Sleep, 15(5):40~14 © 1992 American Sleep Disorders Association and Sleep Research Society
Fundamental Research Spontaneous Ventilation and Respiratory Motor Output During Carbachol-Induced Atonia of REM Sleep in the Decerebrate Cat Department of Animal Biology, School of Veterinary Medicine; and Center for Sleep and Respiratory Neurobiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046, U.S.A.
Summary: Microinjections of carbachol into the pons induce a state that resembles rapid eye movement (REM) sleep in intact cats and, in decerebrate, artificially ventilated cats, produce postural atonia accompanied by a powerful depression of the respiratory motor output. In this study, pontine carbachol was used in decerebrate, spontaneously breathing cats to assess the effects of mechanical and chemical respiratory reflexes on the magnitude and pattern of the carbachol-induced depression of breathing, and tOi determine whether the depression is altered in those animals in which rapid eye movements are present. Phrenic nerve activity and tidal volume were only transiently depressed at the onset of the carbachol-induced postural atonia, whereas the decrease in respiratory rate and the depressions of hypoglossal and intercostal activities persisted until the response was reversed by a pontine microinjection of atropine 15-101 minutes after the onset of carbachol response. Ventilation was reduced to 70% of control during the steady-state conditions. The irregularity of breathing, characterized by the inter-quartile ranges of the distributions of the peak phrenic nerve activity and respiratory timing, did not increase following pontine carbachol. Neither vagotomy nor vigorous eye movements were associated with increased breathing irregularity. This contrasts with the irregular breathing (with minor average chan~:es in ventilation) typical of natural REM sleep. We propose that the carbachol-injected decerebrate cat provides a useful model of the depressant effects that neural events associated with REM sleep may have on breathing. Key Words: Atonia-Carbachol-Control of breathing- PonsREM sleep- Vagus.
Cholinergic mechanisms within the dorsal pontine characterization of the behaviors that can be elicited tegmentum have been repeatedly implicated in the in the decerebrate preparation. In a recent study (5), we showed that cholinergic generation of rapid eye movement (REM) sleep (1,2). Recent demonstrations that these mechanisms can be stimulation within the dorsal pontine tegmentum of pharmacologically activated in an acutely decerebrate decerebrate cats produced a powerful depression ofthe cat by microinjections of carbachol into the pontine respiratory motor output that developed in parallel to tegmentum (3-5) provide a novel animal model to the atonia of postural muscles. A large decrease in study the neural mechanisms related to the postural respiratory rate accompanied a stereotyped suppresatonia of REM sleep and, perhaps, other REM sleep sion of the phrenic, hypoglossal and intercostal nerve phenomena. This new line of investigation of REM activities. Paralyzed, vagotomized and artificially vensleep-related mechanisms may offer significant advan- tilated cats were used in these experiments, which altages by allowing rigid control over the conditions of lowed us to study the changes in various motor outputs the experiment and use ofa large spectrum of invasive in isolation from feedback mechanisms originating in techniques. To understand the relationship between lung mechanoreceptors and central and peripheral chethe effects seen in this acute animal model and thosl~ moreceptors. The strength of the respiratory deprescaused by carbachol in the chronically instrumented, sion observed in these experiments substantially exbehaving cat or in natural REM sleep requires further ceeded that typically found in either natural REM sleep (6,7) or the REM sleep-like state induced in chronically instrumented, intact animals by pontine carbachol inAccepted for publication June 1992. Address correspondence and reprint requests to Dr. Leszek Kubin, jections (7-9). We ascribed this exaggerated depresCenter for Sleep and Respiratory Neurobiology, 972 Maloney Building, Hospital of the University of Pennsylvania, 3600 Spruce Street, sion, in part, to the absence of feedback mechanisms, Philadelphia, Pennsylvania 19104-4283, U.S.A. which allowed the inhibitory mechanisms to appear in
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Hirokazu Tojima, Leszek Kubin, Hiroshi Kimura and Richard O. Davies
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BREATHING DURING CARBACHOL-INDUCED ATONIA
METHODS The results were obtained from 15 adult cats (2.04.0 kg). They were preanesthetized with ketamine (80 mg im) and diazepam (2 mg im) and, after intubation, anesthetized with halothane and decerebrated at a precollicular level. The halothane anesthesia was then discontinued. Recordings were begun - 9 hours after the injection of ketamine-diazepam and -7 hours after the decerebration. In four cats, both vagi were cut in the midcervical region; in the other animals, they were left intact. A detailed description of the surgical preparation and recording techniques was given previously (5).
