Cortical activation states in sleep and anesthesia. II: Respiratory reflexes

Respiration Physiology 112 (1998) 83 – 94

Cortical activation states in sleep and anesthesia. II: Respiratory reflexes J.D. Hunter, J.Z. McLeod, W.K. Milsom * Department of Zoology, Uni6ersity of British Columbia, 6270 Uni6ersity Bl6d., Vancou6er, B.C. V6T 1Z4, Canada Accepted 16 February 1998

Abstract Under urethane anesthesia, animals exhibit patterns of cortical activity similar to those seen in wake, drowsiness and slow-wave sleep in unanesthetized animals. In the present study, hypoxic and hypercapnic ventilatory reflexes were examined in unanesthetized and urethane-anesthetized golden mantled ground squirrels in states with similar EEG profiles. Synchronized EEG patterns occurred less frequently in both unanesthetized and anesthetized animals during hypoxic (10% O2) and hypercapnic (5% CO2) exposure. Breathing frequency fell significantly during sleep in animals breathing all gas mixtures, while the relative ventilatory sensitivity to hypoxia and hypercapnia increased during sleep. Urethane-anesthetized animals also showed significant falls in breathing frequency and ventilation and increases in relative ventilatory sensitivity to hypoxia and hypercapnia as they moved into states with synchronized EEG patterns. These data suggest that the brain activity states observed under urethane anesthesia mimic sleep/wake in terms of their effect on respiratory function and that changes in breathing pattern and the enhancement of ventilatory responses in states with a synchronized EEG is not due solely to changes in levels of behavioural stimuli. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Anesthesia, sleep, EEG states; Mammals, golden mantled ground squirrel (Spermophilus lateralis); Sleep, states, respiratory relexes

1. Introduction Behavioural and metabolic stimuli, as well as wakefulness per se, provide excitatory inputs to respiration. During slow-wave sleep (SWS), * Corresponding author. Tel.: + 1 604 8222310; fax: +1 604 8222416; e-mail: [email protected]

breathing is presumably governed entirely by metabolic control (Colrain et al., 1987, Orem, 1994). Metabolic control alone also governs breathing during tonic, rapid eye movement (REM) sleep (Phillipson and Bowes, 1986, Orem, 1994). During phasic REM sleep, however, other inputs do modulate respiration and irregular breathing patterns correlated with other phasic

0034-5687/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0034-5687(98)00020-6

84

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

events (i.e. PGO, ponto-geniculo-occipital, waves) characteristic to this state do occur (Netick and Foutz, 1980, Hendricks et al., 1991, Orem, 1994). The influence of sleep on reflex ventilatory responses to hypoxia and hypercapnia is variable between species and studies. Some investigators report decreased responses or no change in dogs, cats and goats (Phillipson et al., 1978, Sullivan et al., 1979, Santiago et al., 1981, Parisi et al., 1992), while increased responses have been noted in the rat (Pappenheimer, 1977). The variability which exists in the available data may represent speciesspecific responses, or differences in experimental protocol. In particular, they may reflect differences in the level of behavioural drive present in awake animals. Similar changes in cardiovascular and respiratory pattern have been reported in golden mantled ground squirrels moving between natural arousal states and states with similar EEG profiles under urethane anesthesia (Hunter and Milsom, 1998). To further explore the extent to which the arousal states observed under urethane anesthesia mimic sleep/wake in terms of their effect of respiratory function, the present study sought to describe the changes in hypoxic and hypercapnic ventilatory responses during sleep in these ground squirrels and to compare them with those that occur during the transition between analogous arousal states, based on EEG criteria, in urethane-anesthetized animals. Since no changes in levels of behavioural stimuli will be associated with the changes in the state of central neural activity in the later case, this may also allow us to elucidate the effects of changes in state, per se, on ventilatory sensitivity in rodents.

2. Methods Golden mantled ground squirrels (Spermophilus lateralis) were used in this study. Animals were housed individually in polycarbonate cages (25 cm × 20 cm ×45 cm) in an environmental chamber, which was kept at room temperature (20 9 2°C) throughout the duration of the study. Light conditions were maintained as 12 h light and 12 h dark per day. Food and water were supplied ad

libitum. Food was withheld for 4 h prior to surgery and all experiments utilizing urethane anesthesia.

