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CO2 chemosensitivity in the cane toad
Respiratory Physiology & Neurobiology 156 (2007) 266–275
Chronic hypoxia attenuates central respiratory-related pH/CO2 chemosensitivity in the cane toad Jessica McAneney, Stephen G. Reid ∗ Centre for the Neurobiology of Stress, Department of Life Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4 Canada Accepted 19 October 2006
Abstract This study examined the effects of chronic hypoxia (CH) and mid-brain transection on central respiratory-related pH/CO2 chemosensitivity in cane toads (Bufo marinus). Toads were exposed to 10 days of CH (10% O2 ) following which in vitro brainstem-spinal cord preparations, with the mid-brain attached, were used to examine central pH/CO2 chemosensitivity. A reduction in artificial cerebral spinal fluid (aCSF) pH increased fictive breathing frequency (fR) and total fictive ventilation. CH reduced fictive fR and total fictive ventilation, compared to controls. Mid-brain transection caused an increase in fictive fR, at the lower aCSF pH levels, in both control and CH preparations. In the CH preparations, mid-brain transection restored fictive breathing to control levels. In both groups, mid-brain transection eliminated fictive breath clustering. The data indicate that CH attenuates central pH/CO2 -sensitive fictive breathing but a mid-brain transection in the middle of the optic lobes abolishes this attenuation. The results suggest that CH induces inhibition of central pH/CO2 chemoreceptor function via descending inputs from the mid-brain region. © 2006 Elsevier B.V. All rights reserved. Keywords: Amphibian; Control of breathing; Hypercapnic ventilatory response; In vitro brainstem-spinal cord preparation; Mid-brain transection
1. Introduction Exposure to chronic hypoxia in mammals results in ventilatory acclimatization to hypoxia, which is manifest as an increase in resting ventilation, a decrease in arterial pCO2 and, usually, an augmentation of the acute hypoxic ventilatory response (Dempsey and Forster, 1982; Bisgard and Neubauer, 1995; Powell et al., 1998, 2000; Bisgard, 2000). Anuran amphibians (frogs and toads) are, in general, more hypoxia-tolerant than mammals (Storey and Storey, 1986; Pinder et al., 1992; Boutilier, 2001). A recent study (McAneney et al., 2006) on cane toads (Bufo marinus) demonstrated that exposure to chronic hypoxia (10 days; 10% O2 ) did not alter resting ventilation but did cause an attenuation of the acute hypoxic ventilatory response when toads were exposed to 5% O2 . Gheshmy et al. (2006) recently demonstrated that exposure of cane toads to another long term respiratory challenge (chronic hypercapnia; 10 days; 3.5% CO2 ) produced an augmentation of central pH/CO2 chemoreceptor function (i.e., pH/CO2 -sensitive fictive
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1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.10.002
breathing) measured using in vitro brainstem-spinal cord preparations. Anuran amphibians have a host of respiratory control systems that, for the most part, are similar to those found in mammals (Milsom, 1991; Kinkead, 1997). These include central pH/CO2 sensitive chemoreceptors located on the ventrolateral surface of the medulla (Smatresk and Smits, 1991; Milsom, 2002; Taylor et al., 2003a) and peripheral O2 /CO2 -sensitive chemoreceptors located in the carotid labyrinth (West et al., 1987). The respiratory responses to peripheral and central chemoreceptor stimulation are interactive with the level of O2 influencing the acute hypercapnic ventilatory response and the CO2 level influencing the acute hypoxic ventilatory response (West et al., 1987; Smatresk and Smits, 1991; Reid, 2006). CO2 -sensitive olfactory chemoreceptors located in the nasal epithelium inhibit breathing when stimulated with high levels of CO2 (Kinkead and Milsom, 1996; Coates, 2001). CO2 -sensitive pulmonary stretch receptors (PSR) located in the walls of the lungs (Milsom and Jones, 1977; Kuhlman and Fedde, 1979) can stimulate or inhibit breathing depending upon whether the PSR feedback is phasic or tonic and the degree of lung inflation. Adult anurans are discontinuous breathers with the primary breathing pattern consisting of single breaths or the occasional
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doublet/triplet under low levels of respiratory drive. When respiratory drive is elevated (i.e., during acute hypoxia or hypercapnia) breathing becomes episodic in which clusters of breaths are separated by periods of apnea. A number of studies have demonstrated that fluctuations in blood-gas levels are not necessary for episodic breathing in these animals (West et al., 1987; Kinkead et al., 1994, 1997; Milsom et al., 1999; Reid et al., 2000). Reid et al. (2000) demonstrated, using isolated bullfrog brainstem-spinal cord preparations with the mid-brain attached, that a mid-brain transection abolished episodic fictive breathing and produced a pattern of relatively continuous fictive breathing. Based on the observations that (1) chronic hypoxia abolished the acute hypoxic ventilatory response in vivo (McAneney et al., 2006), (2) chronic hypercapnia increased the magnitude and sensitivity of central pH/CO2 -sensitive fictive breathing in vitro (Gheshmy et al., 2006) and (3) the respiratory responses to O2 and CO2 chemoreceptor stimulation are interactive (West et al., 1987; Reid, 2006) we hypothesized that exposure to chronic hypoxia would lead to an attenuation of central pH/CO2 sensitive breathing (i.e., attenuate the changes in breathing normally associated with changes in cerebral spinal fluid pH/CO2 ). While, some anurans encounter chronic hypoxia during the winter months in underground burrows (terrestrial species) or underwater (aquatic species) (Pinder et al., 1992), the cane toad (B. marinus) endures long periods of apnoea and arterial hypoxia during estivation (Coelho and Smatresk, 2003). Coelho and Smatresk (2003) report a reduction in arterial PO2 from 90 to 15–20 mm Hg following 1 h of apnea. Given this, we hypothesized that respiratory control systems would be altered in order to reduce lung ventilation during chronically hypoxic conditions when enhanced lung ventilation is unlikely to be advantageous either due to environmental constraints or reduced metabolic rate. Furthermore, given that mid-brain inputs are involved in the regulation of breath clustering, we hypothesized that input from the mid-brain may also mediate any chronic hypoxia-induced decrease in central pH/CO2 -sensitive breathing. To address these hypotheses, cane toads were made chronically hypoxic (10% O2 ) for 10 days. In vitro brainstem-spinal cord preparations (with the mid-brain attached) were used to assess the pH/CO2 -sensitivity of fictive breathing before and after a mid-brain transection. In these preparations, motor output from a respiratory nerve was used as the index of breathing (fictive breathing). 2. Materials and methods
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mittee and conform to the guidelines established by the Canadian Council for Animal Care. 2.2. Exposure to chronic hypoxia Toads were placed, for a 10-day period, into a Plexiglas chamber (35 cm × 25 cm × 10 cm) within which the inspired O2 level was maintained at 10% using a Pro-Ox 110 control unit (Biospherix; Redfield, NY) (McAneney et al., 2006). An O2 electrode within the chamber monitored the level of O2 . When the O2 level rose above 10%, the Pro-Ox delivered a small amount of N2 to lower the level back to 10%. Routine measurements of CO2 (CD-3A CO2 analyzer, AEI Technologies; Pittsburgh, PA) confirmed that inspired CO2 levels remained at approximately 0.03%. The chamber was maintained at room temperature and exposed to a 12 h:12 h cycle of light and darkness. McAneney et al. (2006) demonstrated that the chronic hypoxia-induced blunting of the in vivo acute hypoxic ventilatory response occurred after 3 days of chronic hypoxia and remained for the entire 10-day experimental period of hypoxia. Given this, we were confident that, in the current study, the 10-day period of chronic hypoxia was of sufficient duration to observe any changes in central chemosensitivity that may arise due to exposure to chronic hypoxia. A period of 9–10 days of chronic hypoxia is also the general time frame required to induce ventilatory acclimatization to hypoxia in mammals (rats; Aaron and Powell, 1993; Reid and Powell, 2005). 2.3. The in vitro brainstem-spinal cord preparation Toads were anaesthetized by emersion in a solution of 3aminobenzoic acid ethyl ester (MS222, 1 g/L; Sigma) buffered to pH 7.0 with sodium bicarbonate (Reid and Milsom, 1998; Reid et al., 2000; Gheshmy et al., 2006). Animals were kept in the anaesthetic until the eye-blink and toe-pinch reflexes were eliminated. Using a Dremmel Tool, an incision was made in the skull rostral to the optic lobes. The cranial case was removed with rongeurs/bone shears and placed onto a Sylgard-coated dissecting dish. The brain was exposed and superfusion with ice-cold oxygenated artificial cerebral spinal fluid (aCSF) was initiated; the rostral forebrain was then removed (see Fig. 1). The transection to remove the rostral forebrain was performed in the middle of the cerebral hemispheres. Based on the Hoffmann (1973) atlas of the toad brain, this transection site would correspond to
2.1. Experimental animals Cane toads (B. marinus) (N = 36; 200–300 g) were obtained from a commercial supplier (Boreal Scientific, St. Catharine’s, Ontario) and maintained in a fibreglass tank at room temperature (20–22 ◦ C). Toads were supplied with both terrestrial and aquatic habitats consisting of moist peat moss and trays of dechlorinated water, respectively. The photoperiod was maintained at 12 h light and 12 h dark. Animals were fed live crickets once per week. Holding conditions and experimental protocols were approved by the University of Toronto Animal Care Com-
Fig. 1. A lateral view of the toad brain illustrating the level of transections (dotted lines) performed in this study. The rostral- and caudal-most transections were used to generate the in vitro preparation. The mid-brain transection through the optic lobes was the experimental manipulation. NI indicates the location of the nucleus isthmi. V, trigeminal nerve root; VII, facial nerve root; IX, glossopharyngeal nerve root; X, vagus nerve root.
