of Medicine, Uni ersity of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5 b Department of Zoology, Uni ersity of British Columbia, 6270 Uni ersity Bl d., Vancou er, BC, Canada V6T 1Z4 Accepted 13 December 1999 Abstract This study used in vitro brainstem-spinal cord preparations from the American bullfrog, Rana catesbeiana, to examine the influence of central descending inputs on breathing pattern formation. In preparations with an episodic pattern of fictive breathing, a transection slightly caudal to the optic chiasma produced a continuous breathing pattern and increased the overall frequency of fictive breathing. Following a transection to isolate the medulla, the frequency of fictive breathing decreased and the incidence of other forms of motor output increased. Further transections between the trigeminal and vagus nerve roots resulted in variable and asynchronous discharge from each nerve. These results suggest that a primary respiratory rhythm is produced within the medulla but descending influences stimulate breathing and promote episodic breathing. It would appear that multiple elements of the respiratory control system, including tegmental and medullary sites, play a role in shaping the burst pattern of motor output associated with each breath and that slower rhythms of longer burst duration are generated by more caudal hindbrain sites Elsevier Science B.V. All rights reserved. Keywords: Amphibians, bullfrog (Rana catesbeiana); Brainstem, pattern of breathing; Control of breathing, central pattern generation; Pattern of breathing, episodic breathing, gasping, central descending inputs 1. Introduction The normal pattern of breathing in amphibians such as the bullfrog (Rana catesbeiana) consists of randomly distributed breaths or occasional doublets that occur at a rate of about 6 min 1 * Corresponding author. Tel.: ; fax: address: reidsg@hotmail.com (S.G. Reid) (Milsom, 1991; Kinkead, 1997). When respiratory drive is elevated by hypoxia or hypercapnia, breathing becomes more regular and breaths frequently occur in episodes. Although vagotomy alters breathing pattern by increasing tidal volume and increasing the occurrence of breathing episodes, the episodes still consist primarily of two or three breaths when the animals breath air, and increase substantially when the animals are made hypoxic or hypercapnic (e.g. West et al., 1987; Kinkead and Milsom, 1996, 1997) /00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 198 S.G. Reid et al. / Respiration Physiology 120 (2000) Recently, the use of reduced preparations, such as the in vitro brainstem-spinal cord preparation (e.g. Kinkead et al., 1994; McLean et al., 1995a,b) and the in situ decerebrate, paralysed and unidirectionally ventilated animal (e.g. Kogo and Remmers, 1994; Kogo et al., 1994; Kinkead and Milsom, 1997), has facilitated the investigation of various mechanisms (central and peripheral) involved in producing the complex patterns characteristic of amphibian respiration (Kinkead, 1997). In the isolated brainstem of both tadpoles and adult frogs, as well as in decerebrate, unidirectionally ventilated preparations, spontaneous rhythmical bursting activity, representative of the efferent motor output underlying lung ventilation, can be recorded from the trigeminal, facial, glossopharyngael, vagal and hypoglossal nerve roots (Sakakibara, 1984; Kinkead et al., 1994; Kogo et al., 1994; McLean et al., 1995a; Torgerson et al., 1997). Studies utilising such preparations have identified various sites (Oka, 1958; McLean et al., 1995b; Kinkead et al., 1997), cell types (Kogo and Remmers, 1994) and mechanisms (Kinkead et al., 1994; Kogo et al., 1994; Galante et al., 1996) involved in producing or modulating central respiratory rhythm or breathing pattern formation in amphibians (see review by Milsom et al., 1999). However, in addition to motor output indicative of fictive breathing, reduced preparations can also exhibit various other forms of motor output whose function is unclear. Galante et al. (1996) and Liao et al. (1996) observed complex forms of discharge from in situ and in vitro preparations of a R. catesbeiana tadpole while Reid and Milsom (1998) described ten forms of other motor output (i.e. not normal fictive breaths) from the adult bullfrog in vitro preparation. Despite the advances that have been made, the mechanisms involved in clustering breaths together into episodes have yet to be identified (Kinkead, 1997; Milsom et al., 1997). West et al. (1987) first demonstrated that fluctuations in blood gas levels were not necessary to produce an episodic breathing pattern. Kinkead et al. (1997) initially hypothesised that the nucleus isthmi, a mesencephalic structure located between the roof of the midbrain and the cerebellum, was involved in clustering breaths together into episodes. However, the results of their study (Kinkead et al., 1997) indicate that while the nucleus isthmi provides tonic excitatory input to respiratory centers and is involved in the integration of chemosensory information, it is not responsible for the production of breathing episodes. None-the-less, episodic breathing is absent in many in vitro preparations of the amphibian brainstem-spinal cord (McLean et al., 1995a,b; Reid and Milsom, 1998) and it has been suggested (Milsom et al., 1997) that episodic breathing may result from an interaction between positive and negative descending inputs to the medullary respiratory centers arising from other midbrain or higher sites. The goal of this study was to use progressive rostral to caudal transections of the in vitro bullfrog brainstem-spinal cord to examine the effects of central descending inputs on breathing pattern formation in this species and address this hypothesis. 