2.1. Surgical preparation Animals were anesthetized with sodium pentobarbital (Somnotol; 11.25–18.33 mg/100 g). The animal’s head and neck were shaved, treated with a depilatory cream, thoroughly cleaned with distilled water and finally sterilized with ethanol. Animals were then placed in a Kopf stereotaxic device for the duration of the surgery. The details of this surgery have been described previously (Hunter and Milsom, 1998). Briefly, the surface of the skull and muscles at the back of the neck were exposed and four stainless steel EEG electrodes, a thermal re-entrant tube, EKG (electrocardiogram) electrodes and EMG (electromyogram) electrodes were attached. The wire leads from the EKG and EMG electrodes were fed under the skin to the top of the head, and all eight electrodes were inserted into amphenol pin strips affixed to the skull surface with dental acrylic. Upon completion of surgery, animals were given antibiotic injections of ampicillin sodium (Penbritin-250, 6.25 mg/100 g) and analgesic injections of meperidine hydrochloride (Demerol, 1 mg/100 g), administered as needed. Incision areas were treated with a topical antibiotic (Flamazine, 2% silver sulfadiazine).

2.2. Experimental protocol Animals were allowed at least 2 weeks to recover from the surgery before being used in recording experiments. One half of the animals (n=7) received intraperitoneal injections of urethane (ethyl carbamate, approximately 1.35 g/kg) while the other half (n= 7) were subjected to the same handling procedures, but received no injection. Each animal was placed in a clear plexiglass chamber set inside a darkened environment chamber. Air flow through the chamber was 1 litre/ min. The contact pins in the animal’s headpiece were connected to long wire leads, and a thermocouple was inserted into the cranial re-entrant

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

tube. The EEG, EMG and EKG signals were amplified (Grass model 7P511 K), and recorded on a polygraph recorder (Grass model 79E). The thermocouple was connected to a digital monitor (Sensortek). A sample tube connected the animal chamber to O2 and CO2 analyzers (Beckman) to allow continuous monitoring of gas concentrations within the chamber. The gas analyzers were calibrated before each experiment using a precision gas supply (Radiometer (Copenhagen) GMA2) for CO2 and room air for O2. Once the wire leads and probe were in place, unanesthetized animals were left in the recording chamber to stabilize for approximately 1 h. Recordings from anesthetized animals typically began 30 min after animals had been placed in the chamber. Ventilation was measured in unanesthetized, unrestrained animals, using the plethysmographic (barometric) technique (Jacky, 1980). The pressure signal was detected by a mechanical transducer (Validyne model DP103-18), amplified (Grass lowlevel DC amplifier, model 7P1 22E) and recorded on the polygraph with the other electrophysiological signals. This system was calibrated dynamically as described by McArthur and Milsom (1991). Anesthetized animals were fitted with a facemask containing a pneumotachograph connected to a mechanical transducer (Validyne model DP103-18). The signal from the transducer was amplified (Grass low-level DC amplifier, model 7P122G), transcribed directly on the polygraph and also integrated (Gould integrating amplifier, model 13461570). The integrated signal was also transcribed onto the polygraph and from this integrated signal, tidal volume could be measured directly.

2.3. Data recording and analysis Each recording session lasted between 4 and 8 h. In the unanesthetized animals, recording sessions were generally concluded when animals became restless and were no longer entering bouts of established sleep. Recording sessions in the anesthetized animals were concluded after periods of similar length. Continuous measurement of the EEG, EMG, EKG and respiratory variables were made for the duration of each recording session.

85

On different days the gas flowing through the chamber was normoxic–normocapnic, hypoxic– normocapnic (created by diluting air with nitrogen to produce a mixture containing 10.0% O2), or normoxic–hypercapnic (created by mixing CO2 and air to a final concentration of 5.0% CO2). Sleep states were scored for the unanesthetized animals according to conventional criteria (Rechtschaffen et al., 1968). Standard terms (Czeisler et al., 1980) were used to describe sleep sequences, episodes and patterns. Four arousal states were recognized in unanesthetized animals: wakefulness (W), light sleep (LS), slow-wave sleep (well established slow-wave activity, denoted SWS) and rapid-eye movement (REM) sleep. Under urethane anesthesia, three arousal states were scored: State I (desynchronized cortical activity), State II (intermediate activity) and State III (synchronized activity), with EEG activity resembling that in W, LS and SWS, respectively. Comparisons of these natural and anesthetic states have been described in more detail in a previous study (Hunter and Milsom, 1998). All arousal state data were analyzed in 30 sec epochs and classified according to the predominant state during that epoch. The percentage of total recording time spent in each state was then calculated from these data. Two respiratory variables were measured, breathing frequency (fR) and tidal volume (VT), from which ventilation (V: E) was calculated. Respiratory data are presented as absolute values, and also as values normalized to the normoxic values within each state (illustrating the effect of inspired gas concentration, independent of state effects). Statistical comparisons between arousal states in the same treatment group were performed with a two-way ANOVA and Tukey’s least significant difference (LSD) post-hoc test. A significance level of a= 0.05 was set for each comparison. Statistical comparisons of analogous arousal states (based on EEG criteria) in sleeping and urethane-anesthetized animals under hypoxic and hypercapnic conditions were performed via a three-way ANOVA, followed by a post-hoc LSD test. The significance level was again set at a= 0.05 for both tests. These experiments were an incomplete block design; therefore, one of the

