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Sleep/wakefulness versus urethane anesthesia: analogous arousal states for respiratory control? Hunter, Julia D. 1993

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SLEEP/WAKEFULNESS VERSUS URETHANE ANESTHESIA:ANALOGOUS AROUSAL STATES FOR RESPIRATORY CONTROL?byJULIA DIANE HUNTERB.Sc., The University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESNEUROSCIENCEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAOctober, 1993© Julia Diane Hunter, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of The University of British ColumbiaVancouver, CanadaDate  Octoba, /5/ /993 DE-6 (2/88)fiABSTRACTUnder light urethane anesthesia, animals cycle through patterns of EEG activity whichsuperficially appear like waking (W), light sleep (LS) and slow-wave sleep (SWS) in theunanesthetized animal, referred to as States I, II and DI, respectively. The major question in thisthesis was whether the urethane anesthetic states are analogous to the natural states with similarEEG patterns, at least in terms of respiratory and cardiovascular function. Therefore, the firstobjective of this study was to examine the effects of sleep state on respiratory and cardiovascularvariables, and to compare these effects to those observed during superficially similar arousalstates in urethane-anesthetized animals. In order to further determine whether the arousal statesobserved during urethane anesthesia were analogous to sleep/wake states in terms of respiratoryand cardiovascular control, the effects of sleep state on hypoxic and hypercapnic ventilatoryresponses were assessed and compared to the effects of arousal states with similar EEG patternson these responses in urethane-anesthetized animals. Electroencephalographic (EEG),electromyographic (EMG) and electrocardiographic (EKG) activity were monitored during thedifferent arousal states in sleeping and anesthetized animals under normoxic, hypoxic andhypercapnic conditions. Respiratory variables were also measured under these conditions usingwhole-body plethysmography in unanesthetized animals, and using a face mask andpneumotachograph in the anesthetized animals.Golden-mantled ground squirrels spent approximately 33% of the time in wakefulness(W), 31% to 14% in light sleep (LS) and 32% to 48% in slow-wave sleep (SWS). Less than 5%of the time was spent in rapid-eye-movement (REM) sleep. Urethane-anesthetized animals spentapproximately the same amount of time in each arousal state (States I, 11 and III), and theseproportions were the same as those observed in unanesthetized animals in states with similarcortical patterns. Hypoxic (10.0% 02) and hypercapnic (5.0% CO2) conditions reduced theamount of synchronized activity and produced greater amounts of desynchronized activity, butin different ways. These effects, however, were the same in both unanesthetized and urethane-anesthetized animals. In both groups, hypoxic exposure resulted in more LS/State II, while theamount of time spent in W/State I was unaltered, but hypercapnic exposure produced greateramounts of W/State I, while the amount of time spent in LS/State H was unaltered.In all three parts of the present study, sleep states exerted a strong, negative influence onbreathing frequency, but had less consistent effects on tidal volume. In general, the net effectwas that sleep reduced minute ventilation, although in one part of the study, this decrease wasa nonsignificant trend only. These effects of sleep were similar in SWS and REM sleep. Theurethane-anesthetized animals demonstrated exactly the same alterations in respiratory variablesin arousal states exhibiting similar EEG patterns.Unlike many previous reports in other species, the present study revealed that golden-mantled ground squirrels increase their ventilatory sensitivity to hypoxia and hypercapnia duringsleep. The urethane-anesthetized animals also showed these same increases in ventilatorysensitivity to hypoxia and hypercapnia as they moved into arousal states with synchronizedcortical activity (State III).Like respiration, heart rate was also affected by sleep/wake states in the golden-mantledground squirrel. As animals moved into deeper sleep states (ie. SWS from W and LS), heart ratedecreased. Additionally, more variability in the heart rate was observed during sleep, due to agreater preponderance of respiratory sinus arrhythmia. The sinus arrhythmia was most evidentduring REM sleep. Urethane anesthesia increased heart rate well above the levels observed inivthe unanesthetized animals; this increase was due in part to the abolishment of respiratory sinusarrhythmias. However, in spite of this increase, heart rate still decreased in arousal states withsynchronized EEG patterns in the urethane-anesthetized animals, Thus, the increase in heart rateappeared to be a tonic effect of the anesthesia, but although superimposed on this backgroundincrease, the effect of arousal state was still evident.In conclusion, the changing cortical patterns observed under light urethane anesthesiamimic sleep/wake states with respect to natural oscillations between arousal states, their influenceon respiratory and cardiovascular control under normoxic, hypoxic and hypercapnic conditions,as well as the changes in state induced by hypoxic and hypercapnic exposure. Theseobservations support the hypothesis that, in terms of cardio-respiratory function, the various statesseen under urethane anesthesia with EEG activity similar to wakefulness, light sleep and slow-wave sleep, are analogous states.TABLE OF CONTENTSpageAbstractTable of contentsList of Tables^ viList of Figures viiAcknowledgementsIntroduction^ 1Materials and Methods^ 10Results^ 27Discussion 86General Conclusions^ 136Literature Cited 139Appendix^ 155viLIST OF TABLESpageTable I:^Sleep state distribution of various species of the Spermophilus genus^94Table II:^Sleep state distribution in other rodent species^ 95Table ILI:^Respiratory pattern changes during sleep in animal subjects^101Table IV:^Respiratory trends from sleep studies on human subjects 102LIST OF FIGURESviipageFigure 1:Figure 2:Figure 3:Figure 4:Figure 5:Figure 6:Figure 7:Figure 8:Figure 9:Figure 10:Figure 11:Schematic diagram of experimental apparatus for ventilation measurements(using whole-body plethysmographic technique) and recording of electro-physiological variables in unanesthetized animals^ 15Schematic diagram of experimental apparatus for ventilation measurements(using mask and pneumotachograph) and recording of electrophysiologicalvariables in urethane-anesthetized animals. 17Recordings of EEG, EMG, EKG and Respiration during wakefulness (W),slow-wave sleep (SWS) and rapid-eye movement (REM) sleep in anunanesthetized golden-mantled ground squirrel.^ 22Recordings of EEG, EMG, EKG and Respiration during State I(desynchronized EEG activity), State II (intermittent activity) and State III(synchronized EEG activity) in a urethane-anesthetized golden-mantledground squirrel.^ 24Distribution of time spent in the five arousal states scored in unanesthetizedgolden-mantled ground squirrels.^ 29Chronological record of arousal state in an unanesthetized golden-mantledground squirrel.^ 31Distribution of time spent in the three arousal states scored in anesthetizedgolden-mantled ground squirrels. Data for unanesthetized animals insimilar EEG states are included for comparison.^ 33Chronological record of arousal state in a urethane-anesthetized golden-mantled ground squirrel.^ 35The proportion of state changes associated with transitional activity inunanesthetized and urethane-anesthetized animals.^ 38The effect of arousal state on three respiratory variables in golden-mantledground squirrels.^ 40The effect of arousal state on respiration in urethane-anesthetized golden-mantled ground squirrels. 43viiipageFigure 12:^The effect of arousal state on heart rate in unanesthetized animals.^48Figure 13:^The effect of arousal state on heart rate in urethane-anesthetized animals^50Figure 14:^The effect of arousal state on respiratory sinus arrhythmia in unanesthetizedanimals.^ 52Figure 15:^The effect of hypoxia and hypercapnia on arousal state distribution inunanesthetized animals.^ 55The effect of hypoxia and hypercapnia on respiration (breathing frequency,tidal volume and minute ventilation) and on the associated state-dependentchanges in these respiratory variables in unanesthetized golden-mantledground squirrels.^ 58The effect of hypoxia and hypercapnia on respiration (breathing frequency,tidal volume and minute ventilation) within each arousal state inunanesthetized animals^ 60The effect of arousal state on the hypoxic and hypercapnic ventilatoryresponses in unanesthetized animals.^ 62The effect of hypoxia and hypercapnia on arousal state distribution inurethane-anesthetized animals. 67The effect of hypoxia and hypercapnia on the proportion of state changesassociated with transitional activity in urethane-anesthetized animals.^69The effect of hypoxia and hypercapnia on respiration (breathing frequency,tidal volume and minute ventilation) and on the associated state-dependentchanges in these respiratory variables. 72The effect of hypoxia and hypercapnia on respiration (breathing frequency,tidal volume and minute ventilation) within each arousal state in urethane-anesthetized animals. 74The effect of arousal state on the hypoxic and hypercapnic ventilatoryresponses in urethane-anesthetized animals^ 76The effect of hypoxia and hypercapnia on heart rate and on arousal state-dependent changes in heart rate under urethane anesthesia.^81Figure 16:Figure 17:Figure 18:Figure 19:Figure 20:Figure 21:Figure 22:Figure 23:Figure 24:ixpageFigure 25:Figure 26:Figure 27:Figure 28:Figure 29:The combined effect of arousal state and alterations in inspired gasconcentration (hypoxia and hypercapnia) on heart rate in urethane-anesthetized animals.^ 83The effect of urethane anesthesia and inspired gas composition (hypoxiaand hypercapnia) on respiratory sinus arrhythmia.^ 85Comparison of the time which unanesthetized and urethane-anesthetizedanimals spent in arousal states exhibiting desynchronized, transitionaland synchronized EEG patterns.^ 122The alteration in arousal state distribution during hypoxia and hypercapniain urethane-anesthetized and unanesthetized animals.^124Arousal state effects on minute ventilation in the unanesthetized andurethane-anesthetized, normoxic groups, and the effect of hypoxia andhypercapnia on minute ventilation and the associated state changes inthese groups. 129xACKNOWLEDGEMENTS During the course of this study, I have managed to become indebted to a great manypeople. First and foremost, I would like to extend sincere thanks and appreciation to mysupervisor, Dr. Bill Milsom, for his advice, encouragement, and an (almost) unfailing sense ofhumour in spite of "occasional" tardiness on my part. His uncanny ability to make the machineswork simply by approaching them continues to puzzle me.Much appreciation is also owed to my family for providing a supportive background. Inparticular, I would like to thank my father, Dr. Bryant Hunter, for his help and understandingover the years, and for generating and sharing my interest in science. Thanks also to my mother,Doris Hunter, to my brothers, Patrick and James, and to Dan Redekopp for their love, supportand patience.I would also like to thank all the other Milsom lab members for providing advice,assistance and being in the same situation. Thanks especially to Supriti Bharma for herfriendship and statistical consultation, to Rhonda Garland for countless supportive hours as wewrote together, and to Mike Harris and Richard Kinkhead for providing plenty of humor andtechnical assistance. I would like to thank the many graduate students of Zoology for makingthis such a great experience. Thanks in particular to Dr. Peter Arthur for listening endlessly tocountless versions of talks and for letting me win at cribbage so often, and to Dr. AgnesLacombe for her friendship and advice. Appreciation is also owed to my committee, Dr. J.Ledsome, Dr. P. Reiner and Dr. J. Steeves, for allowing me to pester them so much, in spite ofextremely busy schedules, during the final stages of the thesis preparation.I have certainly enjoyed being a "Milsornite", and I hope to tour again with "Captain Zotand the Lab Rats" on their upcoming reunion circuit.1SLEEP/WAKEFULNESS VERSUS URETHANE ANESTHESIA: ANALOGOUS AROUSAL STATES FOR RESPIRATORY CONTROL? Respiration functions to maintain acid-base and blood gas homeostasis within anorganism. Necessarily, the system is responsive to exogenous and endogenous fluctuations ofCO2 and 02. The current concept of respiratory motor output generation postulates that a basicrespiratory rhythm develops from a central rhythm generator (CRG) within the brain and that thisrhythm is subsequently modulated at a central pattern generator (CPG), which receives andresponds to afferent information from various mechano- and chemoreceptors (see for review: VonEuler, 1986; Feldman, 1986; Milsom, 1990a, 1990b; Smatresk, 1990). An important additionalinput has also been recognized, that of descending afferent information from suprapontineregions. These various inputs comprise the behavioural, or voluntary, component of respiratorycontrol and include inputs from such diverse structures as the limbic system (emotive reactions),the hypothalamus (thermal and defense responses), the forebrain (thought, attention andanticipation) and the periaqueductal gray region (an area involved in producing analgesia)(Redgate, 1963; Mayer and Liebeskind,1974; Frysinger et a!., 1984; Harper et al., 1984; Waldropet al., 1986; Zhang et al., 1986; Marks et a/., 1987; Ni et al., 1988; Harper et al., 1990; Shea etal., 1990; Waldrop, 1991. See for review: Phillipson, 1978; Orem, 1978; Remmers, 1981;Phillipson and Bowes, 1986). Finally, a host of other inputs including such things as gender andsex hormones also modulate respiratory pattern (Tatsumi et W., 1991), but by poorly understoodmechanisms.Of note for this thesis, the state of consciousness (arousal state) and its effect on2respiration have recently received much attention. The sleeping organism is not simply "switchedoff', but rather moves between distinct and actively regulated states. This movement betweenarousal states (ie. the circadian and ultradian rhythms) is generated by changing levels of activityin various neurotransmitter systems (ie. Lydic et al, 1984; Hobson et al., 1986; Lydic, 1988;Siegel, 1990; Steriade and McCarley, 1990; Steriade, 1992). It has been recognized recently thatchanges in arousal or sleep state are associated with changes in the function of mostphysiological systems (reviewed by Orem and Keeling, 1980; Lydic, 1987).The concept of wakefulness per se acting as a separate respiratory stimulus was firstpostulated by Fink (1961) and has since been greatly expanded (Hugelin and Cohen, 1963;Severinghaus and Mitchell, 1962; Plum and Leigh, 1981; Orem et al., 1977; Orem and Lydic,1978; Orem, 1978). Early studies demonstrated that stimulation of the reticular activating systemproduced arousal (Moruzzi and Magoun, 1949) while bilateral lesions in this region producecoma and sleep-like behaviours (Lindsley et al., 1950). Hugelin and Cohen (1963) first notedthat stimulation of the reticular activating system also resulted in respiratory activation (increasedbreathing frequency, air flow and tidal volume), which provided physiological support for aninteraction between arousal state and respiration. These observations led Orem to initiallypostulate (1986) that the neural substrate for the wakefulness stimulus lay between the caudalpons and posterior hypothalamus. Respiratory neurons which alter their firing patterns inconjunction with changes in arousal state have been identified at several sites, including thepneumotaxic area (Sieck and Harper, 1980), medullary respiratory pump neurons (Orem et al.,1974; Netick et al., 1977; Orem et al., 1985) and various neurons supplying upper airwaymuscles (eg. hypoglossal motor neurons; Richard and Harper, 1991). Recently, Orem and Dick(1983) developed a procedure for classifying respiratory neurons according to their degree of3respiratory modulation. They found that the degree of modulation by arousal state on arespiratory neuron was directly proportional to its degree of modulation by non-respiratory inputs;cells with activity responsive to non-respiratory inputs were most affected by changes in arousalstate. These cells also presumably mediate voluntary inhibition of breathing, based on theobservations that some of these cells are activated intensely when respiration is inhibitedbehaviourally (Orem and Brooks, 1986; Orem, 1987; Orem, 1988; Orem, 1990), and mayrepresent a site of interaction between voluntary and metabolic control. As such, they are alikely anatomical correlate for the input of the wakefulness stimulus.Until 1953, sleep was thought to be a homogenous process, and changes in respiratorycontrol were studied under this premise. In 1953, Aserinsky and Kleitman demonstrated thatsleep is actually composed of two distinct arousal states, with separate mechanisms andphysiology, now known as rapid-eye movent (REM) sleep and non-rapid-eye-movement (NREM)sleep (Aserinsky and Kleitman, 1953). NREM sleep is composed of four states. States 1 and2 are the periods of drowsiness and light sleep. States 3 and 4 are deeper stages of sleep whichare identified by well established periods of slow-wave activity in the EEG. These latter twostates are also known as slow-wave sleep (SWS) (Horne, 1988). SWS is characterized by largeamplitude, slow frequency waves ("delta" waves: 1-4 Hz) in the electroencephalogram (EEG),with decreases in muscle tone (EMG) (Kayed et al., 1979; Brunner et a/., 1990), bodytemperature (Glotzbach and Heller, 1976; Barrett et a/., 1993), metabolism (White et al., 1985)and the activity of various physiological systems (reviewed by Orem and Keeling, 1980;Phillipson and Bowes, 1986; Lydic, 1987). In fact, slow-wave sleep is in part defined as a stateof reduced metabolism (Brebbia and Altschuler, 1965) and the activities of nearly all systems aredepressed in this state (reviewed by Orem and Keeling, 1980; Phillipson and Bowes, 1986; Lydic,41987). In contrast, REM sleep presents a low amplitude, high frequency EEG pattern, similarto that observed in the waking state, accompanied by muscular atonia of skeletal muscles.Thermoregulatory processes are absent (Parmeggiani, 1980; Walker et al., 1983; Glotzbach andHeller, 1985) and metabolic rate appears inconsistent during REM sleep. The depression offunction seen in SWS is maintained in REM sleep, but with phasic alterations now superimposed.Early studies on respiratory control during sleep defmed sleep behaviourally only and thereforepresented mixed results obtained from SWS and REM sleep. Not until several years after thediscovery of the two separate sleep states did studies of respiratory control during sleep accountfor these two states (Birchfield et al., 1959; Bulow, 1963). Since this time, much work has beendone on breathing during sleep, using electroencephalograms to characterize and define sleepstates. Subsequent studies have now analyzed respiratory control during SWS and REM sleep,and found the system to behave very differently under the two conditions.At the behavioural level, breathing patterns have been analyzed as a function of arousalstate in many mammalian species. Cats, dogs, goats, pigs, primates and rodents (Remmers et al.,1976; Orem et al., 1977; Foutz eta!., 1979; Santiago eta!., 1981; Baker eta!., 1981; Phillipsonet al., 1976; DeMesquita and Aserinsky, 1981; Parisi et al., 1987; Scott et al., 1990; Guthrie etal., 1980; Martin et al., 1990; Pappenheimer, 1977; Megirian et al., 1980) show similar changesin respiratory pattern with changes in arousal state. Breathing typically slows and deepens duringSWS, and becomes irregular, rapid and shallow during REM. Similar changes are generally seenin studies on humans, but the results have been much less consistent (Ingvar and Bulow, 1963;Fink et al., 1963; Douglas et al., 1982). Sufficient species variability does exist in the sleepliterature at the quantitative level, however, that observations of breathing pattern as a functionof arousal state cannot