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approximately 1.5 mm anterior to the zero mark (defined as the vertex of the angle formed at the point where the cerebral hemispheres become separate or begin to diverge). Internally this zero point (Hoffmann, 1973) corresponds to the interventricular foramen (foramen Monroi). The forebrain transection goes through the primordial hippocampus, the primordial general pallium, the primordial piriform cortex, the lateral septal nucleus, the striatum, the medial forebrain bundle, the medial septal nucleus and the lateral ventricle. The remaining brain tissue was continually supplied with the aCSF (in mmol/L; NaCl, 103; KCl, 4.05; MgCl2 , 1.38; glucose, 10; NaHCO3 , 29.2; CaCl2 , 2.45; Sigma Chemicals; pH 7.8, i.e., normocapnic CSF; Taylor et al., 2003a,b; Gheshmy et al., 2006). Cranial nerves were cut close to their exit from the skull and the spinal cord was severed at the level of the third spinal nerve (see Fig. 1). The preparation was transferred from the brain case and immobilized with insect pins in a Sylgard-coated dissecting dish continually superfused with oxygenated aCSF. The dura matter surrounding the brain was removed in order to free the cranial nerve roots and the nerve tips were cut to provide a clean surface for recording. The preparation was then pinned, ventral side up, onto a fine stainless steel mesh within a superfused recording chamber. The mesh divided the chamber into upper and lower compartments which ensured simultaneous superfusion of both surfaces of the preparation (Kinkead et al., 1994; McLean et al., 1995a,b; Gheshmy et al., 2006). The preparation was continuously superfused with oxygenated aCSF, at a rate of 10 mL/min, using peristaltic pumps that delivered and removed the aCSF from the chamber. The aCSF was re-cycled (Gheshmy et al., 2006). The preparations were maintained at pH 7.8 and room temperature for 60 min before commencing the experiment. Suction electrodes of various diameters were made from thinwalled capillary glass (1 mm diameter) pulled to a fine tip using a vertical pipette puller (Kopf model 720; Tujunga, CA). The tips were polished using a grinding stone and flame to provide a smooth surface. Using a micro-manipulator, an appropriately sized suction electrode was positioned near the end of the vagus nerve root and the nerve was carefully aspirated into the electrode such that a tight seal was obtained between the nerve and the electrode. In all preparations, recordings were taken of whole nerve discharge from the vagus nerve. In the intact animal, a branch of the vagus nerve innervates the glottis which opens and closes with each breath while another branch innervates buccal pump muscles (Sakakibara, 1984a,b). Since these preparations are devoid of any afferent input and breathing is an inherently rhythmic process generated in the brainstem, all rhythmic (Reid and Milsom, 1998) activity (in vitro motor output) recorded from the vagus nerve was assumed to represent motor output to the respiratory muscles and is therefore an index of breathing termed fictive breathing. This is a well-established preparation for the study of amphibian central respiratory control (Kinkead et al., 1994; McLean et al., 1995a,b; Reid and Milsom, 1998; Reid et al., 2000; Morales and Hedrick, 2002; Wilson et al., 2002; Taylor et al., 2003a,b; Gheshmy et al., 2006). Nerve activity from the suction electrode was amplified (10×) and filtered (30 Hz, high pass; 3 kHz, low pass) using
a DAM50 ac amplifier (World Precision Instruments; Sarasota, FL). The output from the DAM50 was sent to a second ac amplifier (ISO8A, WPI) and amplified a further 100×. The amplified nerve signal from the ISO8A was sent to a moving averager (CWE MA821/RSP; CWE Inc., Ardmore, PA) for integration (time constant = 200 ms) and to an audio monitor (AM Systems Model 3300; Carlsborg, WA). The amplified/filtered nerve signal and integrated signal were monitored and stored using a data acquisition system (Biopac Systems, MP150; Goleta, CA). The sampling rate of analogue to digital conversion was 2000 Hz. Gassing the aCSF with varying levels of CO2 (0–5%; balance O2 ) altered the aCSF pH. The levels of CO2 and O2 , gassing the aCSF, were set using digital mass flow controllers (Smart-Trak 100, Sierra Instruments; Monterey, CA). The pH level of the aCSF was monitored using a pH electrode (VWR) placed within the aCSF reservoir. 2.4. Experimental protocol Following the 1-h stabilization period and the observation of stable levels of neural discharge (fictive breathing), each preparation was exposed to varying levels of aCSF pH (7.6, hypercapnic; 7.8, normocapnic; 8.0, hypocapnic; random order exposure). This pH range approximates that used in previous studies on amphibian brainstem-spinal cord preparations (Kinkead et al., 1994; McLean et al., 1995a,b; Gheshmy et al., 2006). All experiments were performed at room temperature (approximately 22 ◦ C). This is within the temperature range (15–25 ◦ C) reported by Morales and Hedrick (2002) in which fictive breathing is consistently active from in vitro adult bullfrog brainstem-spinal cord preparations. Each pH change was achieved over a period of 15 min. Preparations were allowed to acclimatize to each new pH level for a further 15 min before fictive breathing was monitored for an additional 15 min data analysis period. Following the series of pH changes, the mid-brain was transected slightly caudal to the middle of the optic lobes (Fig. 1). Based on the Hoffmann (1973) atlas of the toad brain, this transection site would correspond to 4 mm posterior to the zero mark (see above). Our mid-brain transection goes through the optic tectum, torus semicircularis, tectum ventricle and tegmentum (see Fig. 4 from Hoffmann, 1973). The transection is immediately rostral to the nucleus isthmi (see Figs. 29 and 30 from Hoffmann, 1973). Following a 30 min stabilization period, the pH changes (7.6, 7.8 and 8.0) were repeated (again in random order). The protocol was performed on brainstem-spinal cord preparations taken from control (N = 18) and chronically hypoxic (N = 18) toads. 2.5. Data and statistical analysis The final 15 min of data at each pH level were analyzed to determine mean values for fictive breathing frequency, breaths/episode, episodes/minute, breath duration (s) and integrated fictive breath area (an index of breath volume). In this study we were recording from the root of the whole vagus nerve. Respiratory-related motor activity in this nerve, in vivo,
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controls the opening and closing of the glottis (via the laryngeal branch of the vagus) as well as the buccal respiratory pump muscles (via the pharyngeal posterior superior nerve) (Sakakibara, 1984a). Sakakibara (1984b) demonstrated that an increase in buccal pressure was positively correlated with integrated trigeminal nerve activity (r = 0.97). The trigeminal nerve (mandibular branch) innervates buccal pump muscles. However, integrated nerve activity from the pharyngeal posterior superior nerve (vagal branch) is also positively correlated with integrated trigeminal nerve activity (r = 0.95; Sakakibara, 1984a,b). This suggests that nerve activity in the pharyngeal posterior superior nerve (a branch of the vagus) should also be correlated with buccal pressure changes (or breath volume). Furthermore, given that the laryngeal branch of the vagus nerve controls the opening and closing of the glottis, it is likely that a larger volume breath requires that the glottis remain open for a longer period of time compared to a smaller volume breath. Given this, it is likely that a larger breath would be associated with a larger amount (i.e., integrated area) of vagal activity. Based on the above observations and assumptions, we believe that it is reasonable to assume that the integrated area of vagal activity is a valid index of breath volume. Furthermore, given that the integrated area takes into account both fictive breath amplitude and duration, we believe that integrated area is a more appropriate measure of breath volume than the amplitude of the fictive breaths. The total fictive ventilation index was calculated as the product of fictive breathing frequency and the integrated breath area. Fictive breaths in a given episode were defined as occurring within 2 s of each other according to general practices in the literature (Kinkead et al., 1994, 1997; Reid et al., 2000; Gheshmy et al., 2006). All statistical analyses were performed using commercial software (SigmaStat 3.0, SPSS). Data are reported as the mean ± one standard error of the mean (SEM). For each condition (normoxic pre-transection, normoxic post-transection, chronically hypoxic pre-transection and chronically hypoxic post-transection) a one-way repeated measures analysis of variance (RM-ANOVA), followed by a Student–Newman–Keuls (SNK) multiple comparison test (MCT), was used to compare the effects of pH on fictive breathing. The effects of chronic hypoxia, mid-brain transection and pH were analysed using a three-way ANOVA followed by a SNK MCT. The limit of significance was 5% (p < 0.05).