86

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

factors included in the three-way ANOVA accounted for subject variation.

3. Results

3.1. Arousal state distribution Altering the inspired concentration of O2 or CO2 did not alter the sleep/wake architecture of unanesthetized golden-mantled ground squirrels appreciably (Fig. 1). Normoxic – normocapnic animals spent significantly less time in LS (14.1%) and significantly more time in SWS (48.4%), compared to W (34.5%). Only 4% of the total recording time was spent in REM sleep (not shown in Fig. 1). The only significant change produced by altering respiratory gases was an increase in the amount of time spent in LS (25.9%) in hypoxia. While hypercapnic and hypoxic exposures did not produce significant changes in the amount of time spent in other states, the amount of time spent in SWS was no longer significantly greater than that spent awake. Under normoxic conditions, urethane-anesthetized animals spent approximately the same amount of time in established arousal states (I and III) and significantly less time in the transitional State II (Fig. 1). Both hypoxia and hypercapnia tended to desynchronize the EEG; animals spent significantly less time in State III compared to State I.

ing frequency dropped further with movement from SWS into REM sleep (59% of W). Hypoxic exposure prompted a large increase in fR in all arousal states, ranging from 192% to 258% of normoxic values (Fig. 3). Again, the effect of arousal state was still evident under hypoxic conditions; fR decreased significantly during SWS and REM sleep (85% and 84% of W values), although

3.2. Breathing frequency In unanesthetized, normoxic – normocapnic animals, breathing frequency decreased progressively from 53 breaths/min in W to 34 breaths/min in SWS (Figs. 2 and 3). Frequency remained depressed at 36 breaths/min in REM sleep. Hypercapnic exposure produced significant increases in fR in all states but REM sleep; hypercapnic frequencies ranged from 122% (REM) to 148% (LS) of normoxic values (Fig. 3). Arousal state-dependent effects on fR, however, were not affected by hypercapnia; fR still decreased progressively as animals moved from W through LS (81% of W values), and into SWS (64% of W values). Breath-

Fig. 1. The effect of hypoxia (10.0% O2; open bars) and hypercapnia (5.0% CO2; hatched bars) on arousal state distribution in both unanesthetized (above; n = 7) and urethaneanesthetized (below; n =7) golden-mantled ground squirrels. Black bars represent data from normoxic – normocapnic animals. + indicates values significantly different from normoxia in the same state; * indicates values significantly different from waking/State I values within the same gas treatment.

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

87

EEG under normoxic–normocapnic conditions (Fig. 4). Hypercapnic exposure did not increase fR significantly in any state (Fig. 5), but the effect of arousal state on fR was still observable. Breathing was significantly slower in States II and III compared to State I (70% and 61% of State I values, respectively). In contrast, hypoxia elicited a significant increase in fR over normoxia in all states; fR ranged from 149% to 186% of normoxic values (Fig. 5). Again, the effect of arousal state was still evident in the hypoxic anesthetized group; decreases in fR were noted in both States II and III (83% and 72% of State I values), although this was only significant in State III.

3.3. Tidal 6olume

Fig. 2. The effect of hypoxia (10.0% O2; open bars) and hypercapnia (5.0% CO2; hatched bars) on respiration (breathing frequency, tidal volume and ventilation) as a function of state in unanesthetized golden-mantled ground squirrels (n= 7). Black bars represent data from normoxic–normocapnic animals. * indicates values significantly different from waking with the same inspired gas; + indicates values significantly different from normoxia in the same state.

the decrease in fR during LS (92% of W values) was very slight. Urethane-anesthetized animals also demonstrated a progressive decrease in fR from 54 breaths/min (State I) to 31 breaths/min (State III) as they moved from the state with a desynchronized EEG into the state with a synchronized