In all animals, the activity of the C5 branch of one phrenic nerve and either the activity of a dorsal cervical motor nerve branch (C4) or the neck muscle electromyogram (EMG) were recorded (20-2,000 Hz bandwidth). The electrooculogram (EO G) was recorded (0.1-20 Hz bandwidth) with two steel wires inserted bilaterally under the skin overlying the postorbital processes of the zygomatic arches. In addition, in five animals, the activity of the genioglossal branch of the hypoglossal nerve and, in another five animals, the intercostal muscle EMG containing either inspiratory, expiratory or both types of activities were recorded. In an additional three animals, genioglossal and intercostal activities were recorded simultaneously. The animals were placed in a stereotaxic instrument and the anterior cerebellar vermis exposed for microinjections in the pons.
The tracheal CO2 was continuously monitored with an infrared gas analyzer (Godard). Because thes~de cerebrate, spontaneously breathing animals were often hypocapnic in control conditions, O 2 (30-50%) and CO 2 (1-3%) were added in some experiments to the inspired gas mixture to elevate the hypoglossal and/or intercostal activities to a measurable level. Airflow was measured using a pneumotachograph (Fleish 00) and differential pressure transducer. The inspiratory volume was obtained by electronic integration of the airflow signal (CWE PI-830). These variables, the moving averages of the nerve or EMG activities and an event marker were monitored on a chart recorder (Gould T A 2000). The raw nerve and EMG signals and other variables were stored on magnetic tape (H-P 3968A) for subsequent analysis. Solutions of carbachol (Sigma) in saline (10 or 88 mM) with 2% pontamine blue dye were placed in glass micropipettes having tip diameters of 30-40 /-tm. The pipettes were positioned in the dorsal pontine tegmentum using visual landmarks and stereotaxic coordinates, aiming for the coordinates of P3.5, H-4.0 and L2.0 (1,2,12). The injections were made using pressure pulses (NeuroPhore BH-2), and the volume ejected was directly determined by measuring the movement of the meniscus in the pipette with a pocket microscope and reticle. The injected volume of carbachol ranged from 140 to 1,000 nl (mean ± SD: 420 ± 280 n1), corresponding to 0.25-16.0 /-tg. In most animals, after a sufficient period of observation following the carbachol response, atropine S04 (Sigma) (equimolar with carbachol and at a volume of 1-4 times that of the preceding carbachol injection) was injected into the same pontine site. This reversed the carbachol effects. Subsequently, in three animals, a second injection of carbachol was then made in a symmetrical site in the contralateral pons (5) and these three responses were also included in our database. In two of these three animals, the first response to pontine carbachol had to be excluded from analysis for technical reasons. In seven animals, after conclusion of the experiment, the brain stem was removed, fixed in 10% formalin and cryoprotected with 30% sucrose, and the pontine region was cut in 50 /-tm slices in the coronal plane. Every fourth slice from the region containing the blue dye was mounted and counterstained with neutral red, and the center of the injection was subsequently redrawn on a standard cross-section of Berman's atlas (13).