2. Materials and methods 2.1. Experimental animals Bullfrogs (R. catesbeiana) ( 250 g; n=30) were obtained from a commercial supplier (Cyr s Biological Company, Ponchatoula, LA) and transported to the University of British Columbia. Animals were maintained in fiberglass tanks supplied with a constant flow of dechlorinated City of Vancouver tapwater to provide a moist environment. Room temperature was approximately 20 C and the photoperiod was maintained at 12L:12D. Frogs were fed live locusts at least once a week. The animals were allowed to acclimate to laboratory conditions for a minimum of 1 week before experimentation The in itro brainstem-spinal cord preparation Bullfrogs were anaesthetized by partial submersion in an aqueous solution of ethyl-m-aminobenzoate (MS 222; 0.6 g l 1 ) buffered to ph 7.0 with sodium bicarbonate. A surgical level of anesthesia (i.e. elimination of withdrawal and eye blink refl-

3 S.G. Reid et al. / Respiration Physiology 120 (2000) exes) was obtained in approximately 30 min. A longitudinal incision was made in the skin above the cranium and the skin removed. Using a dental drill, a small hole was made in the skull rostral to the optic lobes and the cranial case was removed with bone shears and placed onto a Sylgardcoated dissecting dish. The brain was exposed, superfusion with oxygenated Ringers was initiated, and the rostral forebrain was removed. The remaining tissue was continually superfused with oxygenated, bicarbonate-buffered Ringers (in mmol l 1 ; NaCl, 75.0; KCl, 4.5; MgCl 2, 1.0; NaH 2 PO 4, 1.0; NaHCO 3, 40.0; CaCl 2, 2.5; glucose, 5.0; ph 7.8). A ph of 7.8 was selected as it approximates the ph of amphibian plasma at room temperature (West et al., 1987; McLean et al., 1995a). Cranial nerves were cut close to their exit from the skull and the spinal cord was severed at the level of the second spinal nerve. The brainstemspinal cord was lifted from the cranial case and pinned in a dissecting dish where the membranes (dural, arachnoid, meninges) were removed in order to free the cranial nerve roots. The tips of the nerves were cut in order to provide a clean, undamaged surface for recording. The preparation was then pinned ventral side up on a fine stainless steel mesh within a superfusion chamber. The mesh divided the chamber into upper and lower compartments, which facilitated constant superfusion of both surfaces (Kinkead et al., 1994; McLean et al., 1995a,b). Once the brainstemspinal cord was secure within the chamber (approximately 30 min following the commencement of surgery), the preparation was superfused, utilizing a flow through system, (5 ml min 1 ) for at least 1 h to allow it to stabilise. All experiments were performed at room temperature. The small diffusion distances within the brainstem, coupled with its anoxia/hypoxia tolerance, allow this preparation to remain viable for many hours (Perry et al., 1995) Ficti e breathing Utilizing a micro-manipulator, a suction electrode, synthesized from thin-walled capillary glass (inner diameter 1 mm) was positioned near the end of one severed nerve and the nerve rootlet was gently aspired into the electrode. Numerous electrodes, with a wide range of tip diameters, were fashioned in order to provide an assortment of electrodes such that one of appropriate size was always available to obtain a tight seal on different nerve roots from animals of different size. Care was taken to ensure that the nerve fit tightly into the electrode permitting the formation of a good seal. Once the nerve rootlet was firmly in place within the electrode, whole nerve discharge from the trigeminal and/or vagus nerve(s) was recorded to provide an index of fictive breathing. Nerve discharge was observed in these preparations within 1 6 h post-dissection. This delay presumably reflects the time required for residual anaesthetic to diffuse from the tissue Experimental protocol Once stable levels of neural discharge were observed, motor output was then recorded for a period of min. All recordings were made with the ph of the superfusate between 7.7 and 8.0. In this preparation, under these conditions, fictive breathing and other forms of neural discharge (see below) are unaffected by changes in superfusate ph (Reid and Milsom, 1998). In those preparations exhibiting an episodic pattern of fictive breathing, a transection was made, using fine scissors, immediately rostral to the optic chiasma. After a sufficient period of time elapsed to record a stable pattern of discharge, a further transection was made immediately caudal to the optic chiasma (Fig. 1). Again, following a stable recording period, further transections were made at the rostral border of the medulla and then between the trigeminal and vagus nerve roots, at the approximate level of the facial/acoustic nerve root (Fig. 1). During the transection process, care was taken not to disrupt the seal between the nerve root and the suction electrode(s). Not all preparations exhibited an episodic pattern of fictive breathing. Nevertheless, in those preparations that did not exhibit episodic breathing, the transections were still performed in an identical fashion.