necessarily be extrapolated from one species to another.5Respiratory control has been divided into behavioural (including voluntary) and autonomic(metabolic) control (Phillipson and Bowes, 1986). The two systems operate to differing extentsdepending on arousal state, producing the alterations in respiration characteristic of each arousalstate. During wakefulness, behavioural control is capable of overriding metabolic control.Phillipson and Bowes contended that the interaction between these two systems was in the spinalcord (based on direct projections to spinal motorneurons). Orem (1990) contends that the twosystems also interact within the brainstem. Regardless of where the integration occurs, the onsetand establishment of SWS involves a gradual loss of behavioural input and breathing becomesgoverned primarily by metabolic control. This theory is supported by several observations, bothin experimental animals (Phillipson et al., 1976; Remmers et al., 1976; Orem a al., 1977;Sullivan et al., 1978; Foutz et al., 1979; Naifeh and Kamiya, 1981; Colrain a al., 1987; Pack etal., 1992) and in clinical situations (Fink, 1961; Severinghaus and Mitchell, 1962; Guilleminaultet al., 1981; McNicholas a al., 1983). Furthermore, the thalamus, an important area in thegeneration of slow waves, has been shown to contain neurons which develop respiratorymodulation under conditions of increased respiratory drive, ie., which can produce arousal underconditions of increased metabolic activity (Chen et al., 1992).REM sleep, in contrast to SWS, involves an apparent "reactivation" of the cortex. Notsurprisingly, breathing during this state is different than that seen in the other two states.Respiratory control was originally postulated to be independent of metabolic control during REMsleep (Phillipson, 1978), although disparate data existed regarding respiratory responses in thisstate (eg. Remmers a al., 1976; Phillipson a a/., 1976; Orem et al., 1977; Netick and Foutz,1980; DeMesquita and Aserinsky, 1981; Baker a al., 1981; Scott et a/., 1990). REM sleep isdivided into two distinct phases, distinguished as relatively quiet (tonic REM) and active (phasic6REM) periods (Moruzzi, 1963). Phasic REM sleep is the period associated with eye movements,muscular twitches and pontogeniculo-occipital (PGO) waves, and is presumably superimposedon the tonic phase of REM sleep. Many studies, even recently, do not distinguish between thetwo phases of REM sleep when reporting data, even though differences in breathing pattern areobservable between these two states. Phillipson and Bowes (1986) suggest that during tonicREM sleep, breathing is governed by metabolic changes as in SWS, and that respiration ismodulated phasically by other inputs, coincident with other phasic phenomena, during phasicREM sleep. Thus, irregular breathing patterns and reduced respiratory reflexes are correlatedwith the appearance of PG0 waves and other phasic events (Netick and Foutz, 1980; Orem,1980; Hendricks et al., 1990; Kimura et al., 1990; Hendricks et al., 1991). This theory providesan explanation for the data in the literature reporting no differences in respiratory control betweenREM sleep and SWS, and large changes (including the typically observed irregular, shallowbreathing) during REM sleep.The influence of sleep on ventilatory reflexes to hypoxia and hypercapnia is not clear.Some investigators report decreased responses or no change in dogs, cats and goats (Phillipsonet al., 1978; Sullivan et al., 1979; PhilEpson et al., 1980; Santiago et al., 1981; Bowes et al.,1980; Parisi et al., 1992), while increased responses have been noted in the rat (Pappenheimer,1977). These discrepancies may represent species-specific differences, or differences inexperimental protocol (Douglas et al, 1983). In general, the hypoxic and hypercapnic ventilatoryresponses appear to be depressed, compared to wakefulness, but both stimuli still produce largeincreases in ventilation.Like sleep, the induction of anesthesia also results in a loss of behavioural control ofrespiration. Attempting to equate the states of sleep and anesthesia may at first seem to be an7illogical comparison. In fact, anesthesia has even been associated with coma, rather than withsleep. Like coma, anesthesia is often thought of as a homogenous, unchanging state of neurallydepressed activity. This assumption, then, infers that anesthesia and coma are on a sort ofcontinuum separated by the severity of neural depression, and in neither does one see activepatterns of cortical activity like those observed in sleep.Under some anesthetics, however, active cortical patterns are seen, similar to those ofwakefulness and sleep. In particular, under light urethane anesthesia, cortical (EEG) recordingsdisplay at least three very distinct, stable patterns (Grahn et al., 1989; Grahn and Heller, 1989).Superficially, these three patterns (States I, II and III) resemble those of the unanesthetizedanimal when awake, drowsy and in slow-wave sleep, respectively. In addition, these EEG-defined states are sensitive to external stimuli. The stable synchronized cortical pattern (StateIII) of the urethane-anesthetized animal will quickly desynchronize in response to tactile, thermalor auditory stimuli. These are exactly the changes that one would see if an unanesthetized animalin slow-wave sleep was awakened. Also like unanesthetized animals, urethane-anesthetizedanimals oscillate spontaneously between stable bouts of each arousal state, suggesting that thesystems responsible for generating, timing, maintaining and switching the neural activityassociated with these patterns are all intact and operative under anesthesia. Recent work on themolecular actions of urethane at the level of the thalamus (Nuilez et al., 1992) demonstrates thatdelta waves and infrequent spindling in the EEG are the same in State III as in SWS. This datasupports the theory that under urethane anesthesia one sees states which are comparable, in termsof cortical activity, to sleep and wakefulness.The question arises as to whether comparable arousal states (states with similar patternsof cortical activity) seen in anesthetized and unanesthetized animals are analogous in any way.8If the urethane arousal states are analogous to their unanesthetized counterparts, then they shouldexert similar effects on physiological systems. Many anesthetics are known to depress respirationand chemoreflexes significantly (see Nunn, 1990 for review), far more than natural sleep.However, in an extensive review of urethane anesthesia, Maggi and Meli (1986a, 1986b; 1986c)concluded that urethane exerts minimal effects on the respiratory system and barely affectshypothalamic function (which could indirectly alter respiratory function via effects on a numberof reflexes). In addition, urethane anesthesia appears to exert minimal effects on thecardiovascular system (Folle and Levesque, 1976; Hughes et al., 1982; reviewed in Green, 1979).Therefore, evidence suggests that cardio-respiratory control is not affected by urethane anesthesiaper se. What remains unknown, is whether cardio-respiratory control changes during urethaneanesthesia with the changes which occur in cortical patterns.If urethane-induced arousal states prove to be analogous to sleep/wake states for cardio-respiratory control, then the urethane-anesthetized preparation would prove to be particularlyuseful for future research involving brainstem recording of respiratory neurons. At present,attempts to elucidate the relative contribution of various state-dependent respiratory groups withinthe brainstem involve complicated, difficult and time-consuming protocols. In order todemonstrate changes in respiratory neural firing as the arousal state changes, experimentalanimals must be chronically instrumented and trained to sit quietly and fall asleep while boltedinto stereotaxic devices (Orem et al., 1974). With the anesthetized preparation, not only couldanimals be easily immobilized to minimize movement artifacts, greater control and flexibilitywould be afforded experimenters as they could dictate changes in arousal state to some degree.Much work has already been done on respiratory control in the golden-mantled groundsquirrel (Spernwphilus lateralis), and this animal appeared, therefore, to be a good subject choice9for the present study. This animal is interesting because it undergoes large changes inmetabolism during hibernation, which are associated with dramatic changes in respiratory pattern.These studies may be relevant to sleep research because many groups (ie. Walker et al., 1977;Heller et al., 1978; Berger, 1984) consider hibernation to be an extension of SWS.This study concentrated on establishing a possible analogy for respiratory control betweenthe superficially similar arousal states of urethane anesthesia and sleep. Therefore, the focus ofthe first part of this study was to determine the effects of light urethane anesthesia on respiratorypattern (frequency and tidal volume), overall minute ventilation, heart rate and respiratory sinusarrhythmia in the golden-mantled ground squirrel, and to determine whether the changes seen inthese variables with changing arousal state under urethane anesthesia were similar to those seenwith changes between comparable awake/sleep states, as determined by EEG criteria, inunanesthetized animals. To further examine the respiratory control present during these variousarousal states, the second part of this study measured the effects of sleep on the hypoxic andhypercapnic ventilatory responses in the golden-mantled ground squirrel. In the third part of thisstudy, the effect of changing arousal state in urethane anesthetized animals on the hypoxic andhypercapnic ventilatory response was measured and compared to the changes seen inunanesthetized animals in similar EEG states. The overall aim of these studies was to test thehypothesis that the changes in respiratory control associated with different EEG patterns inwakefulness/sleep and under urethane anesthesia are analogous.10MATERIALS AND METHODS I. Animal conditions Golden mantled ground squirrels (Spermophilus lateralis) were used in this study. Theanimals were purchased from a commercial supplier and housed individually or in pairs inpolycarbonate cages (9"x 8" x 17"). The cages were kept on shelves or racks (three to five cagesper shelf) in an environmental chamber, which was kept at room temperature (20 ± 2 °C)throughout the duration of the study. Daily light conditions were maintained on a 12 L:12 Dcycle (12 hours light and 12 hours dark per day). Food and water were supplied ad libitum; therat chow diet was supplemented with sunflower seeds, grapes and apples. Food was withheldstarting four hours prior to surgery and urethane anesthesia experiments.Studies have shown that urethane suppresses bone marrow function in mice, resulting indecreased resistance to infection. These effects are reversible; the recovery period is related tothe administered dosage (reviewed by Field and Lang, 1988). Therefore, animals which had beentreated with urethane were housed in individual cages in the same environmental chamber,separated from the other animals in the colony as much as possible, for the duration of the study.IL Surgical Procedures Animals (252.5 g ± 17.3 g) were anesthetized with sodium pentobarbital (Somnotol;65mg/m1). The effective dosage normally ranged between 1125 and 18.33 mg/100 g. Animalswere assumed to have reached a surgical plane of anesthesia upon suppression of the corneal andpain withdrawal (foot pinch) reflexes. The top of the subject's head and back of the neck werethen shaved, treated with a depilatory cream and thoroughly cleaned with distilled water. The11exposed areas were then sterilized with ethanol.Animals were then placed in a Kopf stereotaxic device. The surface of the skull wasexposed and cleaned with hydrogen peroxide to remove any connective tissue which mightinterfere with the adherence of the headpiece. The incision was continued approximately 2-3 cmdown the neck to expose the neck muscles. All cut areas of skin were treated with the topicalanesthetic Xylocaine (20 mg lidocaine hydrochloride/ml, administered dropwise as required).Four stainless steel EEG electrodes were implanted in the surface of the skull to monitorcortical activity. One was placed above each frontal lobe (right and left) and one was placedabove each occipital lobe. A thermal re-entrant tube was inserted approximately 4.5 mm into themidlateral surface of the skull to monitor brain temperature. A tunnel was produced just underthe skin from the back of the incision to the latero-medial side of the rib cage on each side andEKG (electrocardiogram) electrodes were fed down this tunnel to be glued, with tissue cement,to the muscle wall on either side of the rib cage. The EMG (electromyogram) electrodes weresewn into the dorsal neck musculature (anterior trapezius), which was exposed at the incision site.Postural and submental muscles are standardly monitored for the muscular atonia whichdifferentiates wakefulness from REM sleep; these muscles show REM-associated atonia quicklyand completely. The wire leads from all eight electrodes were fed up to the top of the head. Allelectrodes were soldered to Amphenol terminal connectors. They were inserted into amphenolpin strips which were then affixed upright on the skull surface. A headpiece was built of dentalacrylic surrounding the pin strips, EEG screws and re-entrant tube.The incision was then sewn closed around the headpiece and treated with a topicalantibiotic (Flamazine, 2% silver sulfadiazene) ointment. Upon completion of surgery, subjectswere given antibiotic injections of ampicillin sodium (Penbritin-250, 6.25 mg/100 g) and12analgesic injections of Meperidine Hydrochloride (Demerol, 1 mg/100 g), administered as needed.Animals were then left in individual chambers and continuously monitored for approximatelytwelve hours during recovery from the anesthetic and were then returned to the colony. Thesubjects were allowed at least two weeks to recover from the surgery before being used inrecording experiments. Feeding and behaviour patterns were monitored to ensure that the animalswere recovering properly and not presenting symptoms of discomfort, infection or anorexia.Animals displaying signs of anorexia following surgery were given daily injections of Dextrose(5.0% solution) until they resumed normal eating patterns and body weight.III. Experimental Protocol AI Initial Set-up i.) Unanesthetized Animals Only subjects showing no signs of discomfort and displaying normal behavioural andfeeding patterns were used in the recording studies. The subject was placed in a 1000 cm3 clearwhole-body plethysmograph chamber. Air flow through the chamber was set at 1 litre/min. Thecontact pins in the headpiece on the animal were connected to long wire leads, and athermocouple was inserted into the cranial re-entrant tube. These were then fed out of thechamber to an external panel board through an airtight port in the top of the animal chamber anda port in the side of the environmental chamber. The EEG, EMG and EKG signals were thenfed through amplifiers (Grass model 7P5 11K), and recorded on an EEG & Polygraph DataRecording System (Grass model 79E). The thermocouple was connected to a Sensortek digitalmonitor (Fig. 1). Once the wire leads and temperature probe were in place, the subject was left13in the recording chamber for approximately 1/2 to 1 hour to acclimatize to its surroundings.Anesthetized Animals Prior to recording, animals were anesthetized with urethane (ethyl carbamate,approximately 1.35 g/ kg). Anesthesia was considered sufficient when the pain withdrawal reflexwas suppressed and a stable respiratory pattern was attained.Subjects were placed on wood chip bedding in a 21.5 x 14 cm clear plexiglass chamber(Fig. 2). Front and back ports allowed air to flow through the chamber at a flow rate of 1litre/min. The electrode leads and thermal probe were connected to the animal and led out ofthe animal and environmental chambers in exactly the same way as for unanesthetized subjects.A form-fitting plastic mask was placed over the subject's nose and mouth, and a rubber cuff wasthen pulled over the mask and head to hold the mask in place. The mask was then connectedto a pneumotachograph. The pneumotachograph connections were led out of the chamberthrough airtight ports. Finally, a sample tube was inserted through the top of the chamber andconnected externally to Beckman 02 and CO2 analyzers to allow continuous monitoring of thegas concentrations within the animal chamber. The Beckman analyzers were calibrated beforeeach experiment using a Radiometer GMA2 precision gas supply (type 5021a) for CO2 and roomair for 02.Figure 1: Schematic diagram of experimental apparatus for ventilation measurementsusing the whole-body plethysmographic technique and recording ofelectrophysiological variables in unanesthetized animals. See text for details.1416Figure 2: Schematic diagram of experimental apparatus for ventilation measurementsusing a mask and pneumotachograph and recording of electrophysiological variablesin urethane-anesthetized animals. See text for details.AIR 002 02 NSENSORTEKDIGITALMONffORI FLOWMETER1BECKMAN02 AND CO2ANALYZERINTEGRATOR^ II ^1AMPUFIERSANDCHART RECORDERTHERMALPROBEI MECHANICAL I ^TRANSDUCERANIMALCHAMBEREEG, EMG AND EKGELECTRICAL LEADS'OUTFLOW IEXTERNALPANEL BOARD18ill Ventilation measurements 0 Unanesthetized animals In unanesthetized, unrestrained animals, the plethysmographic (barometric) technique wasused to measure ventilation (Drorbaugh and Fenn, 1955; Jacky, 1978; Epstein and Epstein, 1978;Jacky, 1980). The pressure signal was detected by a Validyne mechanical transducer (modelDP103-18), amplified (Grass low-level D.C. Amplifier, model 7P122E) and recorded on the Grasspolygraph with the other electrophysiological signals (Fig. 1). This system was calibrateddynamically as described by McArthur and Milsom (1991).i0 Anesthetized Animals For the anesthetized animals, the pneumotachograph connected to the face mask wasconnected to a Validyne mechanical transducer model DP103-18 (Fig. 2). The signal from thetransducer was then amplified (Grass low-level D.C. Amplifier, model 7P122G), transcribeddirectly on the Grass polygraph and also integrated (Gould Integrating Amplifier, model 13-46 15-70). The integrated signal was also transcribed onto the Grass polygraph and from this integratedsignal, tidal volume could be measured directly. This system was calibrated immediately at theend of each experiment. The pneumotachograph was removed from the animal's mask, butremained connected to the rest of the system as described above. Known volumes of air, whichapproximated the average breath size, were then pumped through the pneumotachograph. Thepump was also set at various frequencies to ensure that the integrated signal was frequencyindependent.19CI Data Recording Periods and Analysis D Normoxic Ventilation During Awake/Sleep and Urethane AnesthesiaEach recording session lasted between 4 and 8 hours. In the unanesthetized animals,recording sessions were generally concluded when animals became restless and were no longerentering bouts of established sleep. Recording sessions in the anesthetized animals wereconcluded after periods of similar length. Continuous measurement of the EEG, EMG, EKG andrespiratory variables were made during each recording session. The inspired gas for both groupswas room air. Each subject was run under both unanesthetized and anesthetized conditions.Following experiments with urethane anesthesia, animals were allowed at least 11/2 weeks torecover before being used in subsequent experiments.Hvpoxic and Hypercapnic Ventilatory Responses During Awake/Sleep Each animal was run under hypoxic and hypercapnic conditions for one full session. Asunder normoxic conditions, recording sessions lasted 4 to 8 hours. The EEG, EMG, EKG andrespiratory variables were recorded continuously. The hypoxic gas composition was created bydiluting air with nitrogen to produce a mixture containing 10.0% 02. Hypercapnic conditionswere created by mixing CO2 and air to a final concentration of 5.0% CO2. The increased amountof CO2 did not significantly alter the concentration of 02 in this mixture.Hvpoxic