Data analysis
An effective carbachol injection was characterized by the parallel development ofa depression of postural and hypoglossal or intercostal nerve activities (5). The Sleep, Vol. 15, No.5, 1992
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their full strength (5). Apart from the powerful suppression, no irregularities in either respiratory rate or inspiratory firing of the phrenic nerve were observed. This is, therefore, unlike natural REM sleep, during which respiration is characterized by rapid changes in respiratory rate and fractionations of diaphragmatic activity, the frequency and severity of which have been related to the intensity of the characteristic phasic events of REM sleep such as muscle twitches, rapid eye movements and ponto-geniculo-occipital (PGO) waves (6,10,11). In our previous study (5), the use of paralyzed animals precluded the study of eye movements. We now report the effects of pontine carbachol on ventilation and the respiratory motor output in acutely decerebrated, spontaneously breathing cats in which we also recorded the electrooculogram. Thus, we can now relate the strength and pattern of respiratory depression in these animals to the presence of eye movements and compare these results with those obtained from animals with chemical and mechanical respiratory feedbacks absent.
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are presented as the mean ± standard deviation (SD) throughout this report, except where noted. Changes were considered significant if p < 0.05.
RESULTS Changes in breathing associated with carbachol-induced atonia The responses to 16 carbachol injections were analyzed. Three injections (5b, 6 and 7 in Table 1) were made after reversal of the first response with pontine atropine. They produced effects that were not different from the others (5), and all the responses were pooled together for further analysis. Pontine carbachol produced a stereotyped depression of the recorded respiratory nerve and/or EMG activities and a slowing of the respiratory rate that developed in parallel to the atonia of postural activity, as described previously for artificially ventilated cats (5). The mean latency to the onset of the phrenic response was 275 ± 299 seconds, and that for the postural (neck) motor tone was 313 ± 407 seconds (no significant difference, paired t test). The mean delay from the onset of the response to the time of maximal depression of phrenic activity was 99 ± 148 seconds, and that for neck activity was 93 ± 112 seconds (no significant difference, t test). Unless reversed by an atropine injection, the response could last for a prolonged period of time (> 1 hour) as reported for paralyzed and artificially ventilated cats (5). Figure 1 shows two polygraphic records obtained from a vagotomized (part A-cat Sa in Table 1) and a nonvagotomized (part B-cat 10 in Table 1) animal showing the carbachol-induced suppressions of respiratory nerve activities that developed in parallel to the postural atonia. In the response shown in Fig. lA, pontine carbachol induced vigorous horizontal eye movements, whereas no eye movements were observed in the response shown in Fig. 1B. The peak amplitude of the moving average of the phrenic nerve activity was transiently depressed in the cat shown in A (see arrow) and then returned to the control levels. In the example shown in B, the phrenic activity remained almost unchanged. In both animals, the respiratory rate was characteristically decreased and remained reduced for a prolonged period of time. An initial decrease in the amplitude of the phrenic nerve activity of at least 10% was observed at the onset of the response to carbachol in 11 of the 16 trials, the mean initial reduction for all responses being to 86 ± 11 % of control (p < 0.001, t = 4.84, paired t test, n = 16). However, by the time that steady-state conditions were reached, phrenic nerve activity increased in all but three of the responses to a level equal to or higher
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presence of eye movements as indicated by the EOG signal was confirmed by direct observation. The times to the onset of the effect and to reach the maximum effect were measured from the beginning of the injection. For two injections using 88 mM of carbachol, the response commenced immediately after placement of the pipette in the pons, probably due to leakage from the pipette. In these two cases, the time of insertion of the pipette was regarded as the beginning of the injection. The changes in the peak nerve or EMG activities were determined from the moving averages of the corresponding signals and measured with respect to the baseline (established during periods with no activity). Within each respiratory cycle, inspiration was defined as the period from the onset of the increase