Under normoxic–normocarbic conditions, VT increased progressively from W (1.02 ml/100 g body weight), through LS (1.19 ml/100 g), to SWS (1.34 ml/100 g) in unanesthetized animals. During REM sleep, VT (1.15 ml/100 g) was approximately equal to that in LS. Only the VT in SWS was significantly greater than that in W. Goldenmantled ground squirrels did not alter VT in response to hypercapnia in any arousal state (Fig. 2). Tidal volumes ranged from 95 to 117% of normoxic (same state) values (Fig. 3). As in the normoxic–normocapnic group, an effect of arousal state was evident; hypercapnic animals demonstrated a progressive increase in VT from W, through LS (120% of the W value) into SWS and REM sleep (133% and 138% of the W value, respectively). The increases in VT during SWS and REM sleep were both significant. Hypoxia altered both VT and arousal state effects on VT (Fig. 2). Tidal volume was significantly decreased under hypoxia; these decreases ranged from 72% (W) to 57% (SWS) of the normoxic (same state) values (Fig. 3). Exposure to hypoxia also abolished the effect of arousal state; VT no longer increased during sleep. Tidal volumes during LS, SWS and REM were all between 101% and 105% of the awake value. Urethane-anesthetized animals did not increase VT in States II and III, compared to State I (Fig. 4) in normoxic–normocapnic conditions. Tidal volume increased only slightly from 1.04 ml/100 g

88

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

Fig. 3. The effect of hypoxia (10.0% O2; open bars) and hypercapnia (5.0% CO2; hatched bars) on respiration (breathing frequency, tidal volume and ventilation) as a function of arousal state in unanesthetized animals (n =7). Data are normalized to the normoxic values in the same arousal state. Black bars represent data from normoxic – normocapnic animals. + indicates values significantly different from normoxia in the same state.

(body weight) in State I to 1.13 ml/100g in State II and 1.23 ml/100 g in State III. Anesthetized animals responded to hypercapnia in all states, increasing VT 55–67% over normoxic – normocapnic (same state) values (Fig. 5). Despite this significant increase, an effect of arousal state was also evident; VT in States II and III were 17% and 23% greater than State I values (significant in State III). Hypoxia did not alter VT in the urethane anesthetized animals in any state; VT remained fairly constant, ranging from 94% to 104% of normoxic–normocapnic, same state, values (Fig. 5).

3.4. Ventilation Ventilation decreased from 50.8 to 40.6 ml/min/ 100 g as animals moved from W to SWS, with intermediate levels occurring in LS (43.6 ml/min/ 100 g). Levels in REM sleep (39.5 ml/min/100 g) and SWS were the same. However, none of these decreases were significant. Under hypercapnic conditions, golden-mantled ground squirrels increased V: E significantly in all states (32–64% increases over normoxic, same state values; Fig. 3). As in normoxia, no arousal state-dependent

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

89

Fig. 4. The effect of hypoxia (10.0% O2; open bars) and hypercapnia (5.0% CO2; hatched bars) on respiration (breathing frequency, tidal volume and effective ventilation) as a function of state in urethane-anesthetized animals (n =7). Black bars represent data from normoxic – normocapnic animals. * indicates values significantly different from State I values within the same gas treatment. + indicates values significantly different from normoxic values within the same state.

effects were evident under hypercapnic conditions; V: E was constant across arousal states, ranging from 94% to 102% of the awake, hypercapnic response. Ventilation was also increased significantly in response to hypoxia in all arousal states. These increases ranged from 148% to 169% of the normoxic, same state values (Fig. 3). As with the normoxic–normocapnic and hypercapnic animals, hypoxic animals showed no effect of arousal state on V: E (94–88% of waking values). A progressive reduction in V: E was observed in

the urethane-anesthetized animals, which paralleled the decrease in fR (Fig. 4). This decrease was significant in both States II and III. Ventilation dropped from 56.9 ml/min/100 g in State I to 41.0 ml/min/100 g in State II, to 35.0 ml/min/100 g in State III. Hypercapnic exposure in urethane-anesthetized animals elicited significant increases in V: E in all states, ranging from 58% to 97% increases over normoxic–normocapnic (same state) values (Fig. 5). Although V: E was increased overall, the progressive decrease noted under normoxic–nor-

90

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

mocapnic conditions as animals moved from State I to State III was still evident in the hypercapnic group. As hypercapnic animals moved from State I into States II and III, V: E fell to 82% and 77% of the State I value, respectively. Hypoxia elicited a significant increase in V: E in State III only (Fig. 5). Ventilation in hypoxia ranged from no change in State I (107% of normoxic, same state values), to a slight increase in State II (138%), to a large increase in State III (174%). The effect of arousal

Fig. 5. The effect of hypoxia (10.0% O2; open bars) and hypercapnia (5.0% CO2; hatched bars) on respiration (breathing frequency, tidal volume and ventilation) as a function of cortical activation state in urethane-anesthetized animals (n= 7). Data is normalized to normoxic values in the same arousal state. Black bars represent data from normoxic–normocapnic animals. + indicates values significantly different from normoxia in the same state; * indicates values significantly different from State I on the same inspired gas.

state was not maintained under hypoxic conditions; V: E was fairly constant across all three arousal states.