and Hypercapnic Ventilatory Responses During Urethane AnesthesiaThe same protocol was followed here as that described for sleeping animals.L)., Data Analysis 20Sleep states were scored for the unanesthetized animals from EEG and EMG recordingsaccording to conventional criteria (Reschtaffen et al., 1968). Five sleep states were recognized:wakefulness (W), drowsiness or light sleep (LS), slow-wave sleep (well established slow-waveactivity, denoted SWS), a transition state between SWS and REM sleep (REMtr) and rapid-eyemovement (REM) sleep. Although both first and second parts of this study scored LS; differentcriteria were used to distinguish between LS and SWS. In the first part of the study, SWS wasscored very rigorously. Only completely established SWS was scored as such; if anydesynchronized activity was present, the state was scored as LS. In the second part of the study,30 second epochs were scored according to the predominant arousal state (ie. when more thanhalf of the EEG record was synchronized, the epoch was scored as SWS). The EEG profiles ofthe three established states, wakefulness, SWS and REM sleep, are compared in Figure 3.Standard terms (Czeisler et a/., 1980) were used to describe sleep sequences, episodes andpatterns.Under urethane anesthesia, EMG records show a consistent, stable muscle tone.Therefore, arousal state data in these subjects could only be scored from the EEG traces.However, states could be categorized based on EEG patterns (Grahn et al., 1989; Grahn andHeller, 1989). Three of these patterns are similar to those observed in the unanesthetized animal;these states have been termed States I, II and III (Fig. 4). Two additional states were recognizedin the urethane-anesthetized animals, but rarely seen. State V is associated with epileptiformactivity, indicative of deep anesthesia and State IV was a transitional state between thesynchronized State III and the State V.21Figure 3: Recordings of EEG, EMG, EKG and Respiration during wakefulness (W), slow-wave sleep (SWS) and rapid-eye movement sleep (REM) in an unanesthetizedgolden-mantled ground squirrel. See text for details of arousal state scoring.23Figure 4: Recordings of EEG, EMG, EKG and Respiration during State I (desynchronizedEEG activity), State II (intermittent activity) and State 1II (synchronized EEGactivity) in a urethane-anesthetized golden-mantled ground squirrel. See textfor details of arousal state scoring.25All arousal state data were analyzed in 30 second epochs and scored as indicated above.The percentage of total recording time spent in each state was then calculated from these data.Two respiratory variables were measured, breathing frequency (f) and tidal volume (VT), fromwhich minute ventilation (VE) was calculated. Data for the hypoxic and hypercapnic ventilatoryresponses were expressed in several ways to distinguish between the effect of gas and state. Theeffect of inspired gas concentration on breathing variables, independent of arousal state, wasillustrated by normalizing the data to normoxic values in each arousal state. The effect of arousalstate on the breathing variables in normoxia, hypoxia and hypercapnia separately, was illustratedby expressing the data as a percentage of the wakefulness or State I response (for unanesthetizedand anesthetized animals, respectively). Two cardiovascular variables were measured, heart rateand the extent of the respiratory sinus arrhythmia. The sinus arrhythmia was determined bysubtracting the mean expiratory heart rate from the mean inspiratory heart rate under eachcondition in each state. Cardiovascular data was expressed only in absolute terms and as apercentage of normoxic values in wakefulness or State I (for unanesthetized and anesthetizedanimals, respectively) to illustrate the combined effect of gas and arousal state.Statistical comparisons between arousal states in the same treatment group were performedwith a two-way ANOVA and Least Significant Difference (L.S.D.) post-hoc test. A significancelevel of oc = 0.05 was set for each comparison unless otherwise indicated.Comparisons between unanesthetized and anesthetized animals under normoxic conditionswere made between comparable arousal states (ie. wakefulness vs. State I; drowsiness vs. State11 and slow-wave sleep vs. State III), using the Student's T-test (paired design). The significancelevel was again set at 0. = 0.05. Statistical comparisons between arousal states under hypoxicand hypercapnic conditions in both sleeping and urethane anesthetized squirrels were performed26via a three-way ANOVA, followed by a post-hoc L.S.D. test. The significance level was set atcc = 0.05 for both tests, unless otherwise indicated. These experiments were an incomplete blockdesign; therefore, one of the factors included in the three-way ANOVA model accounted forsubject variation.27RESULTS D Normoxic Ventilation During Awake/Sleep and Urethane AnesthesiaD Arousal State Distribution Golden-mantled ground squirrels spent approximately equal amounts of time inwakefulness (W), drowsiness (LS) and slow-wave sleep (SWS) (32.5, 30.9 and 31.7%,respectively; Fig. 5). Only 5% of the total recording time was devoted to REM-relatedbehaviour, 2.5% in REM sleep and 2.3% in the transition (REMtr) between SWS and REM sleep.Figure 6 illustrates an average recording period for a golden-mantled ground squirrel. Thetypical ultradian pattern (oscillation between SWS and REM sleep) emerges from the trace.Bouts of each arousal state were relatively stable, and animals tended to move from wakefulnessinto SWS through a drowsy period. REM sleep was always preceded by SWS and was oftenfollowed by brief periods of arousal before animals entered another bout of SWS. The bouts ofSWS preceding REM sleep were longer at the start of a period of sleep (ie. the latency to REMsleep bouts decreased during a period of sleep). Brief arousals into LS rather than completewakefulness, were also often seen during extended bouts of SWS. These were of short duration(usually between 30 seconds and one minute) and were commonly associated with posturaladjustments.Urethane anesthetized animals spent approximately equal amounts of time in States I andIII (41% and 32%, respectively), and significantly less time in State 11 (18%, Fig. 7).Figure 8 illustrates an average recording period for a urethane-anesthetized animal. The28Figure 5: Distribution of time spent in the five arousal states scored in unanesthetizedgolden-mantled ground squirrels. Arousal states are: wakefulness (W), drowsiness(LS), slow-wave sleep (SWS), transition into REM sleep (REMtr) and rapid-eye •movement sleep (REM). * indicates significantly different from waking values.W LS SWS REMtr REM; 4El 14 45c.4CZ 30gCCI);IT4 15ge 0AROUSAL STATEFigure 6: Chronological record of arousal state in an unanesthetized golden-mantledground squirrel. Arousal state notation as in Figure 5.30Ultradian Rhythm of Sleep/ Wake States32Figure 7: Distribution of time spent in the three arousal states scored in anesthetizedgolden-mantled ground squirrels. Arousal states are: State I (I), State II (II) andState DI (III). Data for unanesthetized animals in similar EEG states are includedfor comparison. * indicates significantly different from State I values. + indicatessignificantly different from unanesthetized values, same state.600W/ I^LS/ II^SWS/ InAROUSAL STATE34Figure 8: Chronological record of arousal state in a urethane-anesthetized golden-mantledground squirrel. Arousal state notation as in Figure 4.Oscillations Between Urethane Arousal States36movement between states was similar to that of the unanesthetized animals. Animals movedbetween stable and extended bouts of each arousal state. Brief transitions into State II, or evenState I, were seldom seen during extended periods of State Di. Periods of synchronized activityin the anesthetized animals were interrupted only by extended, stable bouts of desynchronizedactivity.The amount of total recording time spent in synchronized State III in urethane-anesthetized animals was not significantly different from the amount of time spent in SWS in thenonanesthetizetl animals. Although there was a slight increase in the amount of time spent inthe desynchronized State I in the anesthetized animals, it was not significantly different from thatspent in wakefulness in the unanesthetized animals. There was a significant decrease in theamount of time spent in the transitional State II in the anesthetized animals compared to timespent in LS in unanesthetized animals.Figure 9 illustrates that the proportion of time that animals moved through a transitionalstate to enter a new arousal state was the same in both the unanesthetized and anesthetizedanimals. Both groups moved through a transitional state to enter another arousal state about halfthe time (44% of arousal state changes in both groups).Respiratory Variables Breathing Frequency Breathing frequency was clearly affected by arousal state (Fig. 10). It droppedprogressively from 86 breaths/min in W to 39 breaths/min in SWS, with intermediate frequenciesobserved in LS (52 breaths/min). Frequency remained depressed at 36 breaths/min in REMFigure 9: The proportion of state changes associated with transitional EEG activity inunanesthetized and urethane-anesthetized golden-mantled ground squirrels.37CONTROL NORMOXIA0.50E-40.25a..0 0.00a..38TREATMENT GROUPFigure 10: The effect of arousal state on three respiratory variables in golden-mantledground squirrels. Values are presented as absolute values, normalized to bodyweight (per 100g). * indicates significantly different from waking values.39W LS SWS REMAROUSAL STATE1.60.0604020040fl41sleep.Significant decreases in breathing frequency (Fig. 11) were also seen under urethaneanesthesia as the animals moved from the desynchronized EEG state, through the transitionalstate and into the fully synchronized EEG state. As in the unanesthetized animals, this decreasewas also incremental; frequency dropped from 54 breaths/min in State I to 40 breaths/min inState 11 and then to 31 breaths/min in State III. Direct comparison between data obtained fromanesthetized and unanesthetized groups shows that breathing frequencies were not significantlydifferent in the partially and fully synchronized states (LS/II and SWS/III). However, breathingfrequency was significantly lower in the urethane group in State I compared to the unanesthetizedgroup in W.Tidal Volume In unanesthetized animals, there was little apparent effect of arousal state on tidal volume;there were no significant differences between W, LS, SWS and REM sleep. However, thisapparent consistency was due in part to large variations and standard errors; tidal volume waslarger in the drowsy and sleep states compared to W. Tidal volume was 1.10, 1.35, 1.34 and1.32 m1/100 g, in W, LS, SWS and REM, respectively.Tidal volume was also little affected by arousal state under urethane anesthesia (Fig. 11).Although tidal volumes increased progressively from 1.04 m1/100g (State I) to 1.13 m1/100 g(State II) and 1.23 ml/100 g (State III), these changes were not significant. There was much lessvariation (smaller standard errors) in the urethane group compared to the unanesthetized group.42Figure 11: The effect of arousal state on respiration in urethane-anesthetized golden-mantled ground squirrels. Data from unanesthetized animals in similar EEG statesare included for comparison (WA; LS/II and SWS/D1). Values are presented asabsolute values, normalized to body weight (per 100g). * indicates significantlydifferent from State I or waking values, respectively. + indicates significantlydifferent from unanesthetized values, same state.1005^80G.4 S600 IC4020gzZ0.040605 l'" 40I1^20XW/I^LS/II SWS/III43AROUSAL STATE44Direct comparison between states with similar EEG profiles in the urethane-anesthetized andcontrol animals showed no significant differences between the two groups.Minute Ventilation Arousal state affects minute ventilation in the same manner that it alters breathingfrequency (Fig. 10). As animals moved from W, through drowsiness, and into stable SWS,minute ventilation was progressively reduced. This decrease was maintained in REM sleep.Minute ventilation decreased progressively from 62.0 to 48.6, 43.5 and 40.7 ml/min/100 g asanimals moved from W, through LS and into SWS and REM sleep, respectively.Arousal state also affected minute ventilation in the urethane-anesthetized animals and thepattern of change was the same as that observed in the unanesthetized group (Fig. 11). Whenthe urethane-anesthetized animals moved from stable desynchronized activity, through transitionalstates, and into stable synchronized activity, minute ventilation was progressively reduced. Withthe onset and establishment of the synchronized state, minute ventilation dropped from the StateI value of 56.9 to 41.0 (State II) and 35.0 ml/min/100g (State III). Thus, both groups showeda significant decrease in minute ventilation in synchronized cortical states compared to that indesynchronized states. Direct comparisons between states with similar EEG profiles in theanesthetized and unanesthetized groups showed the only significant difference between the twotreatment groups to exist between LS and State II.45Cardiovascular Variables Heart RateHeart rate was also affected by arousal state in the golden-mantled ground squirrel (Fig.12). The average resting heart rate in awake animals was 249 beats/min. This rate decreasedprogressively as animals moved through LS and into SWS (230 and 208 beats/min, respectively).Heart rate in REM sleep (202 beats/min) did not change much from the SWS values. Only theheart rates in SWS and REM sleep were significantly less than that in W.Urethane anesthesia significantly elevated heart rate (Fig. 13).^Compared tounanesthetized animals in states with similar EEG profiles, urethane-anesthetized animalsincreased their heart rate by 47% (State I vs. W), 56% (State II vs. LS) and 65% (State DI vs.SWS). Despite this large elevation in rate by urethane anesthesia, the effect of arousal state onheart rate was similar in both unanesthetized and anesthetized animals. Like the unanesthetizedanimals, heart rate was significantly reduced in the synchronized arousal state (State DI) ascompared to the desynchronized state (State I). Heart rate decreased from 366 beats/min in StateI to 342 beats/min in State LH. Although not significantly different from State I values, State Hvalues (359 beats/min) were intermediate between those recorded in States I and III.Sinus Arrhythmias Sinus arrhythmias were expressed as differences between inspiratory and expiratory heartrate. In the unanesthetized animals, these differences ranged, on average, from 20 to 53beats/min (Fig. 14). The influence of respiration on heart rate became progressively morepronounced with the onset and maintenance of sleep. Although all three sleep states showedgreater amounts of sinus arrhythmia, only REM sleep values were significantly different due to46the large variation in responses. Breath to breath heart rate variation could range from 0 to 80beats/lulu. Under urethane anesthesia, respiratory sinus arrhythmia was completely abolished.47Figure 12: The effect of arousal state on heart rate in imanesthetized animals. * indicatessignificantly different from waking values.W LS SWS REMAROUSAL STATE3004tt^2502004tt;14 150t1004tt504e49Figure 13: The effect of arousal state on heart rate in urethane-anesthetized animals. Datafrom unanesthetized animals with similar EEG profiles are included for comparison(W/I; LS/II and SWS/III). * indicates significantly different from State I or wakingvalues, respectively. + indicates significantly different from unanesthetized values,same state. CONTROLURETHANE•400g^350300i 250.c) 200W 4E-'0 44 150g 6 100504tW/1^LS/II^SWS/IIIAROUSAL STATE51Figure 14: The effect of arousal state on respiratory sinus arrhythmia in unanesthetizedanimals. Values are expressed as the heart rate difference between inspiratory andexpiratory heart rates. * indicates significantly different from waking values.0W LS SWS REMAROUSAL STATE53ID Hypoxic and Hypercapnic Ventilatory Responses During Awake/SleepD Arousal State Distribution In this study, the amount of time that control animals spent in LS (14.1%) wassignificantly less than the time spent in wakefulness (34.5%), while the amount of SWS (48.4%)was significantly greater than that of wakefulness (Fig. 15). Only 4% of the total recording timewas spent in REM sleep; transitional REM states were not scored in this study.Altering the inspired concentration of 02 or CO2 did not appear to greatly affect thesleep/wake architecture of the golden-mantled ground squirrel (Fig. 15). Hypercapnic exposureproduced nonsignificant increases in wakefulness (43%) and decreases in both SWS (39.5%) andREM sleep (0.8%). The amount of SWS was now approximately equal to that of wakefulness,rather than greater. The percentage of time spent in the intermediate state of light sleep wasunchanged (16.7%). Thus, the pattern of distribution of states was unchanged; hypercapnicanimals also exhibited significantly less LS and REM than W and SWS. Hypoxic exposure alsoaltered the distribution of arousal states. A nonsignificant decrease in SWS was noted (41.4%),which was accompanied by a significant increase in the amount of LS (25.9%) rather than anyalteration of time spent awake (31.7%), unlike the hypercapnic group which increased the timespent awake instead of the time spent in LS. REM sleep was again observed infrequently (1.0%).Although the decreased amount of SWS in hypoxia was not significantly different from that seenin normoxia, the amount of SWS was no longer significantly greater than the amount ofwakefulness (unlike the normoxic group).54Figure 15: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on arousal statedistribution in unanesthetized animals * indicates significantly different fromwaking values for the same gas treatment. + indicates significantly different fromnormoxic values, same state.-*-_--_+NORMOXIAHYPER CAPNIAHYPDXIA^',".°.;•:46:1^1Wwa^704E*^60o4 50=g40CC)^30;14g 20CITOo 10e oW LS SWS REMAROUSAL STATE5556II) Respiratory Variables Breathing Frequency Breathing frequency (Fig. 16) decreased progressively as normoxic animals moved fromwakefulness (W), through drowsiness (LS) and into established SWS. This decrease wasmaintained in REM sleep. Breathing frequency dropped significantly from 53 breaths/min in Wto 34 breaths/min in SWS, with intermediate frequencies observed in LS (41 breaths/min).Frequency remained depressed at 36 breaths/min in REM sleep.With hypercapnia (5.0%CO2) (Fig. 16), significant increases in breathing frequencyoccurred in all states except REM sleep; hypercapnic frequencies ranged from 122% (REM) to148% (LS) of normoxic values (Fig. 17). Hypercapnia, however, did not affect the arousal stateeffects on breathing frequency (Fig. 18); light sleep and established sleep states were allassociated with significantly slower breathing rates compared to wakefulness. As under nonnoxicconditions, breathing frequency decreased progressively as animals moved from W through LS(81% of W values), and into sleep. Breathing frequency dropped further with movement fromSWS (64% of W) to REM sleep (59% of W).Hypoxic exposure prompted a large increase in breathing frequency in all arousal states(Fig. 16), ranging from 192-258% of normoxic values (Fig. 17). As in the hypercapnic group,the effect of arousal state was still evident under hypoxic conditions (Fig. 18). Breathingfrequency during SWS and REM sleep declined significantly to 85% and 84% of waking values;during LS, the frequency declined only slightly (92% of W).57Figure 16: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on respiration(breathing frequency, tidal volume and minute ventilation) and on the associatedstate changes in these respiratory variables in unanesthetized golden-mantled groundsquirrels (absolute values, per 100g body weight). * indicates significantly differentfrom waking values, same gas treatment. + indicates significantly different fromnormoxic values, same state.0tPj)*MINUTE VENTILATION^TIDAL VOLUME^BREATHING FREQUENCY(ml /100g /min)^(m1/100g)^(Breaths /min)log^Utft..] ^00^tn^toy^0^th^tio59Figure 17: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on respiration(breathing frequency, tidal volume and minute ventilation) within each arousal statein unanesthetized animals. Data are normalized to the nonnoxic values in the samearousal state, per 100g body weight. + indicates significantly different fromnormoxic values, same state.t 2751 250225200175150i 12510075i 50250150125100.