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Fig. 2. Fictive breathing (vagal motor output) recorded from isolated brainstemspinal cord preparations (at pH 7.6) taken from (A) a normoxic control toad (pre-mid-brain transection), (B) the normoxic control toad (post-mid-brain transection), (C) a chronically hypoxic toad (pre-mid-brain transection), and (D) the chronically hypoxic toad (post-mid-brain transection). In all cases the upper trace represents the raw electroneurogram (eng X) recorded from the vagus nerve root while the lower trace ( eng X ) represents the integrated trace.
tions in which (in the intact animal) the floor of the mouth rises and falls with the glottis closed. 3.1. Fictive breathing frequency
3. Results Fig. 2 illustrates fictive breathing recorded at an aCSF pH level of 7.6 in brainstem-spinal cord preparations taken from control and chronically hypoxic animals both prior to and following the mid-brain transection in the middle of the optic lobes. Chronic hypoxia reduced fictive breathing frequency prior to mid-brain transection (compare Fig. 2A and C). Mid-brain transection converted discontinuous (episodic) fictive breathing to a pattern of relatively continuous fictive breathing (compare Fig. 2A with B and C with D). The small bursts of activity in Fig. 2B are likely indicative of non-ventilatory buccal oscilla-
In the normoxic control preparations prior to mid-brain transection, fictive breathing frequency increased as aCSF pH was lowered from 8.0 to 7.6 (Fig. 3A; p < 0.001). Mid-brain transection augmented this increase at pH 7.6 (Fig 3A; p = 0.044). In the preparations taken from chronically hypoxic toads, there was no increase in fictive breathing frequency as aCSF pH was lowered prior to mid-brain transection (Fig. 3B; p = 0.149). Midbrain transection caused a significant increase in fictive breathing frequency in the chronically hypoxic preparations (Fig. 3B; p = 0.002) as aCSF pH was lowered from 8.0 to 7.8 and 7.6. The fictive breathing frequency values in the pre-transection chron-
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per episode. Prior to mid-brain transection, the number of fictive episodes per minute increased, slightly but significantly, as aCSF pH was lowered in the control (Fig. 4A; p < 0.001) but not the chronically hypoxic (Fig. 4B; p = 0.087) preparations. Midbrain transection resulted in a significant increase in the number of fictive episodes per minute in both the control (Fig. 4A; pH 7.6; p < 0.001) and chronically hypoxic (Fig. 4B; pH 7.6–7.8; p < 0.001) preparations. Prior to transection, the number of fictive episodes per minute in the chronically hypoxic group was reduced, albeit not significantly (p = 0.056), compared to the control group. The post-transection fictive episodes per minute values in the control and chronically hypoxic groups were not different (p = 0.496). In the control (Fig. 4C) and chronically hypoxic (Fig. 4D) groups, changes in aCSF pH had no effect on the number of fictive breaths per episode prior to (control, p = 0.333; chronic hypoxia, p = 0.565) or following (control, p = 0.999; chronic hypoxia, p = 0.820) mid-brain transection. In both groups, midbrain transection caused a significant reduction in the number of fictive breaths per episode with the value falling to approximately one in all cases (controls and chronically hypoxic, p < 0.001). Chronic hypoxia had no effect on the number of fictive breaths per episode either before (p = 0.310) or after (p = 0.137) mid-brain transection. 3.3. Integrated fictive breath area, duration and the total fictive ventilation index
Fig. 3. Fictive breathing frequency (fictive breaths per minute) recorded from isolated brainstem-spinal cord preparations taken from (A) normoxic control toads (n = 18) and (B) chronically hypoxic toads (n = 18). Open circles (A) and open squares (B) represent the values prior to mid-brain transection. Closed circles (A) and closed squares (B) represent the values following mid-brain transection. Lower case letters (a and b) indicate differences, within any given group, between the different aCSF pH levels. A number sign (#) indicates a significant difference between the pre- and post-transection values. A plus sign (+) indicates a significant difference between the normoxic control values in panel A and the corresponding chronically hypoxic value in panel B. The data are reported as the mean ± S.E.M. A partial data set of the pre-transection fictive breathing frequency data in both panels (this figure only) has been briefly reported in a recent review article (Reid, 2006).
ically hypoxic preparations were reduced (Fig. 3B) at pH 7.6 (p = 0.007) and 7.8 (p = 0.087), compared to the values in the pre-transection control preparations shown in Fig. 3A. The values in the control and chronically hypoxic groups were the same post-transection (p = 0.496). 3.2. Fictive episodes per minute and fictive breaths per episode Fictive breathing frequency is the product of the number of fictive episodes per minute and the number of fictive breaths
With the exception of a post-transection increase in fictive breath duration at pH 7.8 in the control group (Fig. 5C; p = 0.004), changes in aCSF pH, mid-brain transection and chronic hypoxia had no statistically significant effect (p > 0.05) on either the integrated area of the fictive breaths (Fig. 5A and B) or fictive breath duration (Fig. 5C and D). The total fictive ventilation index (fictive breathing frequency × integrated fictive breath area) increased in the control (Fig. 6A) and chronically hypoxic (Fig. 6B) groups, as aCSF pH was lowered, both prior to (control, p = 0.006; chronic hypoxia, p = 0.036) and following (control, p = 0.010; chronic hypoxia, p = 0.047) mid-brain transection. There was no effect of midbrain transection, on total fictive ventilation, in the control preparations (p = 0.819). Following mid-brain transection in the chronically hypoxic group there was a significant increase in the total fictive ventilation index at aCSF pH levels of 7.8 and 7.6 (Fig. 6B; p = 0.002). Exposure to chronic hypoxia reduced the pre-transection levels of the total fictive ventilation index (compare the pre-transection values in Fig. 6A and Fig. 6B; p = 0.007) at aCSF pH values of 7.8 and 7.6. The total fictive ventilation index values in the control and chronically hypoxic groups were the same post-transection (p = 0.730). 4. Discussion One of the major findings of this study was that chronic hypoxia abolished the increase in fictive breathing (frequency and total ventilation index) associated with a decrease in aCSF pH. The results of this study are consistent with those of
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Fig. 4. The number of fictive breathing episodes per minute (A and B) and fictive breaths per episode (C and D) recorded from isolated brainstem-spinal cord preparations taken from normoxic control toads (A and C) and chronically hypoxic toads (B and D). Symbols are the same as described for Fig. 3.
McAneney et al. (2006) in that chronic hypoxia has reduced an acute ventilatory chemoreflex (in this case central pH/CO2 sensitive fictive breathing). In other words, chronic hypoxia has reduced/blunted the in vitro equivalent of the central pH/CO2 chemoreceptors’ contribution to the acute hypercapnic ventilatory response. There was no effect of chronic hypoxia on the integrated area of the fictive breaths; all of the modulatory effects of chronic hypoxia influenced fictive breathing frequency. In vivo, breathing room air (resting conditions) and 4.0–4.5% CO2 (acute hypercapnia) leads to arterial pH levels of approximately 7.8–8.0 and 7.6, respectively (Kinkead and Milsom, 1996). The chronic hypoxia-induced blunting of fictive breathing (frequency and total ventilation) was pH-sensitive. In both cases, the chronically hypoxic values were reduced at aCSF pH levels of 7.8 and 7.6 but not at pH 8.0. These data suggest that chronic hypoxia does not alter breathing under hypocapnic conditions (i.e., fictive breathing at pH 8.0) but does alter fictive breathing during the in vitro equivalent of normocapnia (aCSF pH 7.8) and during an elevation in the CO2 -mediated drive to breathe (i.e., aCSF pH level of 7.6). The observation that the effects of chronic hypoxia are pH sensitive is also consistent with that of McAneney et al. (2006) who demonstrated that chronic
hypoxia abolished the in vivo acute hypoxic ventilatory response but did not alter resting ventilation. The second major finding of this study was that the chronic hypoxia-induced abolition (blunting) of pH/CO2 sensitive fictive breathing was eliminated by mid-brain transection. In other words, mid-brain transection restored pH/CO2 -chemosensitivity. The mid-brain transection also eliminated the clustering of fictive breaths (the number of fictive breaths/episode was approximately one under all conditions following the transection) and caused an increase in fictive breathing frequency. This is consistent with Reid et al. (2000) who demonstrated, using in vitro brainstem-spinal cord preparations from bullfrogs (Rana catesbeiana), that descending inputs, from the mid-brain to the medulla, were involved in the clustering of breaths into episodes. The post-transection increase in fictive breathing frequency indicates that descending inputs from the mid-brain normally inhibit or reduce breathing frequency (and the overall level of breathing), under both normocapnic and hypercapnic conditions. The chronic hypoxia-induced blunting of pH/CO2 -senstive fictive breathing suggests that these mid-brain inhibitory influences exert a greater effect on fictive breathing following exposure to chronic hypoxia, compared to control preparations. The implication of this observation
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Fig. 5. Integrated area of the fictive breaths (V s; A and B) and fictive breath duration (s; C and D) recorded from isolated brainstem-spinal cord preparations taken from normoxic control toads (A and C) and chronically hypoxic toads (B and D). Symbols are the same as described for Fig. 3.