4 200 S.G. Reid et al. / Respiration Physiology 120 (2000) Data acquisition and analysis Nerve activity from the suction electrode was amplified (filter settings: 50 Hz (high pass) and 10 khz (low pass)), full wave rectified and integrated. The signal was monitored visually using an oscilloscope (Tektronix 5111A) and acoustically with an audio monitor (Grass AM8). Activity was recorded on a chart recorder and representative recordings were saved using a data acquisition system (Windaq, DataQ Systems). The sampling rate of analogue to digital conversion was 2000 Hz. Breathing episodes were designated according to the criteria of Kinkead and Milsom (1997). When breathing was continuous, no values were obtained for breaths per episode or episodes per minute. Breathing frequency was quantified by analyzing the number of fictive breaths per unit time (absolute frequency; breaths/min). The classification of neural discharge as a fictive breath or other discharge was based on comparisons of the in vitro motor output to fictive breathing obtained in a less reduced preparation, the decerebrate, paralyzed and unidirectionally-ventilated in situ preparation. These criteria have been described previously (Reid and Milsom, 1998). Respiratory related discharge (i.e. normal fictive breaths) was considered to be less than 1 sec in duration. Additionally, in order for a burst to be considered a normal fictive breath, it had to exhibit an incrementing ( ramp-like ) onset followed by a similar decrementing offset (see Fig. 1 from Reid and Milsom, 1998). Neural discharge longer than 1 sec and/or exhibiting different forms of onset and/or offset was classified as an other form of discharge. These other forms of discharge have previously been sub-classified on the basis of the ten most common forms observed (Reid and Milsom, 1998) Statistical analysis Fig. 1. Schematic diagrams of the bullfrog brain. (A) A ventral view. (B) A lateral view. In both figures, a dashed line caudal to the optic chiasma illustrates the level at which a transection eliminates fictive breathing episodes and replaces them with a continuous pattern of fictive breathing. In part A (the upper panel) the second dashed line at the rostral border of the medulla indicates the level at which the brainstem was transected to isolate the medulla. Roman numerals in panel B indicate the various cranial nerves. The data are presented as mean values 1 standard error of the mean (SEM). The values before any transection and following the transections rostral and caudal to the optic chiasma were compared using a parametric analysis of variance (ANOVA) followed by Dunnet s multiple comparison test. Given that the data from the isolated medulla was also obtained from the non-episodic preparations, the values for the isolated medulla were compared to the corresponding pre-transection values (i.e. after the transection caudal to the optic chiasma) with a paired t-test. When parametric test assumptions were violated, the data were analysed using a Kruskal Wallis ANOVA on ranks followed by Dunnet s multiple comparison test or a signed rank t-test. An asterisk (*) denotes a significant difference from the value before any transection while a plus sign ( +) denotes a significant difference in the value for the isolated medulla compared with the values following the caudal transection. All statistical testing, including determinations of normality and variance, was performed using commercial software

5 S.G. Reid et al. / Respiration Physiology 120 (2000) Fig. 2. (A) An example of fictive breathing (episodic discharge) and other forms of motor output recorded from the vagus (X) nerve root. (B) An expanded trace illustrating a fictive breathing episode from part A. (C) A further expanded trace illustrating the incrementing and decrementing shape of the neural discharge associated with each fictive breath. In A and B the upper figure illustrates the full wave rectified signal while the lower trace illustrates the integrated ( X) signal. (Sigmastat; Jandel Scientific). The fiducial limit of significance was set at 5%. 3. Results 3.1. Episodic pattern of ficti e breathing Figure 2 illustrates an episodic pattern of fictive breathing recorded from the vagus nerve root in the isolated brainstem-spinal cord preparation. This pattern of fictive breathing is similar to the breathing pattern observed in an intact bullfrog under conditions of elevated respiratory drive although the occurrence of this pattern in vitro is not as consistent as in intact animals. Figure 2B illustrates a fictive breathing episode on an expanded time scale. The ramp-like shape, with incrementing and decrementing phases of neural discharge, indicative of fictive breaths (pulmonary ventilation) is illustrated in Fig. 2C. On the left side of the trace in Fig. 2A there are approximately six large bursts (both in magnitude and duration) of neural discharge that are not normal fictive breaths. These forms of other discharge occur variably between preparations and are insensitive to changes in superfusate ph (Reid and Milsom, 1998). A full description of ten various forms of other discharge has recently been reported with classification based on burst shape, duration and amplitude (Reid and Milsom, 1998). Several of these types of discharge are illustrated in Fig. 3. Type A discharge (Fig. 3A) has a normal incrementing and decrementing

6 202 S.G. Reid et al. / Respiration Physiology 120 (2000) shape, indicative of fictive breaths, but is larger in magnitude and longer in duration than fictive breaths. Type AA discharge (Fig. 3B) consists of biphasic type A discharge with no pause in between the two bursts. Type B bursts exhibit an abrupt onset with a normal decrementing offset (Fig. 3C). Figure 3D illustrates a normal fictive breath for comparison The effect of transections on ficti e breathing episodes In those preparations exhibiting an episodic pattern of fictive breathing, a transection rostral to the optic chiasma did not alter the episodic pattern of fictive breathing. A transection immediately caudal to the optic chiasma, however, converted the episodic pattern into a continuous one with single breaths occurring at relatively evenly spaced intervals. Figure 4 illustrates the effects of such a transection from three separate in vitro Fig. 3. Examples of other motor output from the in vitro brainstem-spinal cord. These forms of discharge have been described previously (Reid and Milsom, 1998). (A) Type A discharge that exhibits an incrementing and decrementing profile similar to normal fictive breaths but is substantially longer in duration and larger in magnitude. (B) Type AA discharge which consists of two type A bursts which blend into one another with no pause between the two phases. (C) Type B discharge exhibiting an abrupt onset followed by a decrementing phase. (D) A normal fictive breath. Fig. 4. Three examples of the effect of transection immediately caudal to the optic chiasma on the pattern of fictive breathing from isolated bullfrog brainstem-spinal cord preparations. (A) Three panels representing a continuous recording of integrated neural discharge from the vagus (X) nerve root. In the upper panel, and the left-hand side of the middle panel, the fictive breaths are clustered into episodes with several single breaths also evident. The arrowhead represents the point at which the brainstem is transected immediately caudal to the optic chiasma. Following this transection, all of the fictive breaths occur as single events rather than being clustered into episodes. (B) A continuous trace illustrating fictive breathing episodes (left side of the trace) replaced by single breaths (right side of the trace) following a transection immediately caudal to the optic chiasma (arrowhead). (C) An episodic pattern of fictive breathing (doublets) before (top panel; pre-transection) a transection at the optic chiasma. Following this transection the pattern is converted into one with relatively evenly spaced single breaths (lower panel; post-transection). preparations. Figure 4A (three panels) represents a continuous trace of fictive breathing recorded from the vagus nerve, with the fictive breaths clustered into episodes (upper panel and the left