^7550250200175150125100755025111. NORMOXIA■-•'111"4 HYPERCAPNIAED HYPDXIA60LS SWS REMAROUSAL STATE61Figure 18: The effect of arousal state on the hypoxic and hypercapnic ventilatory responsesin unanesthetized animals. Data are normalized to waking values (per 100g bodyweight) for normoxic, hypoxic (10.0% 02) and hypercapnic (5.0% CO2) groupsseparately. * indicates significantly different from waking values, same gastreatment.LS SWS REMNORMOXIAHYPERCAPNIAHYPDXIAVg!is11,-4171kANM* *AROUSAL STATE621254 a No0i 75ri 0cAo i 504t44a 25O al 0C.)^175C4 150It 5 1251.40 100•4 75Itt 50CA25We‘W 04et^6,q 1254!^0 100•T•1^VO 75e1 5025063Tidal Volume In normoxic animals, tidal volume increased progressively from W (1.02 m1/100 g bodyweight), through LS (1.19 m1/100g), to SWS (1.34 m1/100g). Although the tidal volume in LSwas not significantly greater than that in W, the increase during SWS was significant. As ananimal moved into REM sleep, tidal volume decreased (1.15 m1/100 g) to values similar to thoseobserved during LS.Golden-mantled ground squirrels did not alter tidal volume in response to hypercapnia inany arousal state (Fig. 16). Tidal volumes ranged from 95 to 117% of normoxic (same state)values (Fig. 17). As in the normoxic group, tidal volume increased progressively in thehypercapnic group as the animals moved from W, through LS and into the two sleep states (SWSand REM sleep), with significant increases in both SWS and REM sleep. Tidal volumesincreased slightly in LS (120% of the W value) and increased further in SWS and REM sleep(133% and 138% of the W value, respectively).Hypoxia altered both tidal volume and the arousal state effects on tidal volume (Fig. 16).Tidal volume was significantly decreased under hypoxia; these decreases ranged from 72% (W)to 57% (SWS) of the normoxic (same state) values (Fig. 17). Exposure to hypoxia also abolishedany apparent effect of arousal state on tidal volume (Fig. 18); under hypoxic conditions, tidalvolume did not increase at all during sleep. Tidal volumes during LS, SWS and REM were allbetween 101% and 105% of the awake value.Minute Ventilation Arousal state did not appear to affect minute ventilation of this second group of normoxicanimals (Fig. 16). However, as animals moved from W, through LS, and into stable SWS,64minute ventilation was progressively reduced. This decrease was maintained in REM sleep.Minute ventilation was decreased from 50.8 to 40.6 ml/min/100 g as animals moved from W toSWS. LS values were intermediate between W and SWS (43.6 ml/min/100g) and REM values(39.5 ml/min/100g) remained the same as those for SWS. However, none of these values weresignificantly different from waking values.Under hypercapnic conditions, golden-mantled ground squirrels increased VE significantlyin all states (32% to 64% increases over normmdc, same state, values; Fig. 17). No arousal stateeffects were evident under hypercapnic conditions either (Fig. 18). Under hypercapnicconditions, minute ventilation was constant across arousal states, ranging from 94% to 102% ofthe awake, hypercapnic response.The changes in minute ventilation induced by exposure to hypoxia approximatelyparalleled those seen in the hypercapnic group. VE was also increased significantly in responseto hypoxia in all arousal states. These increased values ranged from 148% to 169% of thenormoxic (same state) values (Fig. 17). As with normoxic and hypercapnic animals, there wasno arousal state effect on VE in these animals (Fig. 18). Although a very slight, progressivedecline in minute ventilation was observed from W, through drowsiness, to the two sleep states,it was not significant.65Hypoxic and Hvpercapnic Ventilatory Responses During Urethane AnesthesiaD Arousal State Distribution Consistent with the first study, urethane-anesthetized animals spent approximately thesame amount of time in the arousal states exhibiting established cortical activity patterns (StatesI and HI), and significantly less time in the transitional State II. Very little time was spent inState IV and none in State V (Fig. 19).The effect of changing inspired gas concentration on arousal state distribution in urethane-anesthetized animals is also shown in Figure 19. Both hypoxia and hypercapnia tended todesynchronize the EEG (significantly less time was spent in State III compared to State I),although only with hypercapnia was the time spent in State III significantly reduced comparedto normoxic values. The hypercapnic animals spent a greater amount of time in State I, with nochange in the amount of transitional State II. Hypoxic animals also spent significantly less timein State III, but spent the same amount of time in State I. The State II amount was increasedduring hypoxic exposure; although not significantly different from normoxic amounts, it was nolonger significantly less than the State I amount (unlike the normoxic and hypercapnic groups).Figure 20 illustrates the proportion of time that animals moved through a transitional stateto enter a new arousal state. Breathing normoxic gas, urethane-anesthetized animals movedthrough a transitional state to enter another arousal state approximately half the time (44.1% ofarousal state changes). Changing the inspired gas to hypoxic or hypercapnic conditions did notaffect the percentage of time that animals moved through State II to reach another state (47.4%and 46.8%, respectively).66Figure 19: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO 2) on arousal statedistribution in urethane-anesthetized animals. * indicates significantly different fromState I values, same gas treatment. + indicates significantly different from normoxicvalues, same state.70g 600 504c) 4°p4o 30U20ra.C10eoI^II^III^IVAROUSAL STATEFigure 20: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% COO on theproportion of state changes associated with transitional activity in urethane-anesthetized animals.6869NORMOXIA^HYPDXIA HYPERCAPNIATREATMENT GROUP1.000.750.500.250.0070Respiratory Variables Breathing Frequency Progressing from State I, through State II and into State III, breathing frequency decreasedfrom 54 breaths/min, to 40 breaths/nain, and then to 31 breaths/min in normoxic, urethane-anesthetized animals (Fig. 21).In the hypercapnic urethane group, breathing frequency was not significantly differentfrom normoxic values in any state and the effect of arousal state on frequency was still observed(Figs. 21, 22, 23). Breathing was significantly slower in States II, III and IV compared to StateI (70%, 61% and 48% of State I values, respectively). Hypoxia elicited a significant increasein breathing frequency over normoxia in all states in the urethane-anesthetized animals (Fig. 21);the increase in frequency over normoxic values ranged from 49-88% (Fig. 22). As in thehypercapnic group, the effect of arousal state was still evident in the hypoxic =thane group(Fig. 23). Breathing frequency in States II, Ill and IV were 83%, 72% and 56% of State Ivalues, respectively; the breathing frequencies in States DI and IV were significantly lower thanthat in State I.Tidal Volume Urethane-anesthetized animals exhibited nonsignificant increases in tidal volumes in StatesII, DI and IV, compared to State I (Fig. 21). Tidal volume increased slightly from 1.04 m1/100g(body weight) in State Ito 1.13 nil/100g in State II, 1.23 m1/100g in State III, and 1.2 m1/100gin State IV.71Figure 21: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on respiration(breathing frequency, tidal volume and minute ventilation) and on the associatedstate changes in these respiratory variables (absolute values, per 100g body weight)in urethane-anesthetized animals. * indicates significantly different from State Ivalues, same gas treatment. + indicates significantly different from normoxic values,same state.I+*MINUTE VENTILATION^TIDAL VOLUME^BREATHING FREQUENCY^(ml /100g /min)^(m1/100g)^(Breaths /min)om ,..,^ouiba^ba tn .4 0b.) ^U'4^0 r•^P^t"^t'o^us^o^vi^00 tol o ul b 0 col o col o73Figure 22: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on respiration(breathing frequency, tidal volume and minute ventilation) within each arousal statein urethane-anesthetized animals. Data is normalized to normoxic values in the samearousal state, per 100g body weight. + indicates significantly different fromnormoxic values, same state.74NORMOXIA•■..11 HYPERCAPNIAHYPDXIAt 225Z 200ad175w 1504-4 125C../i10075E-14e4 50Wg 25gl 0W^175g 150IS2i 8 125X^100O44t 75X^50tg^25CD 0Z 6 22520044o F45 175150e^1251001 7550250IvAROUSAL STATE75Figure 23: The effect of arousal state on the hypoxic and hypercapnic ventilatory responsesin urethane-anesthetized animals. Data are normalized to State I values (per 100gbody weight) for normoxic, hypoxic (10.0% 02) and hypercapnic (5.0% CO2) groupsseparately. * indicates significantly different from State I values, same gastreatment.NORMOXIAHYPERCAPNIAHYPDXIAII,11P,V,■11•-•:•:11IMII1251000O czz 750z 50O E25O IA 0itt4e*NT01254=t€"4 100ri;I*025I^II^III^wAROUSAL STATE76150125100755025755077Urethane anesthetized animals responded to hypercapnia by increasing tidal volume 55-67% over normoxic (same state) values in all states (Fig. 22). Despite this significant increase,the effect of arousal state on tidal volume was still evident in State III (Fig. 23). Tidal volumeincreased progressively in the hypercapnic group as the animals moved from State I, through lland into State DI. Tidal volumes in States II and ifi were increased by 17% and 23% over StateI values. Hypoxia did not alter tidal volume in the urethane anesthetized animals in any state(compared to the normoxic, anesthetized group). The change in tidal volume ranged from a 6%decrease to a 4% increase over normoxic (same state) values (Fig. 22). Thus, as in normoxia,none of the values were significantly different from the State I value. There was a slight, butinsignificant increase in tidal volume in State ifi (Fig. 23).Minute Ventilation As urethane-anesthetized animals moved from stable desynchronized EEG activity,through transitional states, and into stable synchronized EEG activity, VE was progressivelyreduced (Fig. 21). These decreases were significant in both States ll and Ill, compared to StateI. Minute ventilation dropped from 56.9 ml/min/100g (State I) to 41.0 ml/min/100g in State 11,to 35.0 ml/min/100g (State III). Values were lowest in State IV, at 27.7 ml/min/100g.Hypercapnic exposure in urethane-anesthetized animals elicited significant increases inYE in all states, ranging from 58-97% increases over normoxic (same state) values (Fig. 22).Again, although minute ventilation was increased overall, arousal state continued to exert aneffect (Fig. 23). As observed under normoxic conditions, minute ventilation decreasedprogressively in the hypercapnic group as the animals moved from State I, through II, and intoState DI. Minute ventilation fell to 82% and 77% of the State I value in States II and III,78respectively. Hypoxia elicited significant increases in VE in State III only (Fig. 22). Minuteventilation in hypoxia ranged from no change in State I (107% of nozmoxic, same state values),to a slight increase in State II (138%), to a large increase in State III (174%). The effect ofarousal state was not maintained under hypoxic conditions (Fig. 23); minute ventilation was fairlyconstant across the first three arousal states (States I, II and III). Ventilation in State IV wassignificantly lower than in State I.iii) Cardiovascular Variables Heart Rate Heart rate was significantly elevated by urethane anesthesia (Fig. 24). Despite this largeelevation in rate, an effect of arousal state on heart rate was still observable; significantly lowerheart rates were observed in State III compared to State I. Heart rate decreased from 366beats/min in State Ito 342 beats/min in State III. Although not significantly different from StateI, State II values (359 beats/min) were intermediate between those recorded in States I and III.Compared to State III values, heart rate did not change in State IV (339 beats/min).Under hypercapnic conditions, heart rates ranged from 323 to 336 beats/min. Thehypercapnic group showed a slight drop in heart rate compared to normoxic animals in States Ito III (ranging from 90 to 98% of normoxic values), these changes were significant only in StatesI and II (Fig. 24). Under hypoxic conditions, heart rates ranged from 302 to 307 beats/tnin.These were significantly lower than heart rates observed in normoxic animals in all arousal states;heart rates in States I to III ranged from 84-88% of the normoxic (urethane-anesthetized) values.79Neither hypercapnic nor hypoxic groups showed any influence of arousal state on heart rate (Fig.25).Sinus Arrhythmias Urethane anesthesia completely abolished respiratory sinus arrhythmia regardless ofwhether animals were breathing normo)dc, hypoxic or hypercapnic gas (Fig. 26). However,normoxic urethane-anesthetized animals showed a progressive increase in the amount of sinusarrhythmia as they moved from State I to IV, although the increase was significantly differentonly in State IV. Under both hypoxic and hypercapnic conditions, there was no changes in theamount of heart rate variation; sinus arrhythmia was absent in all arousal states, in both groups.80Figure 24: The effect of hypoxia (10.0% 02) and hypercapnia (5.0% CO2) on heart rate andon arousal state changes in heart rate under urethane anesthesia. Data are expressedas absolute values (per min). indicates significantly different from State I values,same gas treatment. + indicates significantly different from normoxic values, samestate.NORMOXIAV71 HYPERCAPNIAEgg HYPDXIA81C14El*^4004tta^350E••1^300g4et^250W= c.'Em4 200;14 WeO1504ttg100C14 504t4I^11^111^wAROUSAL STATE082Figure 25: The combined effect of arousal state and alterations in inspired gasconcentration (hypoxia and hypercapnia) on heart rate in urethane-anesthetizedanimals Data are normalized to normoxic, state I values. t indicates significantlydifferent from normoxic, State I, value.I^II^III^IVca)PCc.) 100g -404^75W44 •,,404,1 **C., W 50E=1 4et4 E*W ci) 25C.)gW 0a•1111 NORMOXIA(az HYPERCAPNIAIM HYPDXIA83AROUSAL STATEFigure 26: The effect of urethane anesthesia and inspired gas composition (hypoxia andhypercapnia) on respiratory sinus arrhythmia. Data are expressed as the meandifference between inspiratory and expiratory heart rates. Data for unanesthetized,normoxic animals (control) are included for comparison. * indicates significantlydifferent from State I, same gas treatment. + indicates significantly different fromnormoxic values, same state (among urethane-anesthetized groups only). # indicatesa significant difference between the unanesthetized and urethane-anesthetized animals(both normoxic groups) in the same arousal state.84WMMIMEMINIM85r.r4^75 -U4Wg^60 -W;Nlo 2= , 45 -;4 EtilEm4 ''t4t Ng^30 -&logletfr4^15 -=CONTROLNORMOXIAHYPDXIAHYPERCAPNIA*++WI!^LS/II^SWS/Ill REM/IVAROUSAL STATE86DISCUSSION This study confirms that urethane-anesthetized animals undergo spontaneous changes inarousal state as defined by EEG criteria, similar to wake/sleep in unanesthetized animals.Furthermore, it shows that the changes in respiratory and cardiovascular variables associated withspecific EEG patterns are similar for both sleep/wake and urethane anesthetized states. Theconcurrent adjustment of cardio-respiratory function with arousal state changes under both setsof conditions suggests that either there are processes which are common to the control of stateand cardio-respiratory function or that state subsequently exerts an influence on cardio-respiratoryfunction via alteration of the firing profiles of various, state-sensitive neural structures. Althoughmany state-sensitive neurons have been identified (Orem et al., 1974; Sieck and Harper, 1980;Orem et a/., 1985; Krilovvicz et al., 1988; Grahn and Heller, 1989), it remains to be determinedwhether the changes in their discharge patterns are due to, or coincident with, the arousal statechange. Respiratory and cardiovascular alterations tend to occur immediately upon arousal stateconversion (Colrain et al., 1987; Pack et al., 1992), however, suggesting direct and simultaneouseffects. That both immediate and sustained changes in cardio-respiratory function, identical tothose seen in sleep/wake states, are also observed in the urethane-anesthetized animals cyclingbetween states with similar EEG profiles, suggests that whatever is responsible for these changesis unaltered by urethane. This similarity further suggests that the distinct cortical patternsproduced in urethane anesthesia represent arousal states which are analogous to those seen inunanesthetized animals, at least in terms of cardio-respiratory control.87AROUSAL STATES Comparison of Cortical Activity Patterns (EEG) in Sleep and Anesthesia Animals under either the influences of sleep or urethane anesthesia show changing EEG(cortical) patterns ranging from low voltage, high frequency to high voltage, low frequencywaveforms. In this study, five distinct awake/sleep states were defined based on EEG and EMGactivities. Wakefulness (W) was associated with a desynchronized cortical pattern, accompaniedby robust muscle tone (no body movements). During the transitional state (LS) between W andSWS, this desynchronized activity was interrupted by brief periods of low-frequency, highvoltage waves. Muscle tone in this state ranged from fairly robust to diminished. SWS wasidentified by completely synchronized cortical activity, with absolutely no desynchronized activitypresent. Muscle activity during SWS was present, but diminished compared to that of W.Cortical activity appeared very similar during REM sleep and W; both states exhibitedcontinuous, desynchronized activity. Unlike W, however, REM sleep was normally accompaniedby muscular atonia, which allowed positive identification of this state. Although not used todistinguish REM sleep, it was noted that this state was often associated with periods of apneaand prolonged diastole. Like LS, the transition state (REM-ti) between SWS and REM sleep alsoshowed cortical desynchronization interrupted by brief periods of slow-waves; however, muscletone was diminished or absent.Under urethane anesthesia, five distinct cortical patterns were also observed and used todefine arousal states. Three of these EEG patterns closely resembled EEG recordings fromunanesthetized animals. These arousal states have been designated as States 1,11 and III (Grahnet al., 1989; (rahn and Heller, 1989). State I was defined by desynchronized cortical activity,88resembling the EEG of an awake animal. A fully synchronized cortical pattern, appearing verymuch like established slow-wave sleep (SWS), characterized State III. Intermediate corticalpatterns (ie. bouts of slow waves separated by stable desynchronized activity) defined thetransitional State II, and looked very much like those of a drowsy animal in light sleep (LS).State IV was a transition between normal and abnormal electrophysiological activity. Theabnormal electrophysiological activity, denoted as State V, resembled epileptifonn activity; thisstate indicated a very deep plane of anesthesia and was rarely observed. Cortical activity inanimals entering stable, extended periods of State V usually became isoelectric, after which theanimal often did not recover. Anesthetic levels in this study were set to avoid producing anyState V. Because of the tonic EMG present under urethane anesthesia, it is debatable whetherREM-like activity occurred in this preparation. Since it is impossible to differentiate REM-likeactivity from W-like activity without some other criterion such as changing EMG activity, PGOspikes or spectral analysis of the EEG, such activity would have been scored as State I activity.REMtr-like activity, similarly, would have been scored as State II activity. There are indicationsthat such activity does occur in urethane-anesthetized animals (Nufiez et a/, 1991; Heller, pers.comm.) but it is infrequent and of short duration.The