is that chronic hypoxia has enhanced the inhibitory nature of these descending mid-brain influences in order to reduce fictive breathing frequency. Furthermore, the restoration of pH sensitivity following the mid-brain transection suggests that the chronic hypoxia-induced decrease in sensitivity was not the result of metabolic depression. Currently the nature of respiratory-related mid-brain input to the medulla is not known. Indeed the anatomical nature of adult amphibian respiratory control centers has not been investigated other than, for example, studies on respiratory rhythmogenesis (Wilson et al., 2002; Vasilakos et al., 2005), breath clustering (Reid et al., 2000), pH chemosensitivity (Taylor et al., 2003a,b; Noronha-de-Souza et al., 2006), the nucleus isthmi (Kinkead et al., 1997; see review by Gargaglioni and Branco, 2004), respiratory-related neurons (Kogo and Remmers, 1994; McLean and Remmers, 1997) and glutamate/GABA (McLean et al., 1995b). The nucleus isthmi, located slightly caudal to our transection (see Fig. 1), has also been implicated in the control of amphibian breathing (Kinkead et al., 1997; Gargaglioni and Branco, 2004; Milsom et al., 2004) although its primary role appears to be in vision (Gruberg and Udin, 1978). A recent study (Noronha-
de-Souza et al., 2006) suggests that the locus coeruleus (LC; which lies ventral and slightly caudal to the nucleus isthmi) is also involved in amphibian breathing. In that study, lesion of the LC reduced the acute hypercapnic ventilatory response while focal acidification of the LC increased breathing. The K¨ollikerfuse which lies close to the nucleus isthmi and LC (Adli et al., 1999) may also be involved in respiratory control in these animals. Our results indicate that the transection through the optic lobes caused continuous breathing but increased breathing frequency and enhanced CO2 chemosensitivity. It is not clear whether the transection in the current study removed the influence of a specific episode-generating center or if it disrupted a larger network responsible for episode generation. The enhanced breathing frequency and CO2 chemosensitivity suggest that the effects of transection on the breathing pattern (i.e., a shift from episodic to continuous fictive breathing) was not simply the result of altered respiratory drive, or the central integration of respiratory drive, as enhanced drive leads to episodic breathing in these animals (Milsom, 1991). This observation suggests that the transection disrupted a region involved in drive-independent breath clustering.
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Fig. 6. Total fictive ventilation index (V s/min) calculated by multiplying the fictive breathing frequency by the integrated area of the fictive breaths recorded from isolated brainstem-spinal cord preparations taken from (A) normoxic control toads and (B) chronically hypoxic toads. Symbols are the same as described for Fig. 3.
Milsom et al. (1997) present evidence from studies on bullfrogs, hibernating ground squirrels and sleeping elephant seals in support of the notion that, during episodic breathing, alternating positive (excitatory) and negative (inhibitory) descending influences, to the medullary respiratory centers, serve to produce the fast breathing observed during episodes and the apneic periods between episodes, respectively. Further studies on anurans are required to determine the nature of a putative inhibitory (and possibly episode-generating) center, rostral to the nucleus isthmi, as well as any effects of chronic hypoxia on the function of this putative center. However, the results of this study do suggest that chronic hypoxia augments inhibitory inputs from the mid-brain such that breathing frequency is reduced. While the effects of chronic hypoxia on ventilatory chemoreflexes have been well studied in mammals, only a few studies have focused on respiratory plasticity following chronic hypoxia
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in amphibians (Pinder and Burggren, 1983; McAneney et al., 2006) and other lower vertebrates (Burleson et al., 2002; Vulesevic et al., 2006). The results of the current study, in conjunction with those of McAneney et al. (2006), indicate that chronic hypoxia blunts acute ventilatory chemoreflexes rather than augmenting them, as is the general case with mammals (Powell et al., 1998, 2000). This mammalian versus amphibian difference may reflect the different conditions under which amphibians and mammals are likely to encounter chronic hypoxia. Aside from fossorial species, mammals are most likely to encounter chronically hypoxic conditions when they ascend to high altitudes (Bisgard, 2000). Under these conditions, increasing breathing is obviously advantageous. Many terrestrial anuran amphibians, in temperate climates, spend the winter months in underground burrows which are likely to be hypoxic and have a limited supply of air (other than diffusion through the soil and snow) (Breckenridge and Tester, 1961; Pinder et al., 1992). Under these conditions in which air is limited and, due to cold temperatures, metabolic rate is lowered (Boutilier, 2001), the need to ventilate the lungs is likely to be reduced. Given this, it is not surprising that chronic hypoxia leads to a reduction in the ventilatory responses to acute hypoxia in vivo (McAneney et al., 2006) and the in vitro equivalent of acute hypercapnia (this study). However, it is curious that resting breathing was not affected in either case. It is possible that a combination of chronic hypoxia and a reduced ambient temperature may also lead to reductions in resting ventilation that would be expected to occur during winter. The down-regulation of breathing during chronic hypoxia may also assist in minimising water loss. Pinder and Burggren (1983) demonstrated, in adult bullfrogs (R. catesbeiana) that exposure to aerial and aquatic hypoxia (75 mm Hg) caused an increase in blood-oxygen carrying capacity, mediated by an increase in haematocrit and polycythaemia, as well as a decrease in the p50 value. It is possible that anuran amphibians respond to chronic hypoxia by increasing their blood’s capacity to deliver oxygen rather than increasing breathing or augmenting ventilatory chemoreflexes. Indeed anurans that live at high altitude have a smaller red blood cell size, a higher haemoglobin concentration, a larger red blood cell count, and a higher oxygen-haemoglobin binding affinity compared to lowland anurans (Ruiz et al., 1989; Weber et al., 2002). Two recent studies have examined the effects of chronic hypoxia on respiratory chemoreflexes in fish. Burleson et al. (2002) demonstrated that exposure of catfish (Ictalurus punctatus) to 1 week of aquatic hypoxia (water PO2 = 75 mm Hg) augmented the subsequent acute hypoxic ventilatory response to more severe hypoxia. Vulesevic et al. (2006) exposed zebrafish (Danio reiro) to 4 weeks of aquatic hypoxia (water PO2 = 30 mm Hg) and observed a reduction in resting ventilation but no change in the acute hypoxic or hypercapnic ventilatory responses following this exposure. Clearly there is no single pattern of respiratory changes during chronic hypoxia in lower vertebrates. Species-specific differences are likely due to the degree of hypoxia tolerance and the environment in which chronic hypoxia is encountered. Gheshmy et al. (2006) demonstrated that in vivo exposure to chronic hypercapnia (3.5% CO2 ; 9 days) caused an increase
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in central pH/CO2 -sensitive fictive breathing measured using in vitro brainstem-spinal cord preparations from the cane toad. This observation, in conjunction with the results of the current study, indicates that chronic hypercapnia and chronic hypoxia have an opposite effect on central pH/CO2 -sensitive fictive breathing. Furthermore, while mid-brain transection restored the chronic hypoxia-induced blunting of central chemosensitivity observed in this current study, an identical transection (Gheshmy et al., 2006) had no effect on the augmented pH/CO2 sensitive fictive breathing, measured in vitro, following in vivo exposure to chronic hypercapnia. Together, these results could suggest: (1) an active inhibition of central pH/CO2 chemosensitivity, originating from the mid-brain, following chronic hypoxia and (2) an augmentation of central pH/CO2 chemosensitivity, that does not originate from the mid-brain, following chronic hypercapnia. Gheshmy et al. (2006) also examined the effects of chronic hypercapnia on the in vivo acute hypercapnic ventilatory response (exposure to 2.5–5.5% CO2 ) in intact animals. These studies did not reveal a chronic hypercapnia-induced augmentation of the acute hypercapnic ventilatory response in vivo indicating that changes to central pH/CO2 chemoreceptor function, observed in vitro, did not manifest as changes in breathing in the intact animal. This is not necessarily surprising given the myriad of respiratory-related pH/CO2 -sensitive receptors in anuran amphibians that are stimulated (i.e., central, carotid and olfactory chemoreceptors) or inhibited (PSR) by CO2 (Milsom, 2002; Reid, 2006) and the various interactions between different populations of chemoreceptors and PSR (Reid, 2006). It remains to be seen whether or not exposure to chronic hypoxia causes a reduction in the in vivo acute hypercapnic ventilatory response as would be predicted based on the results of this study. 5. Summary The major findings of this study were: (1) that chronic hypoxia abolished the increase in fictive breathing (frequency and total ventilation index) associated with a decrease in aCSF pH and (2) that a mid-brain transection restored the chronic hypoxia-induced reduction in the pH/CO2 -sensitivity of fictive breathing. Together, these observations suggest that influences from the rostral mid-brain inhibit pH-sensitive fictive breathing during chronic hypoxia. The data, together with that reported by Gheshmy et al. (2006) also suggest that the effects of chronic hypoxia and chronic hypercapnia, on central pH/CO2 chemosensitivity, are different. Acknowledgements This study was supported by NSERC of Canada Discovery and Equipment grants to SGR. SGR was supported by a Parker B. Francis Fellowship in Pulmonary Research (Francis Families Foundation) from 2003 to 2006. Funding for equipment was also provided by the Canadian Foundation for Innovation and the Ontario Innovation Trust.