4. Discussion

4.1. Arousal states In the present study, we attempted to select levels of hypoxia and hypercapnia to study that would produce significant ventilatory effects without disturbing normal sleep/wake architecture. In general, this was the case. The only significant change produced by altering respiratory gases in unanesthetized animals was an increase in the amount of time spent in the drowsy state (LS) in hypoxia. As a consequence, animals now spent roughly equal time awake, in light sleep and in established SWS. There was a small, but insignificant trend for animals to spend less time in SWS under both hypoxic and hypercapnic conditions. In the urethane anesthetized animals, this trend became significant; both hypoxia and hypercapnia led to animals spending significantly less time in the state with established slow waves (State III). These results are consistent with the findings of others in another rodent, the rat. Neither Pappenheimer (1977) nor Megirian et al. (1980) reported any significant effect of 5.0% CO2 on SWS in rats. Ioffe et al. (1984), however, found that exposure to 6.0–8.0% CO2 greatly increased the amount of desynchronized EEG activity in rats suggesting that 5% CO2 is just below the threshold for arousal by hypercapnia. The data obtained in the present study for the ground squirrel agree well with this. The effects of hypoxia on arousal state distribution in this study also correlated well with previous studies in rats. Pappenheimer (1977) first noted that hypoxia decreased the total amount of SWS in rats, and changed the normal sleep pattern to a series of brief, incompletely developed episodes. Laszy and Sarkadi (1990) subsequently divided SWS into light (intermittent) and deep (established) states, and found that, not only was the proportion of wakefulness increased, but also that during the sleep periods, intermittent activity

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

predominated over established SWS. Other studies have shown that sleep was interrupted frequently and the transitions between light SWS and wakefulness were greatly increased during hypoxic exposure (Ryan and Megirian, 1982, Hale et al., 1984). The modest effect of hypoxia in the present study suggests that 10% O2 is probably just at the threshold for arousal by hypoxia in the ground squirrel. Few studies provide data regarding hypoxic and hypercapnic effects on cortical activity under anesthesia. Of those that do, the anesthetics used varied considerably. Preliminary observations in urethane-anesthetized rats indicate that hypoxic and hypercapnic exposure produce EEG desynchronization (Tamaki and Nakayama, 1987). While these data suggest that urethane anesthesia does make animals more prone to changing states of cortical activation in response to changes in respiratory gas levels of the magnitude used in the present study, compared to unanesthetized animals, the increased sensitivity was small and the responses of both groups were qualitatively similar.

4.2. Ventilatory responses in awake/sleeping animals Exposure to 10.0% O2 elicited a large increase in fR and small decreases in VT in awake goldenmantled ground squirrels. As a result, V: E increased by only 50%. While the overall increase in V: E on exposure to 10% O2 in awake golden-mantled ground squirrels in the present study was less than that found in some previous studies on this species (McArthur and Milsom, 1991, Webb and Milsom, 1994), 10.0% O2 is right around the threshold for the hypoxic ventilatory response in this species and, thus, this is not surprising. As in the awake state, fR and V: E also were elevated significantly, and VT reduced, in all other arousal states during hypoxia compared to normoxia in that state. In relative terms, the hypoxic sensitivity of the golden-mantled ground squirrel was increased slightly during SWS and REM sleep. The hypoxic ventilatory responses observed in the present study in awake golden-mantled ground squirrels are in accordance with other