7 S.G. Reid et al. / Respiration Physiology 120 (2000) hand side of the middle panel). Several single breaths were also observed in addition to the episodes. Following a transection slightly caudal to the optic chiasma (arrow), the episodes were eliminated and replaced by a relatively evenly Fig. 5. The effect of brainstem transection, both rostral and caudal to the optic chiasma, on the fictive breathing pattern from the isolated bullfrog brainstem-spinal cord preparation. (A) The ratio of the number of single fictive breaths to the number of episodes of fictive breaths. (B) The ratio of the number of single breaths to the total number of fictive breaths contained within episodes. The data are shown as the mean 1 S.E.M. An asterisk (*) denotes a significant difference from the value before any transection. spaced continuous breaths. Figure 4B also illustrates the transition from an episodic pattern of fictive breathing to a continuous one following a transection caudal to the optic chiasma (arrow). Figure 4C illustrates an episodic pattern of fictive breathing, consisting of doublets (Fig. 4C, pretransection) which, following transection caudal to the optic chiasma, was replaced by evenly spaced single breaths (Fig. 4C, post-transection). The loss of episodic fictive breathing following the transection caudal to the optic chiasma is further evident from the data in Fig. 5. This figure illustrates the ratio of the number of single breaths to the number of episodes (Fig. 5A) as well as the ratio of the number of single breaths to the total number of breaths contained within episodes (Fig. 5B). These ratios did not change following the transection rostral to the optic chiasma but increased substantially (six to seven times) following the caudal transection. These data indicate the replacement of breaths within episodes with single breaths following the transection caudal to the optic chiasma. The effect of these transections on the occurrence and timing of fictive breaths is illustrated in Fig. 6. The frequency of episodes did not change following a transection rostral to the optic chiasma but decreased significantly following the caudal transection (Fig. 6A). Although the transection caudal to the optic chiasma eliminated the majority of episodes, an occasional episode was observed and thus the frequency of episodes approached, but never reached, a value of zero. Neither the rostral nor caudal transection had an effect on the number of breaths per episode which, in all cases, was approximately three (Fig. 6B). The caudal transection also caused a decrease in the duration of the apneic period from sec to approximately 10 sec (Fig. 6C). Neither transection influenced the duration of individual fictive breaths (values ranged from to sec; data not shown). Concurrent with the lose of episodicity following the caudal transection was an increase in the absolute frequency of fictive breathing from approximately breaths min 1 (Fig. 6D). Although there was a trend for the frequency of

8 204 S.G. Reid et al. / Respiration Physiology 120 (2000) Fig. 6. The effect of brainstem transection, both rostral and caudal to the optic chiasma and following a transection to leave the medulla in isolation, on the timing and occurrence of fictive breaths and other forms of motor output. (A) The number of episodes per minute. (B) The number of fictive breaths per episode. (C) The apnea duration (time between episodes, episodes and single breaths or individual single breaths; sec). (D) The absolute frequency of fictive breaths (breaths/min). (E) The absolute frequency of other forms of discharge that are likely not respiratory-related (bursts/min). (E) The ratio of other forms of discharge to the number of fictive breaths. The data are shown as the mean 1 S.E.M. An asterisk (*) denotes a significant difference from the value before any transection. A plus sign (+) denotes a significant difference in the isolated medulla group from the previous value (i.e. in the caudal transection group). other non-respiratory-related discharge to increase following the caudal transection, this increase was not statistically significant (Fig. 6E). Additionally, the ratio of the number of other forms of discharge to the number of fictive breaths remained constant following both the rostral and caudal transections (Fig. 6F) Ficti e breathing and other forms of neural discharge from the isolated medulla Figure 7A demonstrates the pattern of continuous fictive breathing observed following a transection caudal to the optic chiasma. Following a transection at the rostral border of the medulla

9 S.G. Reid et al. / Respiration Physiology 120 (2000) (arrowhead in Fig. 7B), motor output was absent for approximately 130 sec. When a motor pattern resumed it consisted of large bursts of neural activity (Fig. 7C) which did not resemble normal fictive breaths (compare Fig. 7D with E). Fictive breaths were also observed from the same preparation following this transection but at a substantially reduced frequency (e.g. Fig. 7C,E, asterisks). Figure 8 illustrates the pattern of motor output from another isolated medulla. In this preparation, fictive breaths were observed along with large gasp-like bursts of discharge (type B). Upon initial inspection (i.e. Fig. 8A), all fictive breaths appear similar. However, when observed on an expanded scale it is apparent that the burst pattern of some of these breaths has been altered. Figure 8B illustrates a normally shaped fictive breath (incrementing then decrementing) observed from the isolated medulla. The burst in Fig. 8C begins with normally incrementing discharge yet terminates abruptly while the burst in Fig. 8D both begins and ends abruptly. Following the transections to isolate the medulla from rostral regions of the brainstem Fig. 7. A continuous trace (A, B and C) illustrating the effect of a transection to leave the medulla in isolation (A) Fictive breathing recorded from the vagus (X) nerve root following a transection immediately caudal to the optic chiasma to remove the fictive breathing episodes. In all cases the upper trace represents the full wave rectified signal while the lower one represents the integrated trace. (B) The effect of a transection (arrowhead) at the rostral border of the medulla to leave the medulla in isolation. Note the lack of discharge immediately following the transection. (C) A drastically altered pattern of neural discharge from the isolated medulla consisting of two long burst that likely are not respiratory-related. (D) An expanded trace illustrating four fictive breaths from part A. (E) An expanded trace illustrating the neural discharge observed in the middle of part C. Note the occurrence of normal fictive breaths (asterisks) in panels C and E.