in the moving average of phrenic nerve activity to the point of rapid decline and expiration as the period from thl~ rapid decline in the moving average of the phrenic nerve activity to the onset of the next burst. To characterize the changes in respiratory timing, the inspiratory duration (T J) and expiratory duration (T E) were determined for control conditions and various stages of the response. (The total respiratory cycle duration was T J + T E') Measurements taken during the carbachol-induced depression were normalized to the corresponding mean control values (immediately before the carbachol injection) and expressed as percent of the control. In many cases, the response to carbachol consisted of two successive phases-a transient depression, during which phrenic nerve activity was maximally depressed, and a subsequent steady state, with ventilation stabilized at a level still reduced but above that of the initial depression. To characterize these changes, the parameters of ventilation were determined for at least 10 consecutive respiratory cycles in each phase and averaged. For 11 experiments, sufficiently long segments of records were available for off-line statistical analysis of the variability of respiratory timing and phrenic nerve magnitude using an IBM PC-AT computer-based data acquisition and analysis software (EGAA and Spread, RC-Electronics). Statistical distributions (histograms) ofTh TE and peak amplitude of the moving average of the phrenic nerve activity were determined from segments of records containing 50-100 respiratory cycles during control conditions and following the carbachol-induced respiratory depression. The interquartile range (IQR), i.e. the range between the lowest and the highest quartile measured from the histogram of the distribution of the analyzed variable, was used to characterize the variability of each of the studied parameters in each condition. Statistical evaluations of the data were performed using a paired or unpaired Student's t test. The data
BREATHING DURING CARBACHOL-INDUCED ATONIA
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TABLE 1. Characteristics oJrespiratory responses to pontine carbachol injections in individual animals Carbachol Animal # ~,
Dose (pg)
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1,000 500 300 280 280 280 260 250 140
no no no no yes yes yes yes no
100 190 40 200 240 320 110 440 270
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16 33 32 22 11 13 36 52 34
98 70 65 50 81 94 59 107 83
Respiratory rate
Period of observation (minutes) 33 64 34 55 70 65 41 67 27
1,000 800 500 500 340 220 140
no 1,080 54 29 30 41 no 20 101 39 30 58 no 130 52 32 52 15 no 180 52 34 78 56 yes 19 13 62 43 800 no 110 15 10 65 30 no 170 18 11 63 55 a Calculated as a product of phrenic nerve activity and respiratory rate. b 5a, b describe two responses evoked in cat 5 by two carbachol injections placed on the opposite sides of the pons, as described in Methods.
1 min
than in the precarbachol state. This secondary increase occurred both in animals that had a transient decrease at the start of the response and in animals in which the transient decrease was absent or less than 10%. On the average, this secondary increase brought the phrenic activity back to a level close to that of the control period (103 ± 20%) and not significantly different from the latter. The average changes in respiratory parameters and blood pressure, determined for the steadystate period after ventilation stabilized following carbachol injection, are shown in Fig. 2 separately for nonvagotomized and vagotomized cats. Separate determinations were made for 11 responses analyzed in
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FIG. 1. A: Response to pontine carbachol in a spontaneously breathing cat (5a in Table 1) in which phrenic nerve activity showed only a transient depression at the onset of the response and strong eye movements occurred in parallel to the carbachol-induced postural and respiratory depression. PHR, HYPO, moving averages of the neural activity of the phrenic and hypoglossal nerves, respec-
tively; FC0 2 , end-tidal CO 2 fraction; EOG, electrooculogram. Carbachol (0.5 Itg) was injected into the pons 8 minutes before the beginning of the record. Note a transient depression of the phrenic nerve activity (arrow) and a decrease of the respiratory rate at the onset of the response. The phrenic nerve activity subsequently quickly recovered (to 104% of control), while the reduction of the respiratory rate was maintained (14 minute-I in control, II minute-I during the steady state following carbachol). As a consequence of the decreased rate, ventilation was reduced and the end-expiratory CO 2 elevated by 0.3%. B: Response to pontine carbachol in a cat (lOin Table I) that did not show eye movements. IC, NECK, moving averages of the EMG activities of expiratory intercostal and neck muscles, respectively. The phrenic nerve activity was reduced to 76% of control 12 minutes after the onset of the response and the respiratory rate decreased from 39 minute-I before, to 30 minute-I after carbachol. The small changes seen on the EOG trace after the carbachol injection did not represent eye movements but rather muscle twitches that occurred transiently before the response to carbachol reached its steady state. Note the parallel changes in the neck and intercostal EMGs during the initial stage of the response. Sleep. Vol. 15. No.5. 1992