91

studies of acute, poikilocapnic hypoxia in awake animals (Walker et al., 1985, Davies and Schadt, 1989, McArthur and Milsom, 1991, Mortola, 1991, Webb and Milsom, 1994, 1994). While there are qualitative differences between studies in the extent to which increases in V: E arise from changes in fR and VT, these are likely due to differences in stimulus intensity. Cragg and Drysdale (1983) found that, in the rat, both V: E and VT were related to PO2 in a hyperbolic fashion, whereas fR was related to PO2 in a linear fashion. With a modest hypoxic stimulus, the main ventilatory response was an increase in fR. As PO2 levels decreased, however, increases in VT became more important to the overall increase in ventilation. The hypoxic ventilatory response during sleep has been studied in few species. Aside from studies on young animals, work has mainly been confined to dogs where the hypoxic sensitivity during SWS is either retained (Phillipson et al., 1978) or reduced (Bowes et al., 1981). The hypoxic ventilatory response during tonic REM sleep was similar to that during SWS, while the response during phasic REM sleep was either depressed (Sullivan et al., 1979) or elevated (Hedemark and Kronenberg, 1982) compared to that seen in SWS. The hypoxic ventilatory response in rats (Pappenheimer, 1977), on the other hand, increased during SWS, as it did in the present study on the golden-mantled ground squirrel. Exposure to 5.0% CO2 elevated V: E in awake golden-mantled ground squirrels due to a significant increase in fR while VT remained constant. Sleeping ground squirrels were equally responsive to 5.0% CO2; the levels of V: E were significantly greater than normoxic values in all sleep states. Exposure to 5.0% CO2 did not alter the effect of state on fR or VT. While the golden-mantled ground squirrels responded to hypercapnia exclusively by increasing breathing frequency in the present study, this species has been observed, under similar experimental conditions, to increase V: E significantly via increases in VT only (Webb and Milsom, 1994) and via equal increases in both fR and VT (McArthur and Milsom, 1991). Similar variations in the relative contributions of changes in fR and

92

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

VT to increases in ventilation have been observed in many species (Hedemark and Kronenberg, 1982, Walker et al., 1985, Cragg and Drysdale, 1983). Studies of sleep effects on the hypercapnic ventilatory reflex in animals are limited and somewhat inconclusive. The hypercapnic ventilatory response was decreased during SWS in both dogs (Sullivan et al., 1979, Bowes et al., 1981) and cats (Santiago et al., 1981), and during REM sleep in dogs (Sullivan et al., 1979), unchanged in goats (Parisi et al., 1992) but increased in rats (Pappenheimer, 1977). Thus, the majority of data indicate that, in general, sleep (SWS and REM sleep) is characterized by a blunted sensitivity to CO2, with O2 sensitivity either decreased, or preserved at waking levels. Respiratory chemosensitivity during REM sleep typically appears to be the same as during SWS, although responses during this state are more irregular. The apparent increase in chemosensitivity during SWS in rodents are an interesting anomaly. The mechanistic basis of these differences which have been recorded in the literature are unclear but may represent differences in the extent to which changes occur in the sensitivity of the chemoreceptors, differences in the magnitude of the wakefulness stimulus which is withdrawn in sleep, or differences in levels of behavioural stimuli present in undisturbed animals in the resting, awake state.

4.3. Ventilatory responses under urethane anesthesia As with unanesthetized animals, urethane-anesthetized animals responded to 10.0% O2 by increasing fR in all states. Hypoxia had no effect on VT and, due to variability in these responses, V: E did not change during State I, showed a small, but insignificant increase in State II and only increased significantly in State III. In anesthetized animals, fR decreased as they moved from the state with desynchronized EEG activity (State I) to the state with well established slow-wave activity (State III) and the extent of the decrease was approximately the same in the normoxic and hypoxic animals. Due to variability

between animals, there was no change in VT or V: E between states under hypoxic conditions. Because V: E decreased as animals entered State III under normoxic conditions, sensitivity to hypoxia increased during State III. Hypercapnia (5.0% CO2) significantly stimulated ventilation in urethane-anesthetized animals in all states. The increased V: E was due only to increases in VT; fR did not change. These data are consistent with those from previous studies in urethane-anesthetized rats and cats (Hughes et al., 1982, Waldrop, 1982, Hayashi and Sinclair, 1991). Arousal state effects on respiration were not altered at all by hypercapnic exposure. All three state-dependent changes observed in the normoxic animals (an increase in VT, and a decrease in fR and V: E) were retained during hypercapnic exposure.