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Chronic hypoxia attenuates central respiratory-related pH/CO2 chemosensitivity in the cane toad Jessica McAneney, Stephen G. Reid ∗ Centre for the Neurobiology of Stress, Department of Life Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, Ontario, M1C 1A4 Canada Accepted 19 October 2006
Abstract This study examined the effects of chronic hypoxia (CH) and mid-brain transection on central respiratory-related pH/CO2 chemosensitivity in cane toads (Bufo marinus). Toads were exposed to 10 days of CH (10% O2 ) following which in vitro brainstem-spinal cord preparations, with the mid-brain attached, were used to examine central pH/CO2 chemosensitivity. A reduction in artificial cerebral spinal fluid (aCSF) pH increased fictive breathing frequency (fR) and total fictive ventilation. CH reduced fictive fR and total fictive ventilation, compared to controls. Mid-brain transection caused an increase in fictive fR, at the lower aCSF pH levels, in both control and CH preparations. In the CH preparations, mid-brain transection restored fictive breathing to control levels. In both groups, mid-brain transection eliminated fictive breath clustering. The data indicate that CH attenuates central pH/CO2 -sensitive fictive breathing but a mid-brain transection in the middle of the optic lobes abolishes this attenuation. The results suggest that CH induces inhibition of central pH/CO2 chemoreceptor function via descending inputs from the mid-brain region. © 2006 Elsevier B.V. All rights reserved. Keywords: Amphibian; Control of breathing; Hypercapnic ventilatory response; In vitro brainstem-spinal cord preparation; Mid-brain transection
1. Introduction Exposure to chronic hypoxia in mammals results in ventilatory acclimatization to hypoxia, which is manifest as an increase in resting ventilation, a decrease in arterial pCO2 and, usually, an augmentation of the acute hypoxic ventilatory response (Dempsey and Forster, 1982; Bisgard and Neubauer, 1995; Powell et al., 1998, 2000; Bisgard, 2000). Anuran amphibians (frogs and toads) are, in general, more hypoxia-tolerant than mammals (Storey and Storey, 1986; Pinder et al., 1992; Boutilier, 2001). A recent study (McAneney et al., 2006) on cane toads (Bufo marinus) demonstrated that exposure to chronic hypoxia (10 days; 10% O2 ) did not alter resting ventilation but did cause an attenuation of the acute hypoxic ventilatory response when toads were exposed to 5% O2 . Gheshmy et al. (2006) recently demonstrated that exposure of cane toads to another long term respiratory challenge (chronic hypercapnia; 10 days; 3.5% CO2 ) produced an augmentation of central pH/CO2 chemoreceptor function (i.e., pH/CO2 -sensitive fictive
∗
Corresponding author. Tel.: +1 416 287 7426; fax: +1 416 287 7642. E-mail address: [email protected] (S.G. Reid).
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breathing) measured using in vitro brainstem-spinal cord preparations. Anuran amphibians have a host of respiratory control systems that, for the most part, are similar to those found in mammals (Milsom, 1991; Kinkead, 1997). These include central pH/CO2 sensitive chemoreceptors located on the ventrolateral surface of the medulla (Smatresk and Smits, 1991; Milsom, 2002; Taylor et al., 2003a) and peripheral O2 /CO2 -sensitive chemoreceptors located in the carotid labyrinth (West et al., 1987). The respiratory responses to peripheral and central chemoreceptor stimulation are interactive with the level of O2 influencing the acute hypercapnic ventilatory response and the CO2 level influencing the acute hypoxic ventilatory response (West et al., 1987; Smatresk and Smits, 1991; Reid, 2006). CO2 -sensitive olfactory chemoreceptors located in the nasal epithelium inhibit breathing when stimulated with high levels of CO2 (Kinkead and Milsom, 1996; Coates, 2001). CO2 -sensitive pulmonary stretch receptors (PSR) located in the walls of the lungs (Milsom and Jones, 1977; Kuhlman and Fedde, 1979) can stimulate or inhibit breathing depending upon whether the PSR feedback is phasic or tonic and the degree of lung inflation. Adult anurans are discontinuous breathers with the primary breathing pattern consisting of single breaths or the occasional
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doublet/triplet under low levels of respiratory drive. When respiratory drive is elevated (i.e., during acute hypoxia or hypercapnia) breathing becomes episodic in which clusters of breaths are separated by periods of apnea. A number of studies have demonstrated that fluctuations in blood-gas levels are not necessary for episodic breathing in these animals (West et al., 1987; Kinkead et al., 1994, 1997; Milsom et al., 1999; Reid et al., 2000). Reid et al. (2000) demonstrated, using isolated bullfrog brainstem-spinal cord preparations with the mid-brain attached, that a mid-brain transection abolished episodic fictive breathing and produced a pattern of relatively continuous fictive breathing. Based on the observations that (1) chronic hypoxia abolished the acute hypoxic ventilatory response in vivo (McAneney et al., 2006), (2) chronic hypercapnia increased the magnitude and sensitivity of central pH/CO2 -sensitive fictive breathing in vitro (Gheshmy et al., 2006) and (3) the respiratory responses to O2 and CO2 chemoreceptor stimulation are interactive (West et al., 1987; Reid, 2006) we hypothesized that exposure to chronic hypoxia would lead to an attenuation of central pH/CO2 sensitive breathing (i.e., attenuate the changes in breathing normally associated with changes in cerebral spinal fluid pH/CO2 ). While, some anurans encounter chronic hypoxia during the winter months in underground burrows (terrestrial species) or underwater (aquatic species) (Pinder et al., 1992), the cane toad (B. marinus) endures long periods of apnoea and arterial hypoxia during estivation (Coelho and Smatresk, 2003). Coelho and Smatresk (2003) report a reduction in arterial PO2 from 90 to 15–20 mm Hg following 1 h of apnea. Given this, we hypothesized that respiratory control systems would be altered in order to reduce lung ventilation during chronically hypoxic conditions when enhanced lung ventilation is unlikely to be advantageous either due to environmental constraints or reduced metabolic rate. Furthermore, given that mid-brain inputs are involved in the regulation of breath clustering, we hypothesized that input from the mid-brain may also mediate any chronic hypoxia-induced decrease in central pH/CO2 -sensitive breathing. To address these hypotheses, cane toads were made chronically hypoxic (10% O2 ) for 10 days. In vitro brainstem-spinal cord preparations (with the mid-brain attached) were used to assess the pH/CO2 -sensitivity of fictive breathing before and after a mid-brain transection. In these preparations, motor output from a respiratory nerve was used as the index of breathing (fictive breathing). 2. Materials and methods
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mittee and conform to the guidelines established by the Canadian Council for Animal Care. 2.2. Exposure to chronic hypoxia Toads were placed, for a 10-day period, into a Plexiglas chamber (35 cm × 25 cm × 10 cm) within which the inspired O2 level was maintained at 10% using a Pro-Ox 110 control unit (Biospherix; Redfield, NY) (McAneney et al., 2006). An O2 electrode within the chamber monitored the level of O2 . When the O2 level rose above 10%, the Pro-Ox delivered a small amount of N2 to lower the level back to 10%. Routine measurements of CO2 (CD-3A CO2 analyzer, AEI Technologies; Pittsburgh, PA) confirmed that inspired CO2 levels remained at approximately 0.03%. The chamber was maintained at room temperature and exposed to a 12 h:12 h cycle of light and darkness. McAneney et al. (2006) demonstrated that the chronic hypoxia-induced blunting of the in vivo acute hypoxic ventilatory response occurred after 3 days of chronic hypoxia and remained for the entire 10-day experimental period of hypoxia. Given this, we were confident that, in the current study, the 10-day period of chronic hypoxia was of sufficient duration to observe any changes in central chemosensitivity that may arise due to exposure to chronic hypoxia. A period of 9–10 days of chronic hypoxia is also the general time frame required to induce ventilatory acclimatization to hypoxia in mammals (rats; Aaron and Powell, 1993; Reid and Powell, 2005). 2.3. The in vitro brainstem-spinal cord preparation Toads were anaesthetized by emersion in a solution of 3aminobenzoic acid ethyl ester (MS222, 1 g/L; Sigma) buffered to pH 7.0 with sodium bicarbonate (Reid and Milsom, 1998; Reid et al., 2000; Gheshmy et al., 2006). Animals were kept in the anaesthetic until the eye-blink and toe-pinch reflexes were eliminated. Using a Dremmel Tool, an incision was made in the skull rostral to the optic lobes. The cranial case was removed with rongeurs/bone shears and placed onto a Sylgard-coated dissecting dish. The brain was exposed and superfusion with ice-cold oxygenated artificial cerebral spinal fluid (aCSF) was initiated; the rostral forebrain was then removed (see Fig. 1). The transection to remove the rostral forebrain was performed in the middle of the cerebral hemispheres. Based on the Hoffmann (1973) atlas of the toad brain, this transection site would correspond to
2.1. Experimental animals Cane toads (B. marinus) (N = 36; 200–300 g) were obtained from a commercial supplier (Boreal Scientific, St. Catharine’s, Ontario) and maintained in a fibreglass tank at room temperature (20–22 ◦ C). Toads were supplied with both terrestrial and aquatic habitats consisting of moist peat moss and trays of dechlorinated water, respectively. The photoperiod was maintained at 12 h light and 12 h dark. Animals were fed live crickets once per week. Holding conditions and experimental protocols were approved by the University of Toronto Animal Care Com-
Fig. 1. A lateral view of the toad brain illustrating the level of transections (dotted lines) performed in this study. The rostral- and caudal-most transections were used to generate the in vitro preparation. The mid-brain transection through the optic lobes was the experimental manipulation. NI indicates the location of the nucleus isthmi. V, trigeminal nerve root; VII, facial nerve root; IX, glossopharyngeal nerve root; X, vagus nerve root.