10 206 S.G. Reid et al. / Respiration Physiology 120 (2000) Fig. 8. An example of various forms of discharge from the isolated medulla. (A) Numerous small bursts indicative of fictive breaths, small bursts of various shapes (see below) and large gasp-like bursts. (B) A normal fictive breath exhibiting an incrementing and decrementing pattern of discharge. (C) A burst similar to a fictive breath with the exception that the decrementing phase has been replaced by an abrupt offset. (D) A small type D-like burst exhibiting an abrupt onset and offset with a plateau phase in between. there was a trend for the duration of apneas (periods of quiescence between fictive breaths) to increase (Fig. 6C). Concomitantly, the absolute frequency of fictive breaths decreased (Fig. 6D) while the absolute frequency of other non-respiratory related discharge increased (Fig. 6E). Additionally, the isolated medulla exhibited an increase in the ratio of the number of other bursts to the number of fictive breaths compared to when descending influences from higher centers were present (Fig. 6F) Transections between the trigeminal and agus ner e roots Figure 9 illustrates various forms of other discharge from an isolated medulla, recorded simultaneously from the Vth and Xth nerve roots. In this case (Fig. 9A), the discharge occurred simultaneously from each nerve root indicating some form of coupling of the motor output. Figure 9B illustrates a continuum (three pairs of traces) of simultaneous recordings from the trigeminal and vagus nerve roots following a further transection between the Vth and Xth nerves. In this case, both nerve roots were isolated from the higher brain centers as well as from each other. In this figure, bursts of motor output from the trigeminal nerve are marked with an asterisk while bursts of discharge from the vagus nerve are marked with an arrow. This figure reveals that both nerve roots are capable of producing motor output, although these bursts do not occur simultaneously nor do they resemble the shape of normal fictive breaths. This result was only observed from a single preparation and all attempts at repetition were unsuccessful. 4. Discussion 4.1. Episodic patterns of ficti e breathing An episodic pattern of breathing occurs in amphibians during periods of elevated respiratory drive (i.e. under hypoxic or hypercarbic/hypercapnic conditions; West et al., 1987; Milsom, 1995). Such a breathing pattern is observed in the intact

11 S.G. Reid et al. / Respiration Physiology 120 (2000) animal (Kinkead and Milsom, 1994) as well as in decerebrate, paralysed, unidirectionally ventilated preparations in which motor output from the trigeminal nerve is used as an index of fictive breathing (Kogo et al., 1994; Kinkead and Milsom, 1996). However, in the in vitro brainstemspinal cord preparation, the occurrence of such a pattern is highly unpredictable (Reid and Milsom, 1998). Given that episodes of fictive breathing can and do occur in isolated in vitro preparations (Kinkead et al., 1994; Reid and Milsom, 1998), it appears as if the mechanisms responsible for the production of breathing episodes are present entirely within the brainstem and are able to function independently of feedback from peripheral sources. Afferent input from lung stretch receptors (Kinkead and Milsom, 1996, 1997; Kinkead et al., 1994) and arterial chemoreceptors (West et al., 1987; Kinkead and Milsom, 1994) can, and does, influence the occurrence of breathing episodes but they are not necessary for their production. The underlying reason(s) for the infrequent and inconsistent occurrence of episodes in these preparations is unclear. It is possible that the central mechanisms responsible for producing breathing episodes do not function optimally in the absence of the afferent input that is normally present in vivo. This suggestion is supported by the observation that, in vitro, stimulation of the cut stump of one vagus nerve (to mimic pulmonary receptor feedback) enhanced the episodicity of fictive breathing in this preparation (Kinkead et al., 1994) The effect of transections on the episodic breathing pattern Fig. 9. (A) A trace of neural discharge recorded simultaneously from the trigeminal (V) and vagus (X) nerve roots following a transection to isolate the medulla. (B) Asynchronous discharge from the Vth and Xth nerves following a transection between these two nerve roots at the approximate level of the VIIth cranial nerve. In this figure, both the trigeminal and vagus nerves are in isolation from higher centers and each other; in essence forming two thick slices. Note that, despite their physical isolation, each nerve is still capable of producing some form of motor output although the rhythm/timing of the discharge is different in both nerve roots. An asterisk (*) marks bursts of discharge from the trigeminal nerve while an arrowhead ( ) marks neural bursts from the vagus nerve root. The results of this study suggest that descending influences from higher brain centers, at the approximate level of the optic chiasma, are involved either directly in, or facilitate, the clustering of breaths together into discrete episodes. Transections slightly rostral to the optic chiasma did not alter the episodic pattern of breathing whereas transections slightly caudal to the optic chiasma essentially eliminated the occurrence of discrete episodes of fictive breaths. The continuous pattern of fictive breathing observed following the caudal transection resembled that observed in previous studies on adult bullfrogs (e.g. McLean et al., 1995a,b; Kimura et al., 1997; Reid and Milsom, 1998). In the few preparations in which this transection did not completely eliminate the occurrence of episodes, the pattern of fictive breathing consisted almost entirely of single breaths with the rare occurrence of a single