4.4. Unanesthetized 6ersus anesthetized animals In a previous study, we found that both unanesthetized and anesthetized animals, when allowed to oscillate spontaneously between arousal states for prolonged periods, spent approximately equal amounts of time in states with completely desynchronized (W and State I) and synchronized (SWS and State III) EEG patterns (Hunter and Milsom, 1998). The modest levels of hypoxia (10% O2) and hypercapnia (5% CO2) used in the present study reduced the amount of time spent in the state with established slow waves (State III) in the anesthetized animals but not the unanesthetized animals. There was a similar, nonsignificant trend in the data for the unanesthetized animals, however, suggesting that the effects on both groups were qualitatively similar. Breathing frequency increased significantly in response to hypoxia in both unanesthetized and urethane-anesthetized golden-mantled ground squirrels in all arousal states. The decrease in respiratory frequency associated with the transition from states with desynchronized cortical activity to states with synchronized cortical activity was still present in hypoxia in both groups. Overall, exposure to 10.0% O2 stimulated V: E in all states in the unanesthetized group, and in State III in the urethane-anesthetized group. These in-

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

creases in fR and V: E were such that there was an increase in relative hypoxic sensitivity as animals moved from desynchronized EEG states (W/State I) to synchronized EEG states (SWS/State III). The data collected from both groups were qualitatively, and, for the most part, quantitatively similar suggesting that changes in cortical activity states under urethane anesthesia mimic changes in vigilance states in unanesthetized animals in terms of their effect on respiratory function. Although both unanesthetized and urethaneanesthetized groups increased V: E in response to hypercapnic stimulation in all states, the respiratory pattern changes employed to produce this increase differed between the two groups. Unanesthetized animals responded with increases in fR but not VT whereas urethane-anesthetized animals responded with a large increase in VT but no change in fR. While the basis for this difference in the present study remains unclear, previous studies have shown that unanesthetized golden-mantled ground squirrels increased VT but did not alter fR under hypercapnic conditions (McArthur and Milsom, 1991, Webb and Milsom, 1994). The effect of arousal state on fR was not altered by hypercapnic exposure in either group. Transition from desynchronized (W/State I) to synchronized (SWS/State III) cortical activity was still accompanied by a decrease in fR and increase in VT. As with the hypoxic ventilatory response, the relative sensitivity to 5.0% CO2 increased in states with a synchronized EEG. Again, the net changes in ventilation in both groups were qualitatively, and, for the most part, quantitatively similar. In conclusion, changes between the arousal states evident under urethane anesthesia are accompanied by similar changes in hypoxic and hypercapnic sensitivity as changes between natural arousal states in waking/sleeping animals with similar EEG patterns. Thus, states which appear superficially similar based on cortical activity patterns and breathing patterns also prove to be similar in terms of ventilatory chemoreflexes. Given that anesthesia removes all contribution of behavioural stimuli to ventilation, the data also suggest that the enhancement of ventilatory responses in states with synchronized EEGs in rodents is not due solely to removal of behavioural stimuli.

93

Acknowledgements This study was supported by the NSERC of Canada.

References Bowes, G., Townsend, E.R., Kozar, L.F., Bromley, S.M., Phillipson, E.A., 1981. Effect of carotid body denervation on arousal response in sleeping dogs. J. Appl. Physiol. 51, 40 – 45. Colrain, I.M., Trinder, J., Fraser, G., Wilson, G.V., 1987. Ventilation during sleep onset. J. Appl. Physiol. 63, 2067 – 2074. Cragg, P.A., Drysdale, D.B., 1983. Interaction of hypoxia and hypercapnia on ventilation, tidal volume and respiratory frequency in the anaesthetized rat. J. Physiol. 341, 477 – 493. Czeisler, C.A., Borbely, A., Hume, K.I., Kobayashi, T., Kronauer, R.E., Schulz, H., weitzman, E.D., Zimmerman, J.C., Zulley, J., 1980. Glossary of standardized terminology for sleep — biological rhythm research. Sleep 2, 287 – 288. Davies, D.G., Schadt, J.C., 1989. Ventilatory responses of the ground squirrel, Spermophilus tridecemlineatus, to various levels of hypoxia. Comp. Biochem. Physiol. 92A, 255 – 257. Hale, B., Megirian, D., Pollard, M.J., 1984. Sleep – waking pattern and body temperature in hypoxia at selected ambient temperatures. J. Appl. Physiol. 57, 1564 – 1568. Hayashi, F., Sinclair, J.D., 1991. Respiratory pattems in anesthetised rats before and after anemic decerebration. Respir. Physiol. 84, 61 – 76. Hedemark, L.L., Kronenberg, R.S., 1982. Ventilatory and heart rate responses to hypoxia and hypercapnia during sleep in adults. J. Appl. Physiol., Respir. Environ. Exer. Physiol. 53, 307 – 312. Hendricks, J.C., Kovalski, R.J., Kline, L.R., 1991. Periodic respiratory muscle patterns and sleep-disordered breathing during rapid eye movement sleep in the English bulldog. Am. Rev. Respir. Dis. 144, 1112 – 1120. Hughes, E.W., Martin-Body, R.L., Sarelius, I.H., Sinclair, J.D., 1982. Effects of urethane – chloralose anaesthesia on respiration in the rat. Clin. Exp. Pharmacol. Physiol. 9, 119 – 127. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardiorespiratory effects. Respir. Physiol. (in press). Ioffe, S., Jansen, A.H., Chernick, V., 1984. Hypercapnia alters sleep state pattern. Sleep 7, 219 – 222. Jacky, J.P., 1980. Barometric measurement of tidal volume: effects of pattern and nasal temperature. J. Appl. Physiol., Respir. Environ. Exer. Physiol. 49, 319 – 325. Laszy, J., Sarkadi, A., 1990. Hypoxia-induced sleep disturbance in rats. Sleep 13, 205 – 217.