extent to which animals exhibited State IV activity could be use to judge the relativedepth of anesthesia in the urethane-anesthetized groups. It was found during the course of theseexperiments that there was a fairly narrow range of dosages which would produce thesynchronized cortical activity; doses which were too high or too low produced primarilydesynchronized activity. The fact that all three urethane-anesthetized groups presented equal, lowamounts of State IV (Fig. 9) suggests that the level of anesthesia was appropriate and constantbetween groups.89The presence of similar, desynchronized cortical activity in wakefulness and State I, andsynchronized cortical activity in slow-wave sleep and State III raises the question of whetherhomologous mechanisms generate and maintain this activity. To evaluate this possible homologyproperly, one would need to understand the mechanisms involved in generating slow-waveactivity in sleep and anesthesia. The precise mechanisms generating slow-waves during deepNREM sleep are still not fully understood. Extensive research has led to the conclusion thatslow-wave generation involves interactions among several brainstem, diencephalic and forebraincell groups. It appears that there may be multiple generators for slow-wave activity that canindependently produce local aspects of this state in either brainstem or forebrain (Siegel, 1990).Of note is the fact that the ascending cholinergic activating system is implicated in producing theEEG desynchrony and associated arousal of waking and that inhibition of this system is requiredto produce slow-waves (Vincent and Reiner, 1987).The pharmacology of urethane suggests that it decreases acetylcholine (ACh) release athigh concentrations (Little et al., 1980). It would be tempting, therefore, to speculate that thiscould account for the slow-wave activity seen in urethane-anesthetized animals, except that notonly were low levels of urethane used in the present study (high levels actually eliminated slow-wave activity), the animals moved between states with desynchronized and synchronized EEGwhile apparently under a constant level of anesthetic.The low-level, tonic activity of the EMG observed in this study, however, may also berelated to the effects of urethane on cholinergic systems. Interestingly, although it decreasesgross ACh release, urethane increases the release of spontaneous ACh quanta at theneuromuscular junction (NMJ) (Quastel and Linden, 1975). In addition, urethane is structurallysimilar to physostigmine, an anti-acetylcholinesterase, and shows some anti-acetylcholinesterase90activity itself. The combined action would increase tonic levels of ACh at the neuromuscularjunction, thereby increasing the muscle tone. However, Quastel and Linden (1975) also foundthat urethane induces blockade of the neuromuscular junction, which might explain why muscleactivity did not change with changes in arousal state in spite of the increased tone.At this point, therefore, the data simply suggest that urethane-anesthetized animals cancycle between desynchronized and synchronized EEG patterns which superficially resemble thoseof unanesthetizecl animals moving between wake and slow-wave sleep. Nuiiez et al. (1992) haverecently recorded thalamic potentials under urethane anesthesia, and conclude that the fieldpotentials, unit discharges and delta oscillations are the same as those under natural sleepconditions. This might suggest that the genesis and cycling between the different cortical activitypatterns are also unaffected by urethane anesthesia but such a conclusion must be taken as verytentative.Arousal State Distribution in Unanesthetized Ground Squirrels In the first part of this study, golden-mantled ground squirrels spent equal amounts of timein wakefulness, drowsiness and SWS. Together, these three arousal states occupied over 90%of the recording time, with the remaining time being divided between REM sleep and thetransition to REM sleep. The REM sleep values probably represent mainly tonic REM sleepperiods. This study was not able to differentiate tonic from phasic REM sleep periods becausethe variables required to make this distinction, such as eye movements or PGO waves, were notrecorded. Since the phenomena associated with phasic REM sleep (such as eye movements)could conceivably create motion-artifacts in the cortical EEG, phasic REM periods may havebeen scored as REMtr. It is conceivable, therefore, that the proportion of time observed in REM91sleep may be an underestimate, but this difference would be very slight and would be correctedby considering REMtr as a part of REM sleep. A transitional state between SWS and REM sleepwas also included in studies by Gottesman and colleagues (Gottesman, 1992; Glin et al., 1991),who termed this the intermediate state (IS) (Table II).Although the second part of the present study also included a drowsy state (LS); differentcriteria were used to define SWS. In the first part of the present study, SWS was scored veryrigorously. Only completely established SWS was scored as such; if any desynchronized activitywas present, the state was scored as LS. When less rigorous criteria were used in the second partof the present study (any 30 second epoch in which more than half of the record wassynchronized was scored as SWS), animals were found to spend 50% more time in SWS and50% less time in LS.Total sleep time reported in the present study is exactly the same as that reported in othersleep studies involving Spermophilus lateralis (Walker et al., 1977), and greater than thatreported for Spermophilus tridecemlineatus (Van Twyver, 1969). Wakefulness values werehighest in the study of Van Twyver (1969) where LS was classified as W, and SWS values weregreatest in the study of Walker et al. (1977), where LS was classified as SWS (Table I).Compared to these other sleep studies, lower proportions of time were spent in SWS in the firstpart of the present study where the scoring was more rigorous and a considerable amount of timewas spent in LS (Table I), but similar amounts of time were spent in W and SWS when the datafrom the second part of the study are compared to those of Walker et al. (1977) for the samespecies. Van Twyver (1969) noted that of the five rodent species which he studied, the groundsquirrel showed two additional states, corresponding to the drowsy and REM-transitional statesof this study, although he did not include either of these two states in his scoring.92Compared to other studies, the present study reports the lowest total amount of REMsleep. While these values are only one third those reported by Walker et al. (1977) for this samespecies, they are not that dissimilar from those reported for hamsters, mice and chinchillas (TableII). The reason for this difference is not clear but most likely stems from the criteria used forscoring REM sleep epochs.It should also be noted, however, that differences exist between these studies in terms ofthe total recording time, and this may affect the reported proportions of time spent in sleep andwakefulness. Van Twyver (1969) recorded continuously for 48 hours, Walker et al. (1977)recorded continuously for 24 hours while in the present study, recordings were made for 8-hourperiods. It is conceivable that the 8-hour recording periods could skew the reported proportionsof wake/sleep, particularly if the experiment ran through a specific portion of the animal'scircadian cycle (ie. through the sleep portion of the circadian cycle). In the latter situation, theserecordings might underestimate the amount of wakefulness. Certainly, the fact that the presentstudy demonstrated much lower proportions of wakefulness than that of Van Twyver (1969), whorecorded for 48-hour periods, implies that such might be the case. However, the present studyattempted to circumvent the problem of skewing arousal state proportions by recording throughapparent portions of the circadian cycle. Golden-mantled ground squirrels were observed to bemost active at dawn and dusk; this behaviour was interpreted as indicative of animals being inthe activity (wake) portion of the circadian cycle. Data recording in the unanesthetized animals,therefore, was begun approximately four to five hours before and concluded four to five hoursafter dusk. The fact that the amount of both wakefulness and of total sleep time was the samein the present study as that reported by Walker et al. (1977), who recorded continuously for 24hours, suggests that the protocol of the present study was sufficient to avoid the problem of93skewed arousal state proportions. In addition, Martin et a/. (1990) employed recording periodsof only four hours in length for hamsters and yet obtained proportions of wakefulness and totalsleep time which were approximately the same as that reported by Van Twyver (1969) forhamsters over a 48-hour period (Table II).Data from studies involving other species suggest that the golden-mantled ground squirrelspends slightly more time sleeping than other rodents, with the exception of the pocket mousewhose total sleep time was an extraordinary 80%.Arousal State Distribution in Urethane-Anesthetized Ground Squirrels Under urethane anesthesia, the amount of time spent in either of the two stable arousalstates (State I and State III) was similar to that spent in W and SWS in the unanesthetizedanimals. The amount of time spent in state ll was significantly less than that spent in LS in part1 of the present study, but similar to that spent in LS in part 2 of the study (Figs. 7 and 15). Theproportion of time that urethane-anesthetized animals moved through this transitional state toenter a new arousal state was exactly the same as in the sleeping animals changing states (Fig.8). Urethane-anesthetized animals, however, changed states, overall, much less often as can beseen from the behavioural records (Fig. 6 and 9). This decreased number of transitions suggeststhat the administration of urethane either attenuated the mechanisms involved in generating andswitching arousal states, or enhanced the mechanisms involved in maintaining stable arousalstates. Furthermore, urethane-anesthetized animals tended to move quickly between establishedarousal states, spending very little time in transition.The time spent in desynchronized activity may also include an analogy of REM sleep.This study could not differentiate a REM-like sleep state; changes in muscle tone were used inTable ISleep state distribution of various species of the Sperraophilus genusSLEEP STUDY TOTAL TIME SPENT IN EACH SLEEP STATE(% of Total Recording Time, TRT)TRT(hrs)W LS SWS REM-t REM TSTSpermophilus lateralis32.6 30.9 31.7 2.3 2.5 67.4 6-8Present study, Part 1Present study, Part 2 34.5 14.1 48.4 N/M 4.0 65.5 6-8Walker et al., 1977 33.6 N/M 54.0 N/M 12.4 66.4 24Spermophilus tridecemlineatus42.5 N/M 43.3 N/M_14.2 57.5 48Van TI,vyver, 1969Abbreviations used in Table I are as follows: TST = total sleep time; TRT = totalrecording time; N/M = not measured; N/R = not reported. The five arousal states aredefmed in Materials and Methods.94Table llSleep state distribution in other rodent speciesSPECIES/ SLPPP STUDYTOTAL TIME SPENT IN EACH SLEEP STATE(% of total recording time, TRT) TRT(firs)W LS SWS REM-ti REM TSTHAMSTERMesocricetus auratus39.9 N/M 46 N/M 14.1 60.1 48Van Twyver, 1969Phodopus sungorus43.4 N/M 50.3 N/M 6.3 56.6 4Martin et cd., 1990RATRattus norvegicus56 N/M 34 1.0 9.0 44.0 N/RGottesman, 1992Van Twyver, 1969 44.8 N/M 44.4 N/M 10.8 55.2 48MOUSEMus musculus45.2 N/M 49.5 N/M 5.3 54.8 48Van Twyver, 1969CHINCHILLAChinchilla laniger47.8 N/M 45.7 N/M 6.5 52.2 48Van Twyver, 1969POCKET MOUSEPerognathus longimembris19.6 N/M 67.45 N/M 12.95 80.4 8Walker et al., 198395Definitions for all abbreviations used in Table II are as indicated at the end of Table I.96this study to distinguish W from REM sleep in unanesthetized animals and such changes inmuscle tone were not seen in anesthetized animals as discussed earlier. Nuilez et al. (1991) havesuggested that some desynchronized activity in urethane-anesthetized rats was comparable toREM sleep based on recordings of unit activity in the RPO (reticularis pontis oralis) nucleusduring periods with spontaneous and sensory-elicited theta rhythms. Theta activity of thehippocampus is associated with REM sleep and muscle atonia, or waking with body movements.It is interesting to note that unanesthetized animals spent 32.6% of the time in W and 4.8% inREM and REMtr sleep (total = 37.4%), while urethane-anesthetized animals were scored asspending 41% of the time in State I, supporting such conjecture.Effects of Hvnoxia and Hvvercannia on Arousal State Distribution Ground Squirrels Both hypoxia and hypercapnia affected the amount of time spent in different arousal statesin unanesthetized and urethane-anesthetized ground squirrels. Normoxic animals in bothurethane-anesthetized and unanesthetized groups spent approximately the same amount of timein established arousal states (W and SWS/States I and BEI), and significantly less time intransitional activity (LS/State II). Under hypercapnic conditions, unanesthetized animals spentless time in SWS and more time awake, while urethane-anesthetized animals spent less time inState HI and more time in State I. However, the reduction in time spent in synchronized activity,relative to time spent in desynchronized activity, was only significant in the latter group.Hypoxia tended to equalize the proportions of time spent in each arousal state, in bothunanesthetized and anesthetized groups; ie. an increase in time spent in LS/State 11 and decreasein time spent in SWS/State HI. Thus, in both unanesthetized and anesthetized animals, the time97spent in LS and State II, respectively, were no longer significantly less than the times spent n theestablished arousal states. Neither hypoxia nor hypercapnia affected the proportion of time thatanimals moved through a transitional state to enter a new arousal state (Fig. 20). Therefore,hypoxia and hypercapnia both decreased the amount of synchronized activity, but in differentways. Under hypercapnic conditions, the switch was to more established desynchronized activity(W and State I), while under hypoxic conditions it was to more transitional activity (LS and StateII). Of significance is the fact that the trends were the same for both unanesthetized andanesthetized animals.Other Sleeping Animals Although much variation exists in data collected from human studies (Gothe et al., 1986),hypercapnia is thought to be a potent arousing stimulus (Phillipson and Sullivan, 1978). Bothadult (Gothe et al., 1982; Berthon-Jones and Sullivan, 1984; Fewell and Baker, 1989) and infant(Haddad et al., 1980) human subjects will arouse to inspired CO2 levels between 2.0 and 4.0%.Haddad et al. (1980) also noted that increased CO2 levels elicited neither ventilatory nor arousalresponses in infants with failure of autonomic ventilatory control. This arousal response isthought to represent a protective ventilatory mechanism when sleeping, and infants with faultyresponses are thought to be more susceptible to SIDS (Sudden Infant Death Syndrome).Although hypercapnia appears to act as an arousal stimulus in humans, the effects are notcompletely clear in rats. Consistent with the present study, neither Pappenheimer (1977) norMegirian et a/. (1980) reported any significant effect of 5.0% CO2 on SWS in rats. The levelof CO2 exposure, however, appeared to be of significance. Ioffe et a/. (1984) found thathypercapnic exposure at 6.0 - 8.0% CO2 greatly increased the amount of desynchronized EEG98activity. The number of wake periods were doubled and the total duration of sleep was decreasedby 28%. During this present study, the levels of CO2 were noted to drift as high as 5.5% CO2.The slight (nonsignificant) arousal response to 5.0% CO2 suggests that golden-mantled groundsquirrels may alter arousal state distribution in response to CO2, but that 5.0% did not providea great enough stimulus.The effects of hypoxia on arousal state distribution in this study also correlated well withprevious studies. Pappenheimer (1977) first noted that hypoxia decreased the total amount ofSWS in rats, and changed the normal sleep pattern to a series of brief, incompletely developedepisodes. Pappenheirner, however, looked only at SWS, and did not distinguish light from deepplanes. Several investigators have since expanded these initial observations, examining theinfluence of hypoxia on all sleep states. Further studies in rats have confirmed these initialfindings, and have also shown that hypoxic exposure greatly increases the amount of transitionalactivity. Laszy and Sardaki (1990) divided the SWS state into light (intermittent) and deep(established) slow wave activity, and subsequently found that not only was the proportion ofwakefulness increased, but also that during the sleep periods, intermittent activity predominatedover established SWS. In accordance with previous studies, these results also showed that sleepwas interrupted frequently and the transitions between light SWS and wakefulness were greatlyincreased (Ryan and Megirian, 1982; Hale et al., 1984). Similar results have been obtained infetal sheep (Boddy et al., 1974; Koos et al., 1987). Hypoxic exposure, however, producesvariable effects on arousal in human subjects (Gleeson et al., 1990 and Gothe et al., 1982).Gothe et a/. (1982) reported that half of their subjects awoke half the time, one quarter of theirsubjects always awoke and one quarter of their subjects never awoke when 02 saturation wasreduced to 75%. In spite of the variability within the human data, the data in sleeping animals99suggests that exposure to hypoxia does increase the amount of time spent in transitional activitybetween synchronized and desynchronized cortical activity. These results correlate quite wellwith the increased amount of transitional LS observed in the unanesthetized animals in this study.Other Anesthetized Subjects The present results correlate fairly well with previous observations of hypercapnic andhypoxic effects on the EEG pattern of anesthetized animals and patients. Under anesthesia, CO2appears to exert a strong influence on EEG patterns. McDowall (1976) noted that hypercapniawould produce "arousal" effects in the EEG profile, while extreme hypocapnia produced large,slow waves. Similarly, Marshall et al. (1965) found that, as over-ventilation decreased CO 2 tohypocapnic levels, high amplitude, low frequency waves appeared in the EEG.Hypoxia also affected EEG profiles of anesthetized patients. Marshall et al. (1965) notedthat with the onset of cerebral anoxia, the EEG trace of anesthetized patients first slowed andincreased in amplitude. As the 02 deprivation became more severe, the trace fmally flattened.McDowall (1976) also found that the amplitude of all EEG frequencies changed as a functionof 02 level, first increasing with hypoxic exposure and then decreasing as the conditions becomemore severe (anoxic). Thus, although hypercapnic anesthetized patients responded in a similarfashion to the anesthetized squirrels, hypoxic patients did not. It should be noted, however, thatthere are few studies which provide data of this sort and the anesthetics used varied considerably.Observations in other urethane-anesthetized animals (rats), although preliminary only, alsoindicated that hypoxic or hypercapnic exposure produces EEG desynchronization (TamaId andNakayama, 1987), consistent with the response observed in golden-mantled ground squirrels.100SLEEP/ANESTHESIA AND BREATHING L NORMOXIC CONDITIONS State-dependent Respiratory Alterations in Wake/Sleep The values for f and VE obtained in the present study in unanesthetized, awake animalswere roughly double those previously reported for euthermic, golden-mantled ground squirrels(McArthur and Milsom, 1991; Webb, 1987). This undoubtedly stems from the fact that previousstudies report data collected from animals resting quietly in dark environmental chambers. Basedon the present study, such animals were probably sleeping 67% of the time. The present studyalso demonstrated that breathing frequency and consistently decreased in sleep (LS, SWS andREM sleep) in the golden-mantled ground squirrel. Therefore, averaging data from acrosssleep/wake states, as these previous studies have done, would produce much lower values for fand VE than those collected definitively during wakefulness.The arousal state-dependent changes in respiratory pattern demonstrated in golden-mantledground squirrels agree with the animal literature available for sleep effects on breathing (TableBI). As in other animals, golden-mantled ground squirrels typically present lower breathingfrequencies and greater tidal volumes in SWS compared to wakefulness. The increase in tidalvolume was, however, only significant in part II of the study. In REM sleep, these animalsnormally show bouts of irregular breathing, but neither average breathing frequency nor tidalvolume differed from SWS.The progressive decrease in breathing frequency with the onset and establishment of SWSappears to be a fairly consistent response between various species, including humans. Tidalvolume responses, on the other hand, show a fair degree of diversity between studies andTable 111Respiratory pattern changes during sleep in animal subjectsInvestigators Subject BreathingFrequencyTidal Volume MinuteVentilationOrem, Netick andDement (1977)Cats REM>W>NREM NREM>W>REM W>NREM>REMRemmers, Bartlettand Putnam (1976)Cats REM=W>NREM NREM>W=REM -Foutz, Netick andDement (1979)Cats - - W>NREM>REMSantiago, Sinha andEdelman (1981)Cats - - W>NREM>REMBaker, Netick andDement (1981)Cats - - W>REM>NREMPhillipson, Muiphyand Kozar (1976)Dogs REM>W>NREM NREM>W>REM REM>W>NREMDeMesquita andAserinsky (1981)Dogs W>NREMREM>NREMNREM>WNREM>REMW>NREM=REMPappenheimer (1977) Rats W>NREM NREM>W W>NREMMegirian, Ryan andSherrey (1980)Rats REM2W>NREM - -Present study(1993)GroundsquirrelW>SWS=REM SWS=REM2W W>SWS2REMMartin, Tannenbaumand DeMesquita (1990)DjungarianhamsterREM>NREM - -Stevenson andMcGinty (1978)Kitten W>REM>NREM - -Scott, Inman andMoss (1990)Piglet W>NREM>REM - -Guthrie, Standaert,Hodson and Woodrum(1980)PrematureprimateNO CHANGE NO CHANGE W2REM2NREM101Table IVRespiratory trends from sleep studies on human subjectsInvestigators Age BreathingFrequencyTidal Volume Minute VentilationGothe et al.