12 208 S.G. Reid et al. / Respiration Physiology 120 (2000) episode. These observations suggest the presence of an episodic center, involved in the clustering of breaths, at a level slightly caudal to the optic chiasma. However, the nature, both anatomically and physiologically, of such a putative center is still a matter of speculation. It is possible that such an episodic center is a discrete anatomical loci or, alternately, it may exist as a diffuse network of neurons whose association is physiological rather than anatomical. Indeed, given that an occasional episode was observed following the caudal transection in several preparations, it would seem as if the clustering of breaths into episodes may be the role of a diffuse physiological network rather than a distinct anatomical center. The effect of various brain transections on breathing pattern have been described in the Japanese bullfrog (Oka, 1958; in Japanese). This study reported that a transection at the caudal border of the optic lobes eliminated normal episodic movements of the buccal cavity (see Fig. 6 from Oka, 1958). It also reported that all transections between the caudal border of the optic lobes and obex produced abnormal respiration (measured as both buccal movements and lung pressure) but never abolished respiratory movements entirely. It is unclear why transections in that study did not result in normal oscillations of the buccal cavity. In the current study, the transections caudal to the optic chiasma that eliminated fictive episodes did not abolish the occurrence of normal fictive breaths. There was a trend for the frequency of other forms of neural discharge to increase but whether or not this other discharge is the neural (motor output) equivalent of the abnormal buccal oscillations observed by Oka (1958) is hard to judge. Regardless, that episodic breathing was eliminated in the previous study by Oka (1958) supports the suggestion that an episodic center is involved in the clustering of breaths into episodes Positi e and negati e modulation of breathing pattern Given that the clustering of breaths together into episodes can occur both in vitro, it appears as if the mechanisms responsible for producing breathing episodes are located within the brain and do not require input from peripheral chemoreceptors or mechanoreceptors. It has been suggested (Milsom et al., 1997) that the production of breathing episodes is controlled by positive and negative inputs descending into the brainstem from higher brain centers. Under this scenario, periods of apnea may arise from negative modulation whereas episodes occur when a positive influence is expressed or the negative influence is removed or overridden. The hypothesis that episodic breathing is produced by both positive and negative modulation appears to be supported by work on in situ, unidirectionally ventilated bullfrogs. If frogs are unidirectionally ventilated via a steady flow of gas that enters one lung and exits the other lung, both pulmonary and arterial blood gas tensions can be held constant. Under these conditions, breathing is still episodic (West et al., 1987). Since there are no phasic changes in pulmonary or blood gas levels, the generation of breathing episodes appears to be an intrinsic property of the respiratory control system rather than a result of blood gas fluctuations. Indeed the observation that breathing episodes can be produced by an in vitro preparation (Kinkead et al., 1994; Reid and Milsom, 1998) supports the suggestion that the production of breathing episodes is an intrinsic property of the brainstem. In the current study, the disappearance of prolonged periods of apnea, following the transection caudal to the optic chiasma, suggests that there is a powerful descending negative influence associated with episodic breathing. Although West et al. (1987) demonstrated that apnea-induced fluctuations in blood gas levels do not cause the production of breathing episodes, it is possible that fluctuations in blood gas levels (i.e. reduced p O2 and elevated p CO2 ), acting via chemoreceptors, could modulate or determine the frequency of breathing within episodes in the intact animal (Milsom, 1991). However, in most animals that exhibit an episodic pattern of breathing, an increase in respiratory drive due to external hypoxic or hypercarbic stimuli generally leads to an increase in breathing frequency through an increase in both the number of breaths per