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Eye movement group 1 16 2 8 0,8 3 4 0.5 5ab 0.5 5bh 0.5 6 0.47 7 0.45 0.28 8 No eye movement group 16 9 10 13 11 8 12 8 13 0.6 14 0.44 15 0.25
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11 nonvagotomized, and five responses in four vagotomized, animals. The peak phrenic nerve activity and tidal volume were not significantly different from control in either group of animals, whereas the peak flow and respiratory rate in animals with vagi intact and blood pressure in both animal groups remained significantly reduced. No significant differences in the magnitudes of any aspect of the response were found between the animals with vagi intact and cut (I test), nor did the vagotomy affect the variability of respiration following pontine carbachol (see below). The lack of significance in the carbachol-induced chang1es in the peak flow and respiratory rate in the anima.ls with vagi cut could be due to a lower number of observations in this group. In contrast to the changes in phrenic nerve activity, the hypoglossal and intercostal nerve activities (or corresponding EMGs) remained significantly suppressed for a prolonged period of time. After ventilation stabilized following carbachol injection and phrenic nerve activity was close to or above the control level in most cases, as described above, hypoglossal activity was depressed to 21 ± 15% of control (n = 7), inspiratory intercostal activity to 63 ± 29% (n = 5), and expiratory intercostal activity was completely abolished in all three experiments in which expiratory activity was present in control conditions. The respiratory rate also remained significantly depressed; the mean control rate was 40.0 ± 21.0 minute-I and it fell to 25.5 ± 12:.1 minute-I after carbachol (p < 0.001, t = 4.20, paired t test, n = 16). These observations were made over a Sleep, Vol. 15, No.5, 1992
time period ranging from 15 to 101 minutes following the maximal initial depression (mean: 50 ± 21 minutes). Atropine was then injected into the pons to reverse the carbachol effects or a substantial spontaneous recovery from the carbachol effects became apparent. Subsequent to the microinjections of atropine, the respiratory rate and all nerve activities returned to controllevels, as has been described earlier (5). Ventilation, calculated as the product of the peak phrenic nerve activity (arbitrary units) and respiratory rate, was reduced to 57.1 ± 20.6% of control during the transient maximal depression and then, due to the subsequent increase in peak phrenic activity, recovered to 70.3 ± 19.7% after ventilation stabilized (n = 16). The mean end-tidal CO2 rose by 0.8 ± 0.6% following pontine carbachol, as determined for the nine experiments for which adequate calibrations were available for the whole period of analysis. The decrease in respiratory rate was due to prolongations of both T J and T E' After all but one injection, TI increased (mean control = 0.8 ± 0.5 seconds; carbachol = 1.1 ± 0.6 seconds; p-< 0.001,1 = 8.00, paired t test, n = 16) and after all but two injections, T E increased (mean control = 1.1 ± 0.6 seconds; carbachol = 1.8 ± 1.1 seconds; p < 0.001, 1 = 4.12, paired t test, n = 16). Although the absolute change in T E was larger, the relative change of T J was not different from that of T E. Figure 3A shows the effect of vagotomy on the average peak phrenic nerve activity and respiratory timing before and after carbachol. Figure 3B shows the carbachol-induced changes in the irregularity of the
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respiratory cycle (as assessed by IQRs ofT[ and T E) in vagotomized and nonvagotomized cats. The data used in Fig. 3 are from four responses in vagotomized and seven responses in nonvagotomized animals that were suitable for statistical analysis of breathing irregularities, as explained in Methods. As expected, the control T[ and T E were longer in vagotomized than in nonvagotomized animals, and in both animal groups they were prolonged further during the carbachol-induced depression. Vagotomy had no effect on the IQRs of the respiratory timing in either control or post-carbachol conditions (t test).