94

J.D. Hunter et al. / Respiration Physiology 112 (1998) 83–94

McArthur, M.D., Milsom, W.K., 1991. Ventilation and respiratory sensitivity of euthermic Columbian and goldenmantled ground squirrels (Spermophilus columbianus and Spermophilus lateralis) during the summer and winter. Physiol. Zool. 64, 921–939. Megirian, D., Ryan, A.T., Sherrey, J.H., 1980. An electrophysiological analysis of sleep and respiration of rats, breathing different gas mixtures: diaphragmatic muscle function. Electroencephalogr. Clin. Neurophysiol. 50, 303– 313. Mortola, J.P., 1991. Hamsters versus rats: ventilatory, responses in adults and newborns. Respir. Physiol. 85, 305– 317. Netick, A., Foutz, A.S., 1980. Respiratory activity and sleep– wakefulness in the deafferented, paralyzed cat. Sleep 3, 1–12. Orem, J., 1994. The wakefulness stimulus for breathing. In: Saunders, N.A., Sullivan, C.E. (Eds.), Sleep and Breathing, 2nd ed. Marcel Dekker, New York, pp. 113–155. Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J. Physiol. 266, 191–207. Parisi, R.A., Edelman, N.H., Santiago, T.V., 1992. Central respiratory carbon dioxide chemosensitivity does not decrease during sleep. Am. Rev. Respir. Dis. 145, 832–836. Phillipson, E.A., Bowes, G., 1986. Control of breathing during sleep. In: Cherniack, N.S., Widdicombe, J.G. (Eds.), Handbook of Physiology, Section 3: The Respiratoy System, vol. II: Control of Breathing, part 2. American Physiological Society, Washington, DC, pp. 649–689.

.

Phillipson, E.A., Sullivan, C.E., Read, D.J.C., Murphy, E., Kozar, L.F., 1978. Ventilatory and waking responses to hypoxia in sleeping dogs. J. Appl. Physiol. 44, 512 – 520. Rechtschaffen, A., Kales, A., Berger, R.J., Dement, W.C., Jacobson, A., Johnson, L.C., Jouvet, M., Monroe, L.J., Oswald, I., Roffward, H.P., Roth B., Walter R.D., 1968. A Manual of Standardized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects. US Government Printing Office, Washington, DC. Ryan, A.T., Megirian, D., 1982. Sleep – wake patterns of intact and carotid sinus nerve sectioned rats during hypoxia. Sleep 5, 1 – 10. Santiago, T.V., Sinha, A.K., Edelman, N.H., 1981. Respiratory flow – resistive load compensation during sleep. Am. Rev. Respir. Dis. 123, 382 – 387. Sullivan, C.E., Murphy, E., Kozar, L.F., Phillipson, E.A., 1979. Ventilatory responses to CO2 and lung inflation in tonic versus phasic REM sleep. J. Appl. Physiol. 47, 1304 – 1310. Tamaki, Y., Nakayama, T., 1987. Effects of air constituents on thermosensitivities of preoptic neurons: hypoxia versus hypercapnia. Pflugers Arch. 409, 1 – 6. Waldrop, T.G., 1982. Posterior hypothalamic modulation of the respiratory response to CO2 in cats. Pflugers Arch. 418, 7 – 13. Walker, B.R., Adams, E.M., Voelkel, N.F., 1985. Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J. Appl. Physiol. 59, 1955 – 1960. Webb, C.L., Milsom, W.K., 1994. Ventilatory responses to acute and chronic hypoxic hypercapnia in the ground squirrel. Respir. Physiol. 98, 137 – 152.