(1982)young adult WaNREM W2.NREM W.NREMShea et a/.(1990)adult NREM>W 1) No Change2) NREM>W1) NREM>W2) W>NREMWeil et al.(1984)adult NREM>WREM>WW>NREMW>REMW>NREMW>REMGothe et al.(1981)adult W>NREM W>NREM -Skatrud andDempsey(1983)adult (only2 subjects)1) W>NREM2) NREM>W1) W>NREM2) NREM>W1) W>NREM2) NREM>WCurzi-Dascalovaet al.(1981)infant NREM>REM - -Krieger et al.(1990)adult NO CHANGE W>REM=NREM W>REM=NREMWhite et a/.(1981)adult NO CHANGE W>NREM=REM W>NREM=REMIngvar andBillow (1963)adult - - W>NREMRigatto et al.(1980)infant(preterm)REW_NREM REW_NREM REMk.NR.EMColrain et al.(1990)young adult - - W>NREMKahn et al.(1992)infant NO CHANGE - -102103subjects. Tidal volume changes during sleep appear to be more influenced by such things asexperimental protocol (Douglas et a/., 1983), and intra- and inter-subject variability thanfrequency alterations.Overall minute ventilation was decreased in both sleep states (SWS and REM) comparedto wakefulness (W). The decrease, however, was significant in the first part of the study, but notin the second (controls for hypoxic and hypercapnic responses in sleep). The state-dependentdecrease in VE in the first normoxic group stemmed from a large drop in breathing frequency,which offset a smaller, nonsignificant increase in VT. The second normoxic group showed asmaller decrease in frequency, which was not great enough to offset a significant increase in VT,resulting in a nonsignificant change in VE•In many animal species studied to date (Table III), minute ventilation has also consistentlybeen found to decrease during SWS (Orem et al., 1977; Pappenheimer, 1977; Phillipson et al,1978; Baker et al., 1981; DeMesquita and Aserinsky, 1981; Santiago et al., 1981; Megirian etal., 1980). The influence of REM sleep on breathing, however, is less consistent. For example,kittens show their highest breathing frequencies in wakefulness, intermediate values in REM sleepand lowest values in SWS (Stevenson and McGinty, 1978). Piglets also show the highestfrequencies in wakefulness, but intermediate values in SWS and the lowest in REM sleep (Scott,Inman and Moss, 1990). In cats, Orem et al. (1977) and Santiago et al. (1981) both found thegreatest minute ventilation in wakefulness and the lowest in REM sleep. Baker eta!. (1981) alsofound cats to exhibit the highest values in W, but these investigators found the lowest values inSWS. DeMesquita and Aserinsky (1981) found dogs also exhibited the greatest levels of minuteventilation during wakefulness, but the values in sleep were not different between REM andSWS. In contrast to most other studies, Phillipson eta!. (1976) found dogs to exhibit the greatest104levels of minute ventilation in REM sleep, although SWS again was associated with the lowestvalues. The only species which failed to demonstrate arousal state effects on breathing was thepremature primate; interestingly, these results are very similar to those obtained in human studies(see below).Thus, differences between animal studies, including the present one, appear to revolvemainly around the values obtained for REM sleep. The changes associated with moving intoSWS from W appear to be fairly consistent. Like the other two states, REM sleep is not acompletely homogenous state, it consists of phasic and tonic phases, yet values reported for REMsleep seldom distinguish between these two phases. Indeed, in the present study, even thoughwaking values were standardized by using subjects at quiet rest and SWS values werestandardized by using only periods of well-established slow-wave activity, the different stagesof REM were not distinguished. Fairly large differences in the values of the respiratory variablesare seen in these two phases of REM sleep. The typically irregular breathing reported for REMsleep is usually associated with the phasic phase of REM sleep (Hendricks et a/, 1991; Netickand Foutz, 1980), while the tonic phase is associated with regular and SWS-like breathing(Sullivan et al, 1979; Phillipson, 1978; Phillipson and Bowes, 1986). The respiratory and cardiacvalues obtained for REM sleep in the present study probably reflect tonic, rather than phasic,REM sleep. This distinction would account for the lack of difference noted between the SWSand REM sleep values for any of the respiratory variables.The influence of arousal state on respiratory pattern in human subjects has also beenextensively investigated (Table IV). However, unlike the data accumulated for animal subjects,the existing literature for human respiration in sleep is extremely equivocal and contradictory,probably due mainly to differences in experimental technique (Douglas et al, 1983; Krieger et105al, 1990). In human adults, all possible respiratory responses to arousal state changes have beenobserved. Gothe et al. (1981, 1982) found that frequency, tidal volume and minute ventilationwere all greater in wakefulness than in SWS. Shea et al. (1990) and Weil et a/. (1984) bothfound frequency, tidal volume and minute ventilation to be greater in SWS than in W. Kriegeret al. (1990) and White et al. (1985) found frequency to be independent of arousal state, whileboth tidal volume and minute ventilation were greater in W, but not different between the twosleep states. Although the studies of Ingvar and Billow (1963) and Colrain et al. (1990) reportedonly changes in minute ventilation, these investigators also found a greater minute ventilation inW than in SWS sleep. The results of Skatrud and Dempsey (1983) typify the variation observedin these human studies; these investigators looked only at two subjects and found exactlyopposite results. One subject showed a greater tidal volume and lower frequency and minuteventilation in SWS compared to W, while the second subject showed a lower tidal volume anda higher frequency and minute ventilation in SWS compared to W. The data for infants has beensimilarly ambiguous. Curzi-Dascalova et al. (1981) found higher frequency values in SWScompared to REM, while Rigatto et a/. (1980) found lower or equal frequencies in SWScompared to REM sleep values. Rigatto et a/. also noted that the tidal volume increased andthe minute ventilation dropped in SWS compared to REM sleep.Much work has gone into defining the mechanisms underlying arousal state-dependentalterations in respiration. Whether arousal state-dependent changes in various physiologicalsystems are direct changes, or secondary responses to other alterations, is somewhat unclear.In general, breathing in wakefulness is considered to be governed by both behavioural andmetabolic control. The behavioural system involves a wide variety of inputs from voluntary toemotive. Anatomically, such structures as the cerebral cortex and limbic structures have been106implicated, projecting down via corticobulbar and corticospinal tracts. The behavioural influencesproject directly to both brainstem respiratory nuclei and to the spinal cord motomeurons andappear to interact with the metabolic system directly and indirectly. Fink (1961) first postulatedthe idea that wakefulness itself could serve as a stimulus for breathing. Since this time, a numberof investigators have examined this concept. (Billow, 1963; Orem et al., 1977; Phillipson, 1978;Sullivan et al., 1978; Foutz et al., 1979; Orem, 1990). Many investigators now believe that itis the progressive loss of the wakefulness stimulus which produces the respiratory changesassociated with SWS, as opposed to mechanisms specific to SWS. Several studies (Naifeh andKarniya, 1981; Colrain et al., 1987; Pack et al., 1992) have correlated respiratory changes withalteration in EEG pattern. All have found that respiration changes immediately with the onsetof State 1 and 2 NREM sleep (drowsiness and light sleep, identified as LS in the present study).These researchers maintained that the immediate and distinct correlation between respiration andEEG frequency supported the hypothesis that the loss of wakefulness exerts a direct effect onrespiration, and indicated that centres involved in respiration and arousal state determinationinteract at the level of the CNS.Slow-wave sleep is thus considered to be governed primarily, if not exclusively, by themetabolic demands of the body; the behavioural (voluntary) component has been removed in thisstate. In fact, during both SWS and light sleep, normal respiratory rhythmogenesis appears tobe dependent on the CO2 stimulus (Skatrud and Dempsey, 1983; Datta et al, 1991). Theseobservations are also supported by clinical evidence (Fink, 1961; Guilleminault et al., 1981;McNicholas et al, 1983). Thus, respiratory function in SWS is not considered to be fundamentallydifferent from that in wakefulness, just lower in magnitude due to the loss of behavioural input.Breathing during REM sleep was first considered to be independent of metabolic control.107Recently, however, it has been noted that the tonic and phasic phases of REM sleep representfundamentally different states with respect to breathing and other physiological systems.Phillipson (1978; Phillipson and Bowes, 1986) noted that breathing variables in tonic REM sleepare often not different from SWS, while those recorded during phasic REM sleep are associatedwith increased frequency and minute ventilation, compared to SWS (Sullivan et al, 1979). Thus,PhilEpson suggests that respiratory changes in tonic REM may reflect metabolic control, whereasthe changes seen in phasic REM may be governed by nonmetabolic control (which may reflectbehavioural control).State-Dependent Respiratory Alterations Under Urethane Anesthesia In general, respiratory changes coincident with arousal state alterations in urethane-anesthetized animals appeared to parallel those observed in unanesthetized animals changingbetween states of wakefulness and natural sleep. Significant decreases in breathing frequencyoccurred with the onset (State II) and establishment (State HI) of synchronized cortical activity.Similarly, as EEG patterns became synchronized, minute ventilation also decreased significantly.These alterations were equivalent to those observed in the unanesthetized golden-mantled groundsquirrels with the development of synchronized activity in LS and SWS.Under urethane anesthesia, respiratory changes appeared to occur in close association withthe alterations in EEG activity. Even as intermittent activity appeared in the EEG, respiratoryvalues immediately changed to intermediate levels between State I and State DI These datasuggest that common mechanisms may be involved in the state changes and state-dependentrespiratory changes seen in the urethane-anesthetized animals as in the unanesthetized, controlanimals. The fact that respiratory changes were also intermediate in the transitional state of108urethane-anesthetized animals is indirectly supportive of the hypothesis that some stimulus tobreathing frequency and minute ventilation exists during periods of desynchronized corticalactivity in these animals, which is lost gradually as cortical activity becomes synchronized.Completely synchronized activity may be associated with complete loss of this stimulus and thelowest frequency and minute ventilation was indeed observed in this state. Thus, alterations inrespiratory control as a function of, or coincident with, state changes between specific EEGprofiles appear to be the same in urethane-anesthetized and unanesthetized animals.Tidal volume showed small, nonsignificant increases as animals moved from thedesynchronized State I to the synchronized State III. As with the state-dependent changes inbreathing frequency and VE, even these small changes in VT were progressive; intermediatevalues were obtained during the state of transitional EEG activity (State II). The lack ofsignificant change in VT in the urethane-anesthetized group is consistent with the first group ofnormoxic, unanesthetized animals, and both groups show trends in the same direction as thesignificant increases of the second nonnoxic, unanesthetized group. Tidal volumes obtained inthe urethane-anesthetized animals showed much less variability than those recorded in theunanesthetized animals. This most likely reflects the absence of any behavioural effect onventilation in anesthetized animals.II. HYPDXIC AND HYPERCAPNIC VENTILATORY RESPONSES Hypoxic Ventilatory Responses In Awake/Sleeping Ground Squirrels Exposure to 10.0% 02 elicited a large increase in breathing frequency and a smalldecrease in tidal volume in awake golden-mantled ground squirrels. As a result, minuteventilation increased roughly 50%. The overall increase in ventilation under hypoxic conditions109in awake golden-mantled ground squirrels in the present study was approximately half thatreported previously (Webb, 1987; McArthur and Milsom, 1991). Although these latter twostudies most likely averaged data collected during wakefulness and sleep, and hypoxic sensitivitydoes increase during sleep in this species, this could only account in part for the difference.More likely, the difference in the hypoxic responses obtained between the present and latter twostudies suggests that for the animals in the present study, 10.0% 02 was right around, or only justabove the threshold for the hypoxic ventilatory response.As in the awake state, breathing frequency was significantly increased in all other arousalstates during hypoda compared to normoxia in that state. Tidal volume was also reduced in allother states. The net result was an increase in VE in all sleep states. The hypoxic sensitivity ofthe golden-mantled ground squirrel was unchanged, or even increased, during SWS and REMsleep.Exposure to 10.0% 02 tended to attenuate the effect of sleep on all respiratory variables.Although decreases in breathing frequency still occurred in SWS and REM sleep under hypoxicconditions, they were less than during nonnoxia (Fig. 16). Neither tidal volume nor minuteventilation changed at all through the four arousal states.Hypercapnic Ventilatory Responses In Awake/Sleeping Ground Squirrels Exposure to 5.0% CO2 stimulated ventilation significantly in awake golden-mantledground squirrels. As in the hypoxic animals, the increase in VE was due to a significant increasein breathing frequency; tidal volume did not change at all (Figs. 16 and 17). Golden-mantledground squirrels have also been observed, under similar experimental conditions, to increase VEsignificantly via increases in VT only (Webb, 1 987) and via equal increases in both frequency and110VT (McArthur and Milsom, 1991). Fossorial species have been shown to exhibit bluntedhypercapnic ventilatory responses (Boggs et al., 1984) and the sum of data would suggest that5.0% CO2 was just around, or only slightly above, the threshold for the hypercapnic ventilatoryresponse in these animals.As in the awake state, sleeping ground squirrels were equally responsive to 5.0% CO2;the levels of minute ventilation were significantly greater than normoxic values in all sleep states.During LS and SWS, the same pattern of response was observed; breathing frequency wasincreased and tidal volume was unchanged. In REM sleep, neither breathing frequency nor tidalvolume were significantly different from normwdc values during hypercapnia. The increase inbreathing frequency was smaller in this state, and was accompanied by a slight increase in tidalvolume.Exposure to 5.0% CO2 did not alter the state effects on respiratory frequency or tidalvolume. Even under hypercapnic conditions, breathing frequency was significantly depressed inall sleep states, and tidal volumes were significantly greater in both SWS and REM sleep.However, the net effect was that arousal state changes no longer influenced VE during exposureto 5.0% CO2. Under normoxic conditions, the state-dependent decrease in breathing frequencyseen in sleep was larger than the concomitant increase in tidal volume. Although frequencycontinued to decrease during sleep under hypercapnic conditions, it did not offset the tidalvolume increase and thus, minute ventilation did not decrease significantly in SWS. The lackof state effect on minute ventilation during hypercapnia was very obvious; unlike noimoxicconditions (where VE decreased during sleep), ventilation was constant across the four arousalstates. Overall, the fact that VE was not decreased in a sleep-dependent fashion in animals duringexposure to 5.0% CO2 suggests that the sensitivity to hypercapnia was increased during sleep in111the golden-mantled ground squirrel.Hypoxic and Hvpercapnic Ventilatory Responses Compared to Other Species Awake AnimalsThe hypoxic ventilatory responses observed in the present study in awake golden-mantledground squirrels are in accordance with many other studies in awake animals. Previous studiesindicate that the response of golden-mantled ground squirrels to hypoxia is consistently anincrease in breathing frequency, with little or no change in VT (McArthur and Milsom, 1991;Webb, 1987). Many semi-fossorial rodents increase ventilation via increases in frequency, withno change in tidal volume (Davies and Schadt, 1989; Mortola, 1991; Holloway and Heath, 1984;Walker et al., 1985). In the nonfossorial rat, hypoxia stimulates both frequency and tidal volumeequally (Holloway and Heath, 1984), although anesthetized rats respond like semi-fossorialanimals with increases in breathing frequency only (Hayashi and Sinclair, 1991 and Vessal,1988).It is possible that the hypoxic ventilatory responses in the present study were beingdampened by a concomitant hypocapnia, which would decrease the ventilatory drive. Hypocapniaresults from the hyperpnea associated with hypoidc exposure, which frequently produces adecrease in VT. This is true of all studies in which animals are not maintained isocapnic. Giventhe low CO2 sensitivity of these animals, and the fact that the interaction between hypercapniaand hypoxia on the ventilatory response has been found to be minimal in golden-mantled groundsquirrels (McArthur and Milsom, 1991), such an effect should be minimal.That some species do exhibit increases in VT while others do not may reflect a differentialeffect of hypoxia on breathing frequency and tidal volume. Cragg and Drysdale (1983) found112that, in the rat, both minute ventilation and tidal volume were related to P02 in a hyperbolicfashion, whereas frequency was related