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approximately 1.5 mm anterior to the zero mark (defined as the vertex of the angle formed at the point where the cerebral hemispheres become separate or begin to diverge). Internally this zero point (Hoffmann, 1973) corresponds to the interventricular foramen (foramen Monroi). The forebrain transection goes through the primordial hippocampus, the primordial general pallium, the primordial piriform cortex, the lateral septal nucleus, the striatum, the medial forebrain bundle, the medial septal nucleus and the lateral ventricle. The remaining brain tissue was continually supplied with the aCSF (in mmol/L; NaCl, 103; KCl, 4.05; MgCl2 , 1.38; glucose, 10; NaHCO3 , 29.2; CaCl2 , 2.45; Sigma Chemicals; pH 7.8, i.e., normocapnic CSF; Taylor et al., 2003a,b; Gheshmy et al., 2006). Cranial nerves were cut close to their exit from the skull and the spinal cord was severed at the level of the third spinal nerve (see Fig. 1). The preparation was transferred from the brain case and immobilized with insect pins in a Sylgard-coated dissecting dish continually superfused with oxygenated aCSF. The dura matter surrounding the brain was removed in order to free the cranial nerve roots and the nerve tips were cut to provide a clean surface for recording. The preparation was then pinned, ventral side up, onto a fine stainless steel mesh within a superfused recording chamber. The mesh divided the chamber into upper and lower compartments which ensured simultaneous superfusion of both surfaces of the preparation (Kinkead et al., 1994; McLean et al., 1995a,b; Gheshmy et al., 2006). The preparation was continuously superfused with oxygenated aCSF, at a rate of 10 mL/min, using peristaltic pumps that delivered and removed the aCSF from the chamber. The aCSF was re-cycled (Gheshmy et al., 2006). The preparations were maintained at pH 7.8 and room temperature for 60 min before commencing the experiment. Suction electrodes of various diameters were made from thinwalled capillary glass (1 mm diameter) pulled to a fine tip using a vertical pipette puller (Kopf model 720; Tujunga, CA). The tips were polished using a grinding stone and flame to provide a smooth surface. Using a micro-manipulator, an appropriately sized suction electrode was positioned near the end of the vagus nerve root and the nerve was carefully aspirated into the electrode such that a tight seal was obtained between the nerve and the electrode. In all preparations, recordings were taken of whole nerve discharge from the vagus nerve. In the intact animal, a branch of the vagus nerve innervates the glottis which opens and closes with each breath while another branch innervates buccal pump muscles (Sakakibara, 1984a,b). Since these preparations are devoid of any afferent input and breathing is an inherently rhythmic process generated in the brainstem, all rhythmic (Reid and Milsom, 1998) activity (in vitro motor output) recorded from the vagus nerve was assumed to represent motor output to the respiratory muscles and is therefore an index of breathing termed fictive breathing. This is a well-established preparation for the study of amphibian central respiratory control (Kinkead et al., 1994; McLean et al., 1995a,b; Reid and Milsom, 1998; Reid et al., 2000; Morales and Hedrick, 2002; Wilson et al., 2002; Taylor et al., 2003a,b; Gheshmy et al., 2006). Nerve activity from the suction electrode was amplified (10×) and filtered (30 Hz, high pass; 3 kHz, low pass) using
a DAM50 ac amplifier (World Precision Instruments; Sarasota, FL). The output from the DAM50 was sent to a second ac amplifier (ISO8A, WPI) and amplified a further 100×. The amplified nerve signal from the ISO8A was sent to a moving averager (CWE MA821/RSP; CWE Inc., Ardmore, PA) for integration (time constant = 200 ms) and to an audio monitor (AM Systems Model 3300; Carlsborg, WA). The amplified/filtered nerve signal and integrated signal were monitored and stored using a data acquisition system (Biopac Systems, MP150; Goleta, CA). The sampling rate of analogue to digital conversion was 2000 Hz. Gassing the aCSF with varying levels of CO2 (0–5%; balance O2 ) altered the aCSF pH. The levels of CO2 and O2 , gassing the aCSF, were set using digital mass flow controllers (Smart-Trak 100, Sierra Instruments; Monterey, CA). The pH level of the aCSF was monitored using a pH electrode (VWR) placed within the aCSF reservoir. 2.4. Experimental protocol Following the 1-h stabilization period and the observation of stable levels of neural discharge (fictive breathing), each preparation was exposed to varying levels of aCSF pH (7.6, hypercapnic; 7.8, normocapnic; 8.0, hypocapnic; random order exposure). This pH range approximates that used in previous studies on amphibian brainstem-spinal cord preparations (Kinkead et al., 1994; McLean et al., 1995a,b; Gheshmy et al., 2006). All experiments were performed at room temperature (approximately 22 ◦ C). This is within the temperature range (15–25 ◦ C) reported by Morales and Hedrick (2002) in which fictive breathing is consistently active from in vitro adult bullfrog brainstem-spinal cord preparations. Each pH change was achieved over a period of 15 min. Preparations were allowed to acclimatize to each new pH level for a further 15 min before fictive breathing was monitored for an additional 15 min data analysis period. Following the series of pH changes, the mid-brain was transected slightly caudal to the middle of the optic lobes (Fig. 1). Based on the Hoffmann (1973) atlas of the toad brain, this transection site would correspond to 4 mm posterior to the zero mark (see above). Our mid-brain transection goes through the optic tectum, torus semicircularis, tectum ventricle and tegmentum (see Fig. 4 from Hoffmann, 1973). The transection is immediately rostral to the nucleus isthmi (see Figs. 29 and 30 from Hoffmann, 1973). Following a 30 min stabilization period, the pH changes (7.6, 7.8 and 8.0) were repeated (again in random order). The protocol was performed on brainstem-spinal cord preparations taken from control (N = 18) and chronically hypoxic (N = 18) toads. 2.5. Data and statistical analysis The final 15 min of data at each pH level were analyzed to determine mean values for fictive breathing frequency, breaths/episode, episodes/minute, breath duration (s) and integrated fictive breath area (an index of breath volume). In this study we were recording from the root of the whole vagus nerve. Respiratory-related motor activity in this nerve, in vivo,
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controls the opening and closing of the glottis (via the laryngeal branch of the vagus) as well as the buccal respiratory pump muscles (via the pharyngeal posterior superior nerve) (Sakakibara, 1984a). Sakakibara (1984b) demonstrated that an increase in buccal pressure was positively correlated with integrated trigeminal nerve activity (r = 0.97). The trigeminal nerve (mandibular branch) innervates buccal pump muscles. However, integrated nerve activity from the pharyngeal posterior superior nerve (vagal branch) is also positively correlated with integrated trigeminal nerve activity (r = 0.95; Sakakibara, 1984a,b). This suggests that nerve activity in the pharyngeal posterior superior nerve (a branch of the vagus) should also be correlated with buccal pressure changes (or breath volume). Furthermore, given that the laryngeal branch of the vagus nerve controls the opening and closing of the glottis, it is likely that a larger volume breath requires that the glottis remain open for a longer period of time compared to a smaller volume breath. Given this, it is likely that a larger breath would be associated with a larger amount (i.e., integrated area) of vagal activity. Based on the above observations and assumptions, we believe that it is reasonable to assume that the integrated area of vagal activity is a valid index of breath volume. Furthermore, given that the integrated area takes into account both fictive breath amplitude and duration, we believe that integrated area is a more appropriate measure of breath volume than the amplitude of the fictive breaths. The total fictive ventilation index was calculated as the product of fictive breathing frequency and the integrated breath area. Fictive breaths in a given episode were defined as occurring within 2 s of each other according to general practices in the literature (Kinkead et al., 1994, 1997; Reid et al., 2000; Gheshmy et al., 2006). All statistical analyses were performed using commercial software (SigmaStat 3.0, SPSS). Data are reported as the mean ± one standard error of the mean (SEM). For each condition (normoxic pre-transection, normoxic post-transection, chronically hypoxic pre-transection and chronically hypoxic post-transection) a one-way repeated measures analysis of variance (RM-ANOVA), followed by a Student–Newman–Keuls (SNK) multiple comparison test (MCT), was used to compare the effects of pH on fictive breathing. The effects of chronic hypoxia, mid-brain transection and pH were analysed using a three-way ANOVA followed by a SNK MCT. The limit of significance was 5% (p < 0.05).