13 S.G. Reid et al. / Respiration Physiology 120 (2000) episode and the occurrence of breathing episodes. Under these conditions (hypoxic or hypercarbic), there is little or no change in the frequency of breathing within episodes (West et al., 1987; Milsom, 1991) Motor output from the isolated medulla With the exception of ph/co 2 chemoreceptors, the isolated medulla is devoid of all sensory/modulatory input which may influence the formation of breathing within the medullary respiratory centers. Not only are peripheral inputs (i.e. from pulmonary stretch receptors and peripheral chemoreceptors) missing, as is the case in the intact (decerebrate) brainstem-spinal cord preparation, but any influence(s) from centers rostral to the medulla are also absent. Given the absence of these inputs, one might suggest that the pattern of fictive breathing produced by the isolated medulla may be indicative/representative of a basic respiratory rhythm originating from the central respiratory rhythm generator (see review by Feldman et al., 1990) or possibly from multiple rhythm generators in the bullfrog (Kogo et al., 1994). The results of this study demonstrate that the pattern of motor output from the isolated medulla can be highly variable. In addition to normal fictive breaths, the isolated medulla can also exhibit various forms of larger duration motor output. The large bursts of discharge, illustrated in Fig. 8A, resemble phrenic nerve discharge from the neonatal rat indicative of gasping (St. John, 1996) while the long drawn out bursts from the isolated medulla, illustrated in Fig. 7E, resemble the slower frequency, longer duration respiratory activity recorded from caudal sites in lamprey (Thompson, 1985) and chick embryos (Fortin et al., 1995). Such forms of discharge are not limited to the isolated medulla but also occurred when higher brain centers were intact (Reid and Milsom, 1998; Fig. 2, current study). Whether these forms of neural discharge represent an aberrant form of breathing such as apneusis or gasping is a matter of conjecture. The abrupt ( type B or gasp-like) bursts may also result from a disruption of normal synaptic mechanisms responsible for generating respiratory bursts. Kimura et al. (1997) observed that application of the glycine receptor blocker, strychnine (10 20 mol l 1 ), to a modified in vitro preparation from the leopard frog, Rana pipiens, caused discharge from various cranial nerves to change shape and become abrupt and decrementing (i.e. similar to the type B bursts). Whether or not such a mechanism is responsible for producing the other forms of discharge observed from the adult bullfrog brainstem-spinal cord preparation or the isolated medulla is unknown. It is also possible that the presence of these complex and variable forms of motor output are indicative of the fact that the respiratory muscles also serve many other functions such as mastication, swallowing, vocalisation, vomiting and coughing (e.g. Sakamoto et al., 1997) and may receive inputs from a single or activity-specific central rhythm/pattern generator (Tierney, 1996). Thus, it would not be surprising that, like breathing, fictive forms of these activities also are present as motor output from both an isolated medulla and a more intact brainstem-spinal cord preparation. The occurrence of these other forms of discharge, relative to the number of normal fictive breaths, was substantially greater from the isolated medulla than from the more intact brainstem-spinal cord preparation (Fig. 6F). Thus it appears as if central descending influences are not only involved in the production of breathing episodes but also influence the shaping of individual fictive breaths. By analogy with mammalian systems, it is possible that the frog brainstem (medulla) contains eupneic, gasping and apneustic centers (St. John, 1996). If this is indeed the case, it is possible that once the medulla is in isolation, the normal relationship between these centers is disrupted such that the aberrant forms of breathing (gasping- and apneustic-like) begin to occur to a greater degree in relation to the normal fictive breaths Transections between the trigeminal and agus ner es In the developing chick embryo (stage 24), transections between different rhombomers leads to a

14 210 S.G. Reid et al. / Respiration Physiology 120 (2000) different respiratory rhythm recorded from the trigeminal, facial, glossopharyngael and hypoglossal nerves (Fortin et al., 1995; Champagnat and Fortin, 1997). This suggests that each segment (or rhombomer) contains the necessary rhythm generating circuitry to produce a form of respiratory rhythm. In these studies (Fortin et al., 1995), the facial nerve retained the same rhythm before sectioning suggesting that a facial coactivator may play a dominant role in respiratory rhythm generation (Champagnat and Fortin, 1997). Additionally, there was a rostrocaudal trend in which the more rostral levels developed a faster rhythm than the more caudal regions. In the current study, the presence of asynchronous discharge from the Vth and Xth nerve roots was only observed from one preparation despite numerous attempts to repeat this observation. As such, any suggestion of a segmental arrangement of respiratory groups in the adult bullfrog brainstem can only be considered speculative. However, it is possible to envision a scenario where caudal segmental generators produce the other forms of neural discharge but higher centers may over-ride their output under normal circumstances. As illustrated by the isolated medulla, if these higher centers are removed by transection there is a greater occurrence of these other forms of discharge. It would appear that multiple elements of the respiratory control system, including tegmental and medullary sites, play a role in shaping the burst pattern of motor output associated with each breath and that slower rhythms of longer burst duration are generated by more caudal hindbrain sites. Clearly further experiments are required to investigate the possibility of segmental respiratory groups or rhythm generators in amphibians. Acknowledgements This study was supported by Natural Sciences and Engineering Research Council (NSERC) of Canada operating and equipment grants to WKM. SGR was the recipient of an NSERC postdoctoral fellowship. References Champagnat, J., Fortin, G., Primordial respiratory-like rhythm generation in the vertebrate embryo. Trends Neurosci. 