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Effect of eye movements on breathing following pontine carbachol
In nine animals, the carbachol-induced depression of respiratory and postural activity was accompanied by vigorous horizontal eye movements, whereas in the remaining six animals no eye movements were induced. The eye movements, when present, were inSleep, Vol. 15, No.5, 1992
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tense and occurred continuously with a relatively regular amplitude and frequency, rather than in bursts of variable duration and amplitude typical of natural REM sleep in intact cats. Figure 4 shows the distribution of the centers of pontine injection sites for four animals that showed eye movements in response to carbachol injection and three animals that did not. As also noted in another two carbachol studies on intact animals (12,14), there was no clear relationship between the injection site and occurrence of eye movements following carbachol injections. In animals with eye movements, the mean changl~s in phrenic amplitude during either the initial transient depression or the steady-state were not significantly different from those in animals without eye mov,ements. Figures 5A and B show separate histograms of T J and phrenic peak amplitude obtained from one cat (animal 3 in Table 1) in control conditions (open bars) and during a carbachol response with eye movements Sleep, Vol. 15, No.5, 1992
(filled bars). In this cat, carbachol prolonged the mean T J, whereas the IQR ofTJ remained unchanged (40 ms) and increased the peak phrenic nerve activity, whereas the IQR decreased to 82% of control. The mean changes in the peak phrenic nerve activity and respiratory timing in relation to the presence or absence of eye movements are shown in Fig. 5C. The mean data from seven responses in six animals with eye movements and from four responses in four animals without are shown. Neither the peak phrenic nerve activity nor the timing changes induced by carbachol were statistically different between the animals with and without eye movements. Because the intensity of eye movements and phasic REM sleep activity was found to correlate with variability in respiratory drive and timing in natural REM sleep (6,10,11), we determined whether the IQRs of the measured respiratory parameters were larger in those animals that displayed eye movements after the
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carbachol injection than in those without them. The It is based on microinjections of carbachol into the IQRs were determined for those animals for which we pons of decerebrate cats and may be useful for studies had sufficient data for statistical analysis, seven re- of selected aspects of the central neural mechanisms sponses with and four without eye movements. Before of REM sleep. Accordingly, in this study we explored assessing the effect of eye movements or vagotomy on some of the features of the acute model that may be the IQRs, we confirmed (using the Fisher exact test) relevant for studies of REM sleep-related changes in that the two factors, vagotomy and the presence of eye the control of breathing. In contrast to our previous work that described the movements, were independent. For the group with eye movements after the carbachol injection, the IQRs for respiratory suppression by pontine injections of carboth T J and T E tended to decrease slightly compared bachol in decerebrate, paralyzed and artificially vento the control. In contrast, they increased to a variable tilated cats (5), the present report focuses upon the degree in animals without eye movements after car- changes in respiratory motor outputs in spontaneously bachol (see Fig. 5D). The only statistically significant breathing decerebrate cats. Two findings relevant for difference was, however, for the percent change of the the future use of decerebrate cats in studies of REM IQR for T J between the animals with and without eye sleep-related changes in respiration were obtained: 1) movements. In this case, unlike what could be expected The phrenic nerve activity was not depressed in the based on studies during natural REM sleep, the larger steady-state phase of the carbachol response, unlike in variability was seen in animals without eye movements the artificially ventilated cats, whereas the decreases (p < 0.05, t = 2.375, t test). The changes in the IQRs in respiratory rate were similarly profound in the two for the phrenic nerve peak amplitude were evaluated preparations (69% of control in our previous (5) and using the ratio of the peak amplitude before and after 64% in the present study); and 2) the variability ofTJ , carbachol as they were measured in arbitrary units. T E and phrenic peak amplitude did not increase during Following carbachol injection, the IQRs of the phrenic the postural atonia and respiratory depression induced peak amplitude in animals with eye movements re- by pontine carbachol, even in animals in which very mained unchanged (97% of the control), whereas in intense eye movements were induced. There findings the group without eye movements they increased to are in a striking contrast to the pattern of breathing 121 %. These changes were, however, not significant during natural REM sleep (6,10,11). In a recent study of the effects of carbachol on reseither within (paired