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Fig. 2. Fictive breathing (vagal motor output) recorded from isolated brainstemspinal cord preparations (at pH 7.6) taken from (A) a normoxic control toad (pre-mid-brain transection), (B) the normoxic control toad (post-mid-brain transection), (C) a chronically hypoxic toad (pre-mid-brain transection), and (D) the chronically hypoxic toad (post-mid-brain transection). In all cases the upper trace represents the raw electroneurogram (eng X) recorded from the vagus nerve root while the lower trace ( eng X ) represents the integrated trace.
tions in which (in the intact animal) the floor of the mouth rises and falls with the glottis closed. 3.1. Fictive breathing frequency
3. Results Fig. 2 illustrates fictive breathing recorded at an aCSF pH level of 7.6 in brainstem-spinal cord preparations taken from control and chronically hypoxic animals both prior to and following the mid-brain transection in the middle of the optic lobes. Chronic hypoxia reduced fictive breathing frequency prior to mid-brain transection (compare Fig. 2A and C). Mid-brain transection converted discontinuous (episodic) fictive breathing to a pattern of relatively continuous fictive breathing (compare Fig. 2A with B and C with D). The small bursts of activity in Fig. 2B are likely indicative of non-ventilatory buccal oscilla-
In the normoxic control preparations prior to mid-brain transection, fictive breathing frequency increased as aCSF pH was lowered from 8.0 to 7.6 (Fig. 3A; p < 0.001). Mid-brain transection augmented this increase at pH 7.6 (Fig 3A; p = 0.044). In the preparations taken from chronically hypoxic toads, there was no increase in fictive breathing frequency as aCSF pH was lowered prior to mid-brain transection (Fig. 3B; p = 0.149). Midbrain transection caused a significant increase in fictive breathing frequency in the chronically hypoxic preparations (Fig. 3B; p = 0.002) as aCSF pH was lowered from 8.0 to 7.8 and 7.6. The fictive breathing frequency values in the pre-transection chron-
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per episode. Prior to mid-brain transection, the number of fictive episodes per minute increased, slightly but significantly, as aCSF pH was lowered in the control (Fig. 4A; p < 0.001) but not the chronically hypoxic (Fig. 4B; p = 0.087) preparations. Midbrain transection resulted in a significant increase in the number of fictive episodes per minute in both the control (Fig. 4A; pH 7.6; p < 0.001) and chronically hypoxic (Fig. 4B; pH 7.6–7.8; p < 0.001) preparations. Prior to transection, the number of fictive episodes per minute in the chronically hypoxic group was reduced, albeit not significantly (p = 0.056), compared to the control group. The post-transection fictive episodes per minute values in the control and chronically hypoxic groups were not different (p = 0.496). In the control (Fig. 4C) and chronically hypoxic (Fig. 4D) groups, changes in aCSF pH had no effect on the number of fictive breaths per episode prior to (control, p = 0.333; chronic hypoxia, p = 0.565) or following (control, p = 0.999; chronic hypoxia, p = 0.820) mid-brain transection. In both groups, midbrain transection caused a significant reduction in the number of fictive breaths per episode with the value falling to approximately one in all cases (controls and chronically hypoxic, p < 0.001). Chronic hypoxia had no effect on the number of fictive breaths per episode either before (p = 0.310) or after (p = 0.137) mid-brain transection. 3.3. Integrated fictive breath area, duration and the total fictive ventilation index
Fig. 3. Fictive breathing frequency (fictive breaths per minute) recorded from isolated brainstem-spinal cord preparations taken from (A) normoxic control toads (n = 18) and (B) chronically hypoxic toads (n = 18). Open circles (A) and open squares (B) represent the values prior to mid-brain transection. Closed circles (A) and closed squares (B) represent the values following mid-brain transection. Lower case letters (a and b) indicate differences, within any given group, between the different aCSF pH levels. A number sign (#) indicates a significant difference between the pre- and post-transection values. A plus sign (+) indicates a significant difference between the normoxic control values in panel A and the corresponding chronically hypoxic value in panel B. The data are reported as the mean ± S.E.M. A partial data set of the pre-transection fictive breathing frequency data in both panels (this figure only) has been briefly reported in a recent review article (Reid, 2006).
ically hypoxic preparations were reduced (Fig. 3B) at pH 7.6 (p = 0.007) and 7.8 (p = 0.087), compared to the values in the pre-transection control preparations shown in Fig. 3A. The values in the control and chronically hypoxic groups were the same post-transection (p = 0.496). 3.2. Fictive episodes per minute and fictive breaths per episode Fictive breathing frequency is the product of the number of fictive episodes per minute and the number of fictive breaths
With the exception of a post-transection increase in fictive breath duration at pH 7.8 in the control group (Fig. 5C; p = 0.004), changes in aCSF pH, mid-brain transection and chronic hypoxia had no statistically significant effect (p > 0.05) on either the integrated area of the fictive breaths (Fig. 5A and B) or fictive breath duration (Fig. 5C and D). The total fictive ventilation index (fictive breathing frequency × integrated fictive breath area) increased in the control (Fig. 6A) and chronically hypoxic (Fig. 6B) groups, as aCSF pH was lowered, both prior to (control, p = 0.006; chronic hypoxia, p = 0.036) and following (control, p = 0.010; chronic hypoxia, p = 0.047) mid-brain transection. There was no effect of midbrain transection, on total fictive ventilation, in the control preparations (p = 0.819). Following mid-brain transection in the chronically hypoxic group there was a significant increase in the total fictive ventilation index at aCSF pH levels of 7.8 and 7.6 (Fig. 6B; p = 0.002). Exposure to chronic hypoxia reduced the pre-transection levels of the total fictive ventilation index (compare the pre-transection values in Fig. 6A and Fig. 6B; p = 0.007) at aCSF pH values of 7.8 and 7.6. The total fictive ventilation index values in the control and chronically hypoxic groups were the same post-transection (p = 0.730). 4. Discussion One of the major findings of this study was that chronic hypoxia abolished the increase in fictive breathing (frequency and total ventilation index) associated with a decrease in aCSF pH. The results of this study are consistent with those of
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Fig. 4. The number of fictive breathing episodes per minute (A and B) and fictive breaths per episode (C and D) recorded from isolated brainstem-spinal cord preparations taken from normoxic control toads (A and C) and chronically hypoxic toads (B and D). Symbols are the same as described for Fig. 3.
McAneney et al. (2006) in that chronic hypoxia has reduced an acute ventilatory chemoreflex (in this case central pH/CO2 sensitive fictive breathing). In other words, chronic hypoxia has reduced/blunted the in vitro equivalent of the central pH/CO2 chemoreceptors’ contribution to the acute hypercapnic ventilatory response. There was no effect of chronic hypoxia on the integrated area of the fictive breaths; all of the modulatory effects of chronic hypoxia influenced fictive breathing frequency. In vivo, breathing room air (resting conditions) and 4.0–4.5% CO2 (acute hypercapnia) leads to arterial pH levels of approximately 7.8–8.0 and 7.6, respectively (Kinkead and Milsom, 1996). The chronic hypoxia-induced blunting of fictive breathing (frequency and total ventilation) was pH-sensitive. In both cases, the chronically hypoxic values were reduced at aCSF pH levels of 7.8 and 7.6 but not at pH 8.0. These data suggest that chronic hypoxia does not alter breathing under hypocapnic conditions (i.e., fictive breathing at pH 8.0) but does alter fictive breathing during the in vitro equivalent of normocapnia (aCSF pH 7.8) and during an elevation in the CO2 -mediated drive to breathe (i.e., aCSF pH level of 7.6). The observation that the effects of chronic hypoxia are pH sensitive is also consistent with that of McAneney et al. (2006) who demonstrated that chronic
hypoxia abolished the in vivo acute hypoxic ventilatory response but did not alter resting ventilation. The second major finding of this study was that the chronic hypoxia-induced abolition (blunting) of pH/CO2 sensitive fictive breathing was eliminated by mid-brain transection. In other words, mid-brain transection restored pH/CO2 -chemosensitivity. The mid-brain transection also eliminated the clustering of fictive breaths (the number of fictive breaths/episode was approximately one under all conditions following the transection) and caused an increase in fictive breathing frequency. This is consistent with Reid et al. (2000) who demonstrated, using in vitro brainstem-spinal cord preparations from bullfrogs (Rana catesbeiana), that descending inputs, from the mid-brain to the medulla, were involved in the clustering of breaths into episodes. The post-transection increase in fictive breathing frequency indicates that descending inputs from the mid-brain normally inhibit or reduce breathing frequency (and the overall level of breathing), under both normocapnic and hypercapnic conditions. The chronic hypoxia-induced blunting of pH/CO2 -senstive fictive breathing suggests that these mid-brain inhibitory influences exert a greater effect on fictive breathing following exposure to chronic hypoxia, compared to control preparations. The implication of this observation
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Fig. 5. Integrated area of the fictive breaths (V s; A and B) and fictive breath duration (s; C and D) recorded from isolated brainstem-spinal cord preparations taken from normoxic control toads (A and C) and chronically hypoxic toads (B and D). Symbols are the same as described for Fig. 3.