to Po2 in a linear fashion. With a low hypoxic stimulus(ie. higher PO, the main ventilatory response was an increase in breathing frequency. As theP02 levels decreased, however, tidal volume increases became more important to the minuteventilation increase than an elevation in breathing frequency. Therefore, one might assume thatall studies which produced frequency responses alone applied a lower stimulus (at least at thereceptor site) than studies in which increases in both components of respiratory pattern (frequencyand tidal volume) occurred.Unlike the present study, hamsters and adult humans increased minute ventilation duringhypercapnic exposure via increases in tidal volume only (Holloway and Heath, 1984; Walker etal., 1985; Hedemark and ICronenberg, 1982) Rats, on the other hand, have been shown toincrease either VT only (Holloway and Heath, 1984), or both respiratory frequency and tidalvolume (Walker et al., 1985). The completely fossorial mole rat also responded to hypercapniaby increasing both frequency and tidal volume approximately equally (Arieli and Ar, 1979). Incontrast to these observations, Chapin (1954) observed only a rate increase, and no tidal volumeresponse, in hamsters exposed to hypercapnia. However, the levels of CO2 used in that studywere far more extreme (up to 22% CO2) than in most other studies.According to Cragg and Drysdale (1983), both minute ventilation and tidal volume in ratswere linearly related to Pco2, whereas breathing frequency exhibited a hyperbolic relationshipwith Pø2. Therefore, at the lower end of the Pc02 response curve, changes in tidal volumewould be more important than changes in frequency in increasing minute ventilation. As thePco2 levels increased, the frequency would increase exponentially, becoming the maindeterminant of further increases in minute ventilation. The data of Arieli and Ar (1979) from113the completely fossorial mole rat exhibited these same relationships, although they were notquantified.Given the difference in the relationships between tidal volume and frequency with Pco„that fossorial animals show greater tidal volume responses than frequency responses tohypercapnia, while nonfossorial animals increase both respiratory variables (or only frequency)is not unexpected. Fossorial animals show several adaptations for burrow-dwelling, where theymight encounter chronic hypercapnic exposure. These include a blunted ventilatory response toCO2 fluctuations. Furthermore, although blood adaptations have not been found (RBC number,mean haemoglobin concentration and cell shape were unaltered), an increased [HCO3] (ie. 33mmol/1 in golden-mantled ground squirrels (Burlington et al., 1969) versus 19.8 mrno1/1 in therat (Javaheri et a/., 1980)) and left-shifted 02-dissociation curve have been observed (Baudinette,1973; Bullard et al., 1966; Boggs et al., 1984). Increased [HCO3] would reduce the amount of[H] change which occurred in response to increases in Pcoe Therefore, particularly if the CO2response is primarily central (where the [H1 is purportedly the main/only stimulus), changes inarterial Pc% in the fossorial animals would not represent as much of a stimulus as it would inthe nonfossorial animals. Consequently, those subjects which responded primarily with a tidalvolume increase may not have received as great a CO2 stimulus as subjects which respondedmainly via frequency increases.Sleeping AnimalsIn most other species studied, both hypoxic and hypercapnic ventilatory responses appearto be reduced during sleep. This depression is particularly evident in SWS; REM sleep appearsto be characterized by irregular responses. However, much of the data is disparate and114inconclusive. The majority of studies which have examined respiratory sensitivity to hypoxia andhypercapnia during sleep have been performed on human subjects. The majority of humanstudies on hypercapnic responses during sleep indicate that the sensitivity to CO2 is decreased,the hypercapnic ventilatory response curve (ventilation vs. Pc%) is shifted to the right (highervalues of Pc02), and alveolar hypoventilation with CO2 retention is observed (Robin et al., 1958;Billow, 1963; Ingvar and Billow, 1963; Gothe et al., 1981; 1986; Douglas et al., 1982a; Dempseyet al., 1990; Ingrassia et al., 1991; Bath et al., 1991). Two studies, however, indicate no changein ventilatory responsiveness to CO2 during sleep (Hedemark and Kronenberg, 1982; Weil et al.,1984). Finally, Berthon-Jones and Sullivan (1984) have found gender differences; males werefound to exhibit lower hypercapnic ventilatory responses than females during both sleep states(SWS and REM sleep), yet Well et al. (1984) report no gender difference for either hypoxic orhypercapnic ventilatory responses during sleep.Sleep-induced alterations in 02 sensitivity are similarly varied in the human data. Whiletwo studies found clear and dramatic decreases in the hypoxic ventilatory response during sleep(Douglas et al., 1982b; Dempsey et al., 1990), three similar studies found no change (Hedemarkand ICronenberg, 1982; Gothe et al., 1982; Well et al., 1984). Interestingly, one study (Chin etal., 1989) found a biphasic response to hypoxia in human adults during SWS, consisting of aninitial and rapid increase, followed by a decrease back to normcmic respiratory levels (a reflexnormally present in infants).Data from studies of sleep effects on the hypercapnic ventilatory reflex are no moreconsistent in animals. The response was decreased during SWS in both dogs (Bowes et a/.,1981; PhilEpson et al., 1980 and Sullivan et al., 1979) and cats (Santiago et al., 1981), andduring REM sleep in dogs (Sullivan et a/., 1979). Rats, however, showed an increased sensitivity115to CO2 during SWS (Pappenheimer, 1977), while no change was observed during either SWS orREM sleep in goats (Parisi et W., 1992).Animal studies of the hypoxic ventilatory response during sleep have been fairly limited.Aside from studies on young animals, work has mainly been confined to dogs. Two out of threestudies (Phillipson et al., 1978; Bowes et al., 1980) suggested that hypoxic sensitivity wasretained during SWS, while one study (Bowes et al., 1981) showed a significant decrease in theventilatory response to hypoxia during both SWS and REM. One study in rats (Pappenheimer,1977) found that hypoxic ventilatory responses actually increased during SWS.Thus, the majority of data indicate that, in general, sleep (SWS and REM sleep) ischaracterized by a blunted sensitivity to CO2, with 02 sensitivity either decreased, or preservedat waking values. Respiratory chemosensitivity during REM sleep typically appears to be thesame as during SWS, although responses during this state are more irregular. Upon separatingphasic and tonic REM sleep episodes, Sullivan et al. (1979) found responses during tonic REMsleep to be similar to those during SWS, while responses during phasic REM sleep were lowerthan those seen in SWS. In contrast, Hedemark and Kronenberg (1982) obtained increases inhypoxic sensitivity during REM sleep. The apparent increase in chemosensitivity during SWSin rats is an interesting anomaly, but is consistent with the present findings in the groundsquirrels. These results suggest that further sleep/ventilatory response studies in other rodentsare warranted.The mechanisms underlying the altered responsiveness of the respiratory system to 02 andCO2 during sleep are still unclear. Alveolar hypoventilation, CO2 retention and lower P02 arecharacteristic of SWS (Billow, 1963 and Dempsey et al., 1990). One would expect thecombination of increased respiratory stimuli and lower ventilation levels characteristic of sleep116to indicate reduced chemo-sensitivity. Many investigators believe that it is the loss of thewakefulness stimulus, and associated tonic inputs to the respiratory system, which cause thedecrease in ventilation (Orem et al., 1985; PhilEpson and Bowes, 1986; Ingrassia et al., 1991).Upper airway resistance also increases in sleep (Orem et a/., 1977; Remmers et al., 1978;Remmers, 1981; Hudgel et al., 1984) due to state-dependent alterations in respiratory musclerecruitment. A linear relation exists between upper airway and diaphragmatic muscle recruitmentduring wakefulness (Ônal et al., 1981; Patrick et al., 1982). During sleep, however, thethresholds for different muscle groups (ie. chest wall vs. upper airway) are greatly altered(Weiner et al., 1982; Parisi et a/., 1987; Hudgel, 1990). The phrenic nerve is the main nervesupply to the diaphragm; its activity can therefore be taken as an indication of diaphragmaticmuscle response. The activity of the phrenic nerve suggests that the diaphragm respondsdifferently than the other muscles; its activity continues to increase in a linear fashion in responseto various respiratory stimuli throughout all arousal states (Weiner et al., 1982). Tabachnik etal. (1981) have shown that the work of breathing actually increases during sleep, in spite of thelowered overall ventilation. Therefore, many investigators maintain that it is the increases inupper airway resistance, resulting from loss of tonic and phasic motor output (includingwithdrawal of the wakefulness stimulus) which create the decrease in CO2 responsiveness andCO2 retention (Dempsey et al, 1990; Henke et al, 1990; Skatrud et a/, 1988). By diminishingventilatory output, the increased resistance during sleep also limits the response to subsequentrespiratory challenges.In addition, Parisi et al. (1992) caution that arterial or end-tidal Pc02 may not accuratelyreflect brain levels of CO2 (ie. levels at the central chemoreceptors). Cerebral blood flow andmetabolism are diminished in SWS and tonic REM sleep (Madsen et al, 1991a; 1991b); end-tidal117(expired) CO2, which reflects whole body Pco,, may therefore not match existing levels in thebrain during these two states. This over-estimation of brain Pc% would result in an apparentdecrease in ventilatory response to CO2.The mechanism underlying the ventilatory response to 02, on the other hand, does notappear to be greatly affected by sleep; as in wakefulness, this response still depends on theintegrity of the carotid body (Bowes et al, 1981). Where present, diminishment of the responseduring sleep might also be due to the loss of a (tonic) wakefulness stimulus, possibly actingthrough altered respiratory muscle function.Hypoxic Ventilatory Responses Under Urethane Anesthesia Like the awake (unanesthetized) animals, the urethane-anesthetized animals in State I alsoresponded to 10.0% 02 with an increase in breathing frequency. However, a large variationexisted in these breathing responses (Appendix, Fig. 30), and hypoxic exposure failed to altertidal volume at all in the urethane anesthetized animals (Figs. 21 and 22). Thus, urethane-anesthetized animals did not exhibit any change in VE during State I in response to 10.0% 02.There was a small increase in VE in State II and the only significant increase occurred in StateHI.The overall effect of arousal state on ventilation was attenuated under hypoxic conditionsin the urethane-anesthetized group (Fig. 23). The state-dependent decrease in breathing frequencyappeared unchanged in the hypodc group. Breathing frequency was again significantly less inState Di compared to State I, and the extent of the decrease was approximately the same as inthe normoxic animals. There was a slight increase in tidal volume in State III compared to StateI (Fig. 23); however, as in the normoxic group, this increase was not significant. Four of seven118animals decreased tidal volume during hypcoda and two showed no change. Only one animalincreased tidal volume over normoxic values, and only one animal increased tidal volume in StateIII compared to State I (Appendix, Fig. 31). Thus, hypoxic exposure not only failed to increasetidal volume, it also removed any effect of state on tidal volume. The net result was that theurethane-anesthetized group also failed to exhibit any state effects on VE under hypoxicconditions (Figs. 21 and 23); VE remained constant across the three arousal states (States I to III).This resulted in an apparent increase in sensitivity of animals to hypoxia in State III.Hypercannic Ventilatory Responses Under Urethane Anesthesia Hypercapnia (5.0% CO2) was a significant respiratory stimulus for urethane-anesthetizedanimals in State I. The increased minute ventilation was due only to increases in VT; breathingfrequency did not change. This response is consistent with the ventilatory response reported forhalothane-anesthetized golden-mantled ground squirrels, which also increased VE via VT increasesonly (Osborne and Milsom, 1993). Urethane-anesthetized animals exhibited a 58% increase inVE over normoxic levels, while the halothane group increased VE by only 26%. These twostudies suggest that urethane anesthesia may reduce respiratory sensitivity to CO2 less thanhalothane anesthesia. This result is consistent with previous reports that urethane anesthesia doesnot significantly depress respiration (ie. Maggi and Meli, 1986), whereas halothane does(reviewed by Nunn, 1990).Hypercapnia stimulated ventilation in the same manner in all arousal states (Fig. 22). Inall states, tidal volume was increased and breathing frequency was unaltered compared tonormoxic (same state) values. Consequently, alterations in VE under urethane anesthesia werereflective of changes in VT only (Fig. 21). The alterations in respiratory pattern seen in119hypercapnic, urethane-anesthetized ground squirrels were consistent with three previous studieswhich examined hypercapnic ventilatory responses in urethane-anesthetized rats and cats (Hayashiand Sinclair, 1991; Hughes, 1982; Waldrop, 1991).Arousal state effects on respiration were not altered at all by hypercapnic exposure. Allthree state-dependent changes observed in the normoxic animals (an increase in VT, and adecrease in breathing frequency and VE) were retained during hypercapnic exposure. Ashypercapnic exposure did not alter breathing frequency, the combined effect of gas and state onbreathing frequency in the urethane group (figure 21) represented state effects alone. Comparedto the hypoxic ventilatory response, urethane-anesthetized animals showed much less variationin the breathing frequency response to hypercapnia. Unlike the hypoxic animals, hypercapnicanimals continued to demonstrate state-dependent alterations in VT and VE, in spite of the largeelevations in both variables.UNANESTHETIZED VERSUS URETHANE-ANESTHETIZED ANIMALS Distribution of Arousal States in Unanesthetized and Anesthetized Animals Arousal states in the urethane-anesthetized animals were defined on the basis of thecortical activity, which appeared like that of naturally sleeping and waking animals. Like thealteration between natural states, resulting from circadian and ultradian rhythms, spontaneousoscillation between arousal states was also observed under urethane anesthesia. This movement,independent of external cues, was interpreted as indicating that these arousal states not onlyappeared like natural states, but were also governed in a similar manner, ie. they had a similar120mechanistic basis for generation, maintenance and regulation. This would support the hypothesisthat states associated with similar EEG patterns in wakefulness/sleep and under urethaneanesthesia are analogous.When allowed to oscillate spontaneously between arousal states for periods of eight hours,both unanesthetized and anesthetized groups spent approximately the same amount of time in thetwo states with completely desynchronized and synchronized EEG patterns (W/State I andSWS/State III, respectively) (Fig. 27). They also spent similar amounts of time in the transitionstate (LS/State II) between these two states when data from the urethane-anesthetized animals arecompared to data collected in part II of the present study. However, unanesthetized animals inpart I of the study spent significantly more time in LS compared to the time spent in State II inthe urethane-anesthetized animals. These latter two parts of the study were scored with equalrigor. The decreased time spent in transitional cortical activity in the urethane-anesthetizedanimals may reflect the absence of behavioural input during the desynchronized State I. As theurethane-anesthetized animals moved from desynchronized into synchronized cortical activity,there would presumably be at least one less input to remove. In addition, the period of lightsleep in the unanesthetized animals may be prolonged, making them more responsive topotentially harmful situations, as opposed to the deeper states of sleep in which an organism ismuch more difficult to waken. In spite of the slight difference in transition between states,however, the fact that all groups spent approximately the same amount of time in desynchronizedand synchronized cortical activity also supports the hypothesis.In the unanesthetized and urethane-anesthetized groups, arousal state distributions wereaffected similarly by changes in inspired 02 and CO2, although some of the changes werenonsignificant trends only (Fig. 28). In both groups during hypoxic exposure, the synchronizedFigure 27: Comparison of the time which unanesthetized and urethane-anesthetizedanimals spent in arousal states exhibiting desynchronized (W/State I), transitional(LS/State II) and synchronized (SWS/State III) cortical activity. Arousal states areas defined in Figures 3 and 4. Control groups 1 and 2 are the normoxic,unanesthetized groups from the first and second parts of the study, respectively, andthe urethane group is the anesthetized, normoxic animals. * indicatessignificantly different from W/State I values.121Control, Group 1Control, Group 2Urethane Group1o4 50=04 400Ca) 30W04 20Wm 40 1 0es 0WI!^LS/II^SWS/IIIAROUSAL STATE1227060Figure 28: The alteration in arousal state distribution during hypoxia (10.0% 02) andhypercapnia (5.0% CO2) in urethane-anesthetized and unanesthetized animals.* indicates significantly different from W/State I values, same gas treatment.