t test) or between the animal groups (t test). The mean IQRs of both the timing and am- piration in chronically instrumented, intact cats (8), plitude characteristics of breathing, pooled for all 11 the tidal volume, which is closely related to peak diresponses studied, both with and without eye move- aphragmatic EMG activity or phrenic peak amplitude, ments, did not change significantly after carbachol in- was virtually unchanged from the control (waking) levjections (paired t test). Thus, unlike in natural REM el during the REM sleep-like state. This is similar to sleep, the presence of eye movements was not asso- what we found in the decerebrate, spontaneously ciated with increased respiratory variability during the breathing cats of the present study where, in most ancarbachol-induced postural atonia in decerebrate cats. imals, carbachol caused only a transient depression of the peak phrenic nerve activity, which then recovered to the control level. In contrast, in decerebrate,' vaDISCUSSION gotomized and artificially ventilated cats, a suppresNumerous investigators have found that microin- sion of the peak phrenic amplitude to 66% of control jections of cholinergic agonists into the pontine teg- was observed (5). In all three preparations, the reducmentum of cats produce a state that has many char- tion in respiratory rate was maintained throughout the acteristics of natural REM sleep (1,2). This duration of the carbachol effect. Thus, ventilation was pharmacologically-induced state in intact, chronically decreased in these three types of experiments to 72% instrumented animals is characterized by such signs of of control in intact cats (8); 70% in spontaneously natural REM sleep as a desynchronized EEG, postural breathing, acutely decerebrate cats (present study); and muscle atonia, rapid eye movements, a characteristic 46% in artificially ventilated, acutely decerebrate cats hippocampal theta rhythm and PGO waves. There- (calculated as a product of the peak phrenic nerve acfore, it has been regarded as a valid experimental model tivity and respiratory rate) (5). for the physiological state of REM sleep. Recently, it The lack of a maintained suppression of phrenic was also employed to study the REM sleep-related activity in the present experiments may result, in a changes in ventilation and respiratory motor output large part, from a compensatory increase in respiratory (7-9). As an extension of the studies performed on drive due to a rise in PaC0 2 following the initial venchronically instrumented cats, an acute animal model tilatory depression. This, when compared to the results of the postural atonia of REM sleep was proposed (3). from artificially ventilated cats (5), suggests that the
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aphragmatic activity. This phenomenon may also be explained by stimulation-induced release of acetylcholine in amounts exceeding those occurring during natural REM sleep. Taken together, these data point to the dorsolateral pons as the site where po~tural atonia, cholinergic receptors and mechanisms controlling respiratory rate are closely interrelated. . A characteristic feature of breathing during natural phasic REM sleep in animals and man is profound irregularity of both frequency and tidal volume, with an average increase of the former and a minor change of the latter when compared to non-REM sleep or wakefulness (6). In contrast to breathing during natural REM sleep, we did not find an increased variability in respiratory timing or peak phrenic nerve activity during the carbachol-induced postural atonia in decerebrate cats. In several studies, the irregularity of breathing during natural phasic REM sleep was found to increase roughly proportionally to the intensity of phasic phenomena (6,10,11). In this study, we took advantage of the fact that, following carbachol injections, the postural atonia was not always accompanied by rapid eye movements. This has been reported earlier in chronic animal studies (12,14). Moreover, the presence of eye movements is not clearly related to the injection site (12), and it has been proposed that factors other than the site and the dose determine whether eye movements will occur in association with carbacholinduced atonia (14). In this study, we found also that in decerebrate cats eye movements did not occur in every response and their presence was not clearly related to the site of the injection (Fig. 4). To assess whether breathing irregularities can be related to the presence of such phasic events as eye movements following carbachol injections, we analyzed the variability of breathing separately in cats with and without eye movements. Surprisingly, the variabilities of breathing were slightly smaller in those animals in which vigorous eye movements were evoked than in the animals without eye movements. It must be noted, however, that the pattern of eye movements evoked by carbachol in these decerebrate cats was more regular than that typically observed during both natural REM sleep and carbachol-induced REM sleep in intact cats (1,12). Thus, we cannot exclude the fact that the mechanism of generation of eye movements in our preparation is different from that of natural REM sleep. Such a difference may account for the absence of any increase in the irregularity of breathing coincident with eye movements