is that chronic hypoxia has enhanced the inhibitory nature of these descending mid-brain influences in order to reduce fictive breathing frequency. Furthermore, the restoration of pH sensitivity following the mid-brain transection suggests that the chronic hypoxia-induced decrease in sensitivity was not the result of metabolic depression. Currently the nature of respiratory-related mid-brain input to the medulla is not known. Indeed the anatomical nature of adult amphibian respiratory control centers has not been investigated other than, for example, studies on respiratory rhythmogenesis (Wilson et al., 2002; Vasilakos et al., 2005), breath clustering (Reid et al., 2000), pH chemosensitivity (Taylor et al., 2003a,b; Noronha-de-Souza et al., 2006), the nucleus isthmi (Kinkead et al., 1997; see review by Gargaglioni and Branco, 2004), respiratory-related neurons (Kogo and Remmers, 1994; McLean and Remmers, 1997) and glutamate/GABA (McLean et al., 1995b). The nucleus isthmi, located slightly caudal to our transection (see Fig. 1), has also been implicated in the control of amphibian breathing (Kinkead et al., 1997; Gargaglioni and Branco, 2004; Milsom et al., 2004) although its primary role appears to be in vision (Gruberg and Udin, 1978). A recent study (Noronha-
de-Souza et al., 2006) suggests that the locus coeruleus (LC; which lies ventral and slightly caudal to the nucleus isthmi) is also involved in amphibian breathing. In that study, lesion of the LC reduced the acute hypercapnic ventilatory response while focal acidification of the LC increased breathing. The K¨ollikerfuse which lies close to the nucleus isthmi and LC (Adli et al., 1999) may also be involved in respiratory control in these animals. Our results indicate that the transection through the optic lobes caused continuous breathing but increased breathing frequency and enhanced CO2 chemosensitivity. It is not clear whether the transection in the current study removed the influence of a specific episode-generating center or if it disrupted a larger network responsible for episode generation. The enhanced breathing frequency and CO2 chemosensitivity suggest that the effects of transection on the breathing pattern (i.e., a shift from episodic to continuous fictive breathing) was not simply the result of altered respiratory drive, or the central integration of respiratory drive, as enhanced drive leads to episodic breathing in these animals (Milsom, 1991). This observation suggests that the transection disrupted a region involved in drive-independent breath clustering.
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Fig. 6. Total fictive ventilation index (V s/min) calculated by multiplying the fictive breathing frequency by the integrated area of the fictive breaths recorded from isolated brainstem-spinal cord preparations taken from (A) normoxic control toads and (B) chronically hypoxic toads. Symbols are the same as described for Fig. 3.
Milsom et al. (1997) present evidence from studies on bullfrogs, hibernating ground squirrels and sleeping elephant seals in support of the notion that, during episodic breathing, alternating positive (excitatory) and negative (inhibitory) descending influences, to the medullary respiratory centers, serve to produce the fast breathing observed during episodes and the apneic periods between episodes, respectively. Further studies on anurans are required to determine the nature of a putative inhibitory (and possibly episode-generating) center, rostral to the nucleus isthmi, as well as any effects of chronic hypoxia on the function of this putative center. However, the results of this study do suggest that chronic hypoxia augments inhibitory inputs from the mid-brain such that breathing frequency is reduced. While the effects of chronic hypoxia on ventilatory chemoreflexes have been well studied in mammals, only a few studies have focused on respiratory plasticity following chronic hypoxia
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in amphibians (Pinder and Burggren, 1983; McAneney et al., 2006) and other lower vertebrates (Burleson et al., 2002; Vulesevic et al., 2006). The results of the current study, in conjunction with those of McAneney et al. (2006), indicate that chronic hypoxia blunts acute ventilatory chemoreflexes rather than augmenting them, as is the general case with mammals (Powell et al., 1998, 2000). This mammalian versus amphibian difference may reflect the different conditions under which amphibians and mammals are likely to encounter chronic hypoxia. Aside from fossorial species, mammals are most likely to encounter chronically hypoxic conditions when they ascend to high altitudes (Bisgard, 2000). Under these conditions, increasing breathing is obviously advantageous. Many terrestrial anuran amphibians, in temperate climates, spend the winter months in underground burrows which are likely to be hypoxic and have a limited supply of air (other than diffusion through the soil and snow) (Breckenridge and Tester, 1961; Pinder et al., 1992). Under these conditions in which air is limited and, due to cold temperatures, metabolic rate is lowered (Boutilier, 2001), the need to ventilate the lungs is likely to be reduced. Given this, it is not surprising that chronic hypoxia leads to a reduction in the ventilatory responses to acute hypoxia in vivo (McAneney et al., 2006) and the in vitro equivalent of acute hypercapnia (this study). However, it is curious that resting breathing was not affected in either case. It is possible that a combination of chronic hypoxia and a reduced ambient temperature may also lead to reductions in resting ventilation that would be expected to occur during winter. The down-regulation of breathing during chronic hypoxia may also assist in minimising water loss. Pinder and Burggren (1983) demonstrated, in adult bullfrogs (R. catesbeiana) that exposure to aerial and aquatic hypoxia (75 mm Hg) caused an increase in blood-oxygen carrying capacity, mediated by an increase in haematocrit and polycythaemia, as well as a decrease in the p50 value. It is possible that anuran amphibians respond to chronic hypoxia by increasing their blood’s capacity to deliver oxygen rather than increasing breathing or augmenting ventilatory chemoreflexes. Indeed anurans that live at high altitude have a smaller red blood cell size, a higher haemoglobin concentration, a larger red blood cell count, and a higher oxygen-haemoglobin binding affinity compared to lowland anurans (Ruiz et al., 1989; Weber et al., 2002). Two recent studies have examined the effects of chronic hypoxia on respiratory chemoreflexes in fish. Burleson et al. (2002) demonstrated that exposure of catfish (Ictalurus punctatus) to 1 week of aquatic hypoxia (water PO2 = 75 mm Hg) augmented the subsequent acute hypoxic ventilatory response to more severe hypoxia. Vulesevic et al. (2006) exposed zebrafish (Danio reiro) to 4 weeks of aquatic hypoxia (water PO2 = 30 mm Hg) and observed a reduction in resting ventilation but no change in the acute hypoxic or hypercapnic ventilatory responses following this exposure. Clearly there is no single pattern of respiratory changes during chronic hypoxia in lower vertebrates. Species-specific differences are likely due to the degree of hypoxia tolerance and the environment in which chronic hypoxia is encountered. Gheshmy et al. (2006) demonstrated that in vivo exposure to chronic hypercapnia (3.5% CO2 ; 9 days) caused an increase
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in central pH/CO2 -sensitive fictive breathing measured using in vitro brainstem-spinal cord preparations from the cane toad. This observation, in conjunction with the results of the current study, indicates that chronic hypercapnia and chronic hypoxia have an opposite effect on central pH/CO2 -sensitive fictive breathing. Furthermore, while mid-brain transection restored the chronic hypoxia-induced blunting of central chemosensitivity observed in this current study, an identical transection (Gheshmy et al., 2006) had no effect on the augmented pH/CO2 sensitive fictive breathing, measured in vitro, following in vivo exposure to chronic hypercapnia. Together, these results could suggest: (1) an active inhibition of central pH/CO2 chemosensitivity, originating from the mid-brain, following chronic hypoxia and (2) an augmentation of central pH/CO2 chemosensitivity, that does not originate from the mid-brain, following chronic hypercapnia. Gheshmy et al. (2006) also examined the effects of chronic hypercapnia on the in vivo acute hypercapnic ventilatory response (exposure to 2.5–5.5% CO2 ) in intact animals. These studies did not reveal a chronic hypercapnia-induced augmentation of the acute hypercapnic ventilatory response in vivo indicating that changes to central pH/CO2 chemoreceptor function, observed in vitro, did not manifest as changes in breathing in the intact animal. This is not necessarily surprising given the myriad of respiratory-related pH/CO2 -sensitive receptors in anuran amphibians that are stimulated (i.e., central, carotid and olfactory chemoreceptors) or inhibited (PSR) by CO2 (Milsom, 2002; Reid, 2006) and the various interactions between different populations of chemoreceptors and PSR (Reid, 2006). It remains to be seen whether or not exposure to chronic hypoxia causes a reduction in the in vivo acute hypercapnic ventilatory response as would be predicted based on the results of this study. 5. Summary The major findings of this study were: (1) that chronic hypoxia abolished the increase in fictive breathing (frequency and total ventilation index) associated with a decrease in aCSF pH and (2) that a mid-brain transection restored the chronic hypoxia-induced reduction in the pH/CO2 -sensitivity of fictive breathing. Together, these observations suggest that influences from the rostral mid-brain inhibit pH-sensitive fictive breathing during chronic hypoxia. The data, together with that reported by Gheshmy et al. (2006) also suggest that the effects of chronic hypoxia and chronic hypercapnia, on central pH/CO2 chemosensitivity, are different. Acknowledgements This study was supported by NSERC of Canada Discovery and Equipment grants to SGR. SGR was supported by a Parker B. Francis Fellowship in Pulmonary Research (Francis Families Foundation) from 2003 to 2006. Funding for equipment was also provided by the Canadian Foundation for Innovation and the Ontario Innovation Trust.
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