+ indicates significantly different from normoxic values, same state.123NORkimaAHYPERCAPNIAHYPDXIALS SWS7060504030201000701:4^60rITO^50403020100IllAROUSAL STATE124125activity (SWS/State B:1) was reduced, resulting in an increased amount of transitional activity(LS/State 11). Neither group showed any change in the amount of desynchronized activity(W/State I). Hypercapnic exposure also affected arousal state distribution in the same mannerin unanesthetized and urethane-anesthetized groups. As with hypoxia, hypercapnic animalsdecreased the amount of synchronized activity (SWS/State III). The amount of transitionalactivity (LS/State II), however, was unchanged during exposure to 5.0% CO2, whereas theamount of completely desynchronized activity (W/State I) was increased. Thus, the changeswhich occurred in arousal state distribution when administered the different gases were exactlythe same in both unanesthetized and urethane-anesthetized groups. This similarity also supportsthe hypothesis of analogous arousal states between these two groups.Respiration in Various Arousal States of Unanesthetized and Anesthetized Animals Normoxic AnimalsIn general, urethane anesthesia did not appear to affect respiration. The levels of all threerespiratory variables (f, VT and VE) were the same in both urethane-anesthetized andunanesthetized animals, in corresponding arousal states, as characterized by similar EEG patterns.The arousal state effects observed on breathing in the unanesthetized animals were also observedin the urethane-anesthetized group. In both groups, alteration from desynchronized tosynchronized cortical activity was associated with a decrease in frequency, a nonsignificant trendfor increased tidal volume and a decrease in minute ventilation. This anesthetic produced stablestates and is known to be distributed to, and metabolized in, all tissues equally (Bryan et al.,1957). Therefore, it is unlikely that the respiratory alterations seen with changes in state duringanesthesia were due to fluctuations in the level of anesthetic within specific tissues. Thus, the126respiratory alterations associated with arousal state alterations must have been due to the statechanges themselves.Again, minor inconsistencies occurred when the data from urethane- anesthetized animalswere compared to those from unanesthetiz,ed animals. The trends were identical in all parts ofthe study, but: breathing frequency was significantly less in State I (urethane) compared to W(unanesthetized) in part I of the study, minute ventilation was significantly less in State II(urethane) compared to LS (unanesthetized) in part I of the study, and the fall in VE during sleepwas not significant in part II of the study.The two differences which existed between urethane-anesthetized and unanesthetizedanimals in part I of the study occurred in the two arousal states involving some degree ofwakefulness. Three possible factors could account for the large reduction in breathing frequencyin State I under urethane anesthesia compared to the waking state. As previously discussed, thedesynchronized cortical activity could also include a state analogous to REM. Breathingfrequencies in REM are highly variable, but overall were comparable to SWS. Thus, inclusionof a REM-like state in State I could decrease the observed frequency to rates lower than observedin awake animals. Secondly, the values obtained for waking may include periods of greatlyincreased frequency associated with movements, or other behaviourally-related events. Althoughfrequency was generally taken at periods when the animals appeared to be at quiet rest (ie. nogross body movements or activities), some frequency measurements were recorded after periodsof activity and it is possible that enough time had not elapsed to allow frequency to return tonormal, "quiet waking" values, contributing to higher W values. Finally, urethane anesthetizedanimals do not regulate body temperature (Sharp and Hammel, 1972 and Bryan et al., 1957),which can drop 2-4 degrees in spite of conventional heating techniques. Thus, the observed127differences in frequency could also represent differences in body temperatures between the twogroups.These arguments would also apply to the minor differences observed between ventilatoryrates in State II (urethane anesthesia) and LS in part I of this study. If a REM-like analogy doesexist in this preparation, then a transitional state between this state and the synchronized statemust also exist, and would have been scored as State II. However, the fact that values of f andVE in unanesthetized animals in part II of this study were not different from those observed inanesthetized animals suggests that these minor differences probably stem from individualvariation in groups with small numbers of animals.Overall, the similarity between respiratory variables in the two groups (both absolutelevels and state-dependent effects), also supports the hypothesis that similar EEG patterns inunanesthetized and urethane-anesthetized animals characterize analogous (if not homologous)arousal states with respect to respiratory control.Hypoxic AnimalsBoth unanesthetized and urethane-anesthetized golden-mantled ground squirrels respondedto 10.0% 02 by increasing breathing frequency in all arousal states. Despite these increases, bothgroups continued to exhibit a state-dependent decrease with the transition to synchronized arousalstates (Fig. 29). Overall, the urethane-anesthetized animals did not alter VT in response tohypcoda. Much variability did exist, however, and in fact, VT was reduced in four of sevenanimals (Appendix, Fig. 31), which was consistent with the results obtained in the unanesthetized,hypoxic group (Fig. 16). In neither group were there any state-dependent changes in tidalvolume.Figure 29: Arousal state effects on minute ventilation in the unanesthetized and urethane-anesthetized, normoxic groups (top panel), and the effect of hypoxia (10.0% 02) andhypercapnia (5.0% CO2) on minute ventilation and associated state changes in thesegroups (middle and bottom panels). All three normoxic groups in the top graph areas defined in Figure 27. Unanesthetized animals in the middle graph are from part2 of the study, and anesthetized animals in the lower graph are from part 3 of thestudy. Minute ventilation is expressed as absolute values, per 100g body weight.* indicates significantly different from waking values, same gas treatment.128• am Control, Group 1Control, Group 2iNg Urethane Group75604530150Emi100755010025075SO25LS1LS^SWS129AROUSAL STATE130Overall, exposure to 10.0% 02 stimulated VE in all states in the unanesthetized group, butonly in State III in the urethane-anesthetized group. There was no arousal state effect onventilation during hypoxia in either group (Fig. 29). Consequently, there was an increase inhypoxic sensitivity (Fig. 29) as animals moved from desynchronized states (W/State I) tosynchronized states (SWS/State III). Overall, the data again support the hypothesis that stateswith similar EEG patterns in unanesthetized and urethane-anesthetized animals are analogousstates in terms of respiratory control.Hypercapnic AnimalsBoth urethane-anesthetized and unanesthetized animals responded to 5.0% CO2 withsignificant increases in VE• Overall, urethane-anesthetized animals in State I appeared to be moresensitive to 5.0% CO2 than unanesthetized animals during W. Both groups, however, showedapproximately the same increase in VE in the synchronized arousal states (SWS/State III).Although both unanesthetized and urethane-anesthetized groups increased VE in responseto hypercapnic stimulation, the respiratory pattern changes employed to produce these increasesdiffered between the two groups. Hypercapnia stimulated only breathing frequency in theunanesthetized group, but did not stimulate frequency at all under urethane anesthesia.Unanesthetized ground squirrels did not alter tidal volume at all in response to hypercapnia,whereas urethane-anesthetized animals responded with a large increase in VT (Figs. 16 and 21).The differences in hypercapnic response between anesthetized and unanesthetized animalssuggested that urethane might have affected central integration of hypercapnic stimuli. However,the response of the urethane-anesthetized group was not an anomalous response to hypercapnia.Hypercapnic ventilatory responses reported in the literature for this species show a fair degree131of variation. In fact, two previous studies from this lab, although not controlling for arousalstates, also found that golden-mantled ground squirrels increased tidal volume but did not alterfrequency under hypercapnic conditions (Webb, 1987; McArthur and Milsom, 1991).The effect of arousal state on breathing frequency was not altered by hypercapnicexposure in either group. The decrease in frequency from desynchronized (W/State I) tosynchronized (SWS/State III) cortical activity continued to be evident. Arousal state effects ontidal volume were also not affected by hypercapnia (Figs. 16 and 21); in the unanesthetized andanesthetized groups, tidal volume was elevated in states with a synchronized EEG compared tostates with a desynchronized EEG. Thus, in the urethane group, state-dependent decreases in VEstill occurred, although VE levels in the unanesthetized group were constant across all arousalstates.As with the hypoxic ventilatory response, golden-mantled ground squirrels appeared toincrease their sensitivity to 5.0% CO2 during sleep. Again, in the unanesthetized animals, thiswas a trend during sleep, while in the urethane-anesthetized animals it was a significant increaseduring State In (Fig. 29).Thus, the data from this part of the thesis are also consistent with the proposedhypothesis.132CARDIOVASCULAR CHANGES DURING SLEEP/ANESTHESIA Sleep-dependent Alterations in Heart Rate The effect of sleep on cardiovascular function has also been recognized for some time(see Coote, 1982; Orem and Keeling, 1980 and Lydic, 1987 for review). Blood pressuredecreases in SWS in cats (ref), dogs (ref) and humans (Coccagna et al., 1971), although littlechange is noted in the rat (lwamura et al., 1979), which may be a more comparable animal tothe golden-mantled ground squirrel. Heart rate has been found consistently to decrease fromwakefulness to SWS in many animals, including cats (Orem et al., 1977), rats (Iwamura et al.,1979), marmots (Florant et a/., 1978), piglets (Scott et al., 1990), human adults (Bach et a/.,1991) and human infants (Van Ravenswaaij-Arts et al., 1991). Heart rate decreases in thegolden-mantled ground squirrel in SWS can be added to this list. The heart rate changes are duein part to alterations in the influence on the autonomic nervous system; SWS is accompanied bydecreases in sympathetic activity (Saaman, 1934; Parmeggiani and Morrison, 1990) andalterations in vagal tone (Baust and Bohnert, 1969; Julu et a/., 1993). These changes areaccompanied by an increased preponderance of respiratory related sinus arrhythmias (Schechtmanand Harper, 1992; Trelease et al., 1981; Harper et al., 1978). The increased heart rate variation(due to a greater amount of sinus arrhythmia) in both sleep states in the golden-mantled groundsquirrel is in agreement with these previous studies.Heart rates obtained in the waking state of the unanesthetized ground squirrel correlatewell with observations of heart rate in other, comparable sized rodents. Two other species ofground squirrel (Citellus beecheyi and Citellus tridecemlineatus), moles (Scalopus aquaticus andCondylura cristata) and hedgehogs (Erinaceous europeanus) all have similar heart rates (between133217 and 260 beats/min versus 230 beats/min in the golden-mantled ground squirrel)(Stiumwasser, 1959; Landau and Dawe, 1958; Allison and Van Twyver, 1970; Toutain andRuckebusch, 1975).Urethane anesthesia significantly increased heart rate under all three inspired gasconditions (normcoda, hypoxia and hypercapnia), in all arousal states (Figs. 24 and 25). Undernormal conditions (ie. normoxic, normocapnic and unanesthetized), intrinsic heart rate isgenerated by the sinoatrial (S-A) node, and governed by tonic input from the autonomic nervoussystem (both sympathetic and parasympathetic fibers). Parasympathetic tone is higher thansympathetic tone under normal, resting conditions; therefore, the observed heart rate is slowerthan the intrinsic rate. Urethane anesthesia is known to increase sympathetic nervous activity(Armstrong et al., 1982; Maggi and Meli, 1986a), which would speed up the heart. However,not all investigators have found that urethane anesthesia increases heart rate. Disparateobservations regarding cardiovascular effects of urethane include unchanged (Korner et al., 1968;Matsukawa and Ninomiya, 1989), increased (Giles et al., 1969) and decreased (Maggi et al.,1984; Himori and Ishimori, 1988) heart rate, normotension (Matsukawa and Ninomiya, 1989) andinduction of hypotension (Maggi et al., 1984).In addition to the increase in sympathetic activity, the action of urethane on heart rateprobably also involves impairment of parasympathetic nervous tone. As already discussed,urethane will decrease the release of ACh at moderate and higher concentrations (Little et a/.,1980). Removal of parasympathetic input will leave only tonic sympathetic input (which is alsogreatly increased) to the S-A node. In fact, in studies of human subjects with abolishedautonomic systems, heart rate showed an approximate increase of 50% (from 70 beats/min to 105beats/min). (reviewed by Berne and Levy, 1981). Urethane anesthesia increased heart rate by134a comparable 40% (Fig. 13), suggesting that this anesthetic may impair autonomic function.Depression of parasympathetic tone, particularly in vagal fibres, is also suggested by theelimination of the respiratory sinus arrhythmia under urethane anesthesia. Respiratory sinusarrhythmia is mediated through parasympathetic input, via the vagus nerve and consequently,depression of vagal activity by urethane would remove the ability of the autonomic system toproduce the arrhythmia associated with respiration.Although the heart rate remained elevated under all three inspired gas treatments, animalsexposed to both hypoida and hypercapnia during urethane anesthesia showed a significantdecrease in heart rate. Both hypoxia and hypercapnia also abolished any state-dependent effectson heart rate; consequently, heart rate during hypercapnia was not significantly greater than heartrate during normwda in State III, due to the state-dependent decrease in heart rate in thenormoxic group. Hypoxic and hypercapnic bradycardia have also been documented by Cross andSilver (1963) in urethane-anesthetized rabbits. Cross and Silver showed that peripheralvasoconstriction normally follows hypcoda or hypercapnia; these authors maintained that thevasoconstriction increased arterial blood pressure, resulting in a decreased heart rate.Hypoxic depression of heart rate has also been observed in unanesthetized animals,including fetal sheep (Koos et al., 1987) and woodchucks (Burlington et a/., 1971), suggestingthat this response is not abnormal in the anesthetized preparation. However, hypoxia-inducedincreases in heart rate have also been observed in the woodchuck (Boggs and Birchard, 1989)and humans (Hedemark and Kronenberg, 1982). The present study also demonstrated ahypercapnic bradycardia in golden-mantled ground squirrels, while heart rates increased underhypercapnic conditions in both woodchucks and porcupines (Boggs and Birchard, 1989). It isrelevant, however, to note that in the study of Boggs and Birchard (1989), animals exposed to135less than 5.0% CO2 did not display any heart rate response at all, until they were exposed tolevels greater than 5.0% CO2.136GENERAL CONCLUSIONS The overall goal of this study was to determine whether the changing arousal statesobserved under urethane anesthesia were analogous to natural arousal states with similar EEGpatterns. Arousal states which appeared superficially similar between the two groups werecompared with respect to the proportion of time spent in various states, the changes in respiratoryand cardiovascular variables associated with changes in state, and both the effects of hypoxia andhypercapnia on the proportion of time spent in various arousal states as well as the effect of stateon the respiratory responses to hypoxia and hypercapnia.When allowed to cycle spontaneously through arousal states, both unanesthetized andurethane-anesthetized animals spent similar amounts of time in arousal states withdesynchronized, transitional and synchronized EEGs (Fig. 27). Changes in arousal statedistribution associated with hypoxic and hypercapnic stimulation were unaffected by urethaneanesthesia (Fig. 28). Hypercapnic exposure produced more desynchronized activity, whilehypoxic exposure resulted in more transitional activity, in both unanesthetiz,ed and urethane-anesthetized groups.Urethane anesthesia did not appear to affect respiration under nonnoxic, normocapnicconditions. Differences between respiratory variables (f, VT and VE) for unanesthetized andurethane-anesthetized animals in all states (W/State I, LS/State II and SWS/State III) wereobtained in only two out of twelve comparisons (W/State I breathing frequency and LS/State II'E). All respiratory variables were significantly affected by arousal state in the normoxicurethane-anesthetized animals and these changes were the same as those observed inunanesthetiz,ed animals.The hypoxic response was similar in the two groups, albeit somewhat diminished in the137urethane-anesthetized group. Both groups demonstrated approximately the same overall increasein VE during hypercapnia. Hypoxic and hypercapnic ventilatory sensitivity did not seem to bediminished in the unanesthetized animals during sleep. Rather, there was a trend for sensitivityto increase, although it was not significant. Urethane anesthetized animals similarly appeared toexhibit an increased sensitivity to hypoxic and hypercapnic stimuli in states of synchronizedcortical activity compared to the normoxic values in the same state.On the basis of these results, it seems reasonable to conclude from the present study thatthe arousal states evident under urethane anesthesia are exerting similar effects on respiration asnatural arousal states in waking/sleeping animals with similar EEG patterns. Therefore, stateswhich appeared superficially to be the same based on similar cortical patterns also proved to beanalogous in terms of respiratory control, supporting the main hypothesis of this thesis.Urethane anesthesia did affect heart rate, greatly increasing the rate and abolishingrespiratory sinus arrhythmias. In both unanesthetized and anesthetized groups, however, heartrate was significantly decreased in states of established synchronized activity. Therefore,although urethane tonically increased heart rate, the effects of changing arousal state appearedto be intact under urethane anesthesia. Thus, as with respiration, the arousal states underurethane anesthesia also appeared to be equivalent to natural sleep/wake states with similar EEGpatterns in terms of the state-dependent effects on heart rate.In conclusion, this study has shown that the state produced by urethane anesthesia is notone homogenous state, but rather consists of several distinct arousal states, characterized bydifferences in cortical activity and respiratory and cardiovascular control. In general, with respectto respiratory behaviour, urethane anesthesia appears to produce arousal states which areanalogous to the states of wakefulness, drowsiness and slow-wave sleep in unanesthetized138animals. The one major difference between unanesthetized and urethane-anesthetized animals,that of the increased heart rate under urethane anesthesia, suggests there are major differencesdespite these analogies. 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Experimental Neurology 91:193-207.APPENDIX Individual Responses FrequencyTidal VolumeMinute VentilationHeart RateSinus Arrhythmia155Figure 30: State effects on the breathing frequency of individual subjects for all fourexperimental conditions. The control group consisted of the unanesthetized,normoxic animals. Normoxic, hypoxic (10.0% 02) and hypercapnic (5.0% CO 2)labels indicate the three urethane-anesthetized subject groups.156tISO150100SO•210200soISO200um.0•State I^State II^State DIISO200•State I^State II^State DI157• STATE DEPENDENT FREQUENCYRESPONSES OF INDIVIDUAL SUBJECTSCONTROL^ NORMOXIASW/11^ State I^State II^StateSLEEP STATE AROUSAL STATEHYPERCAPNIA^ HYPDXIAAROUSAL STATE AROUSAL STATE158Figure 31: State effects on tidal volume of individual subjects for all four experimentalconditions. The control group consisted of the unanesthetized, normoxic animals.Normoxic, hypoxic (10.0% 02) and hypercapnic (5.0% CO 2) labels indicate the threeurethane-anesthetized subject groups.State I^State fl^State M. ZAOA0.0SW/W^SYSSLEEP STATEHYPERCAPNIA3.0Si0.0State I^State 11^State M159STATE DEPENDENT TIDAL VOLUMERESPONSES OF INDIVIDUAL SUBJECTSCONTROL^ NORMOXIAAROUSAL STATE AROUSAL STATE160Figure 32: State effects on ventilation responses of individual subjects for all fourexperimental conditions. The control group consisted of the unanesthetized, normoxicanimals. Normoxic, hypodc (10.0% 02) and hypercapnic (5.0% CO2) labels indicatethe three urethane-anesthetized subject groups.CONTROL NORMOXIA7.soCo•To810075SOCo•AROUSAL STATEAROUSAL STATE161STATE DEPENDENT MINUTE VENTILATIONRESPONSES OF INDIVIDUAL SUBJECTSV^SW/V^SWSSLEEP STATE'HYPERCAPNIAState I^State II^StateState I^State II^State ElAROUSAL STATEHYPDXIAState I^State U^State Ill•162Figure 33: State effects on the heart rate of individual subjects for all four experimentalconditions. The control group consisted of the unanesthetized, normcmic animals.Normoxic, hypoxic (10.0% 02) and hypercapnic (5.0% CO2) labels indicate the threeurethane-anesthetized subject groups.STATE DEPENDENT HEART RATESOF INDIVIDUAL SUBJECTSCONTROL^ NORMOXIA163400400300300210We100100MO410BOO300MO300140100w^slot^swsSLEEP STATEHYPERCAPNIA State I^State II^State MAROUSAL STATEHYPDXIAState I^State II^State III State I^State II^State IIIAROUSAL STATE AROUSAL STATE164Figure 34: State effects on the sinus arrhythmia of individual subjects for all fourexperimental conditions. The control group consisted of the unanesthetized, normoxicanimals. Normoxic, hypoxic (10.0% 0 2) and hypercapnic (5.0% CO2) labels indicatethe three urethane-anesthetized subject groups.165STATE DEPENDENT ARRHYTHMIASOF INDIVIDUAL SUBJECTS


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