20, Feldman, J.L., Smith, J.C., Ellenberger, H.H., Connelly, C.A., Liu, G., Greer, J.J., Lindsay, A.D., Otto, M.R., Neurogenesis of respiratory rhythm and pattern: emerging concepts. Am. J. Physiol. 259, R879 R886. Fortin, G., Kato, F., Lumsden, A., Champagnat, J., Rhythm generation in the segmented hindbrain of chick embryos. J. Physiol. Lond. 486, Galante, R.J., Kubin, L., Fishman, A.P., Pack, A.I., Role of chloride-mediated inhibition in respiratory rhythmogenesis in an in vitro brainstem of tadpole, Rana catesbeiana. J. Physiol. Lond. 492, Kimura, N., Perry, S.F., Remmers, J.E., Strychnine eliminates reciprocation and augmentation of respiratory bursts of the in vitro frog brainstem. Neurosci. Lett. 225, Kinkead, R., Milsom, W.K., Chemoreceptors and control of episodic breathing in the bullfrog (Rana catesbeiana). Respir. Physiol. 95, Kinkead, R., Filmyer, W.G., Mitchell, G.S., Milsom, W.K., Vagal input enhances responsiveness of respiratory discharge to central changes in ph/co 2 in bullfrogs. J. Appl. Physiol. 77, Kinkead, R., Milsom, W.K., CO 2 -sensitive olfactory and pulmonary receptor modulation of episodic breathing in bullfrogs. Am. J. Physiol. 270, R134 R144. Kinkead, R., Episodic breathing in frogs: converging hypotheses on neural control of respiration in air breathing vertebrates. Am. Zool. 37, Kinkead, R., Milsom, W.K., Role of pulmonary stretch receptor feedback in control of episodic breathing in the bullfrog. Am. J. Physiol. 272, R497 R508. Kinkead, R., Harris, M.B., Milsom, W.K., The role of the nucleus isthmi in respiratory pattern formation in bullfrogs. J. Exp. Biol. 200, Kogo, N., Remmers, J.E., Neural organisation of the ventilatory activity of the frog, Rana catesbeiana. II. J. Neurobiol. 25, Kogo, N., Perry, S.F., Remmers, J.E., Neural organisation of the ventilatory activity of the frog, Rana catesbeiana. I. J. Neurobiol. 25, Liao, G.S., Kubin, L., Galante, R.J., Fishman, A.P., Pack, A.I., Respiratory activity in the facial nucleus in an in vitro brainstem of the tadpole, Rana catesbeiana. J. Physiol. Lond. 492, McLean, H.A., Kimura, N., Kogo, N., Perry, S.F., Remmers, J.E., 1995a. Fictive respiratory rhythm in the isolated brainstem of frogs. J. Comp. Physiol. A 176, McLean, H.A., Perry, S.F., Remmers, J.E., 1995b. Two regions in the isolated brainstem of the frog that modulate respiratory-related activity. J. Comp. Physiol. A 177,

15 S.G. Reid et al. / Respiration Physiology 120 (2000) Milsom, W.K., Intermittent breathing in vertebrates. Annu. Rev. Physiol. 53, Milsom, W.K., Regulation of respiration in lower vertebrates: role of CO 2 /ph chemoreceptors. In: Heisler, N. (Ed.), Advances in Comparative and Environmental Physiology, Mechanisms of Systemic Regulation: Respiration and Circulation, vol. 1. Springer-Verlag, Berlin, pp Milsom, W.K., Harris, M.B., Reid, S.G., Do descending influences alternate to produce episodic breathing? Respir. Physiol. 110, Milsom, W.K., Reid, S.G., Meier, J.T., Kinkead, R., Central respiratory pattern generation in the bullfrog, Rana catesbeiana. Comp. Biochem. Physiol. A 124 (3), Oka, K., The influence of the transection of the brain upon the respiratory movement of the frog. J. Physiol. Soc. Jpn. 20, Perry, S.F., McLean, H.A., Kogo, N., Kimura, N., Kawasaki, H., Sakurai, M., Kabotyanski, E.A., Remmers, J.E., The frog brainstem preparation as a model for studying the central control of breathing in tetrapods. Braz. J. Med. Biol. Res. 28, 1 8. Reid, S.G., Milsom, W.K., Respiratory pattern formation in the isolated bullfrog (Rana catesbeiana) brainstemspinal cord. Respir. Physiol. 114, Sakakibara, Y., The pattern of respiratory nerve activity in the bullfrog. Jpn. J. Physiol. 34, Sakamoto, T., Nonaka, S., Katada, A., Control of respiratory muscles during speech and vocalisation. In: Miller, A.D., Bianchi, A.L., Bishop, B.P. (Eds.), Neural Control of the Respiratory Muscles. CRC Press, Boca Raton, FL, pp St. John, W.M., Medullary regions for neurogenesis of gasping: noeud vital or noeuds vitals? J. Appl. Physiol. 81, Thompson, K.J., Organization of inputs to motoneurons during fictive respiration in the isolated lamprey brain. J. Comp. Physiol. A 157, Tierney, A.J., Evolutionary implications of neural circuit structure and function. Behav. Proc. 35, Torgerson, C.S., Gdovin, M.J., Remmers, J.E., Ontogeny of central chemoreception during fictive gill and lung ventilation in an in vitro brainstem preparation of Rana catesbeiana. J. Exp. Biol. 200, West, N.H., Topor, Z.L., van Vliet, B.N., Hypoxemic threshold for lung ventilation in the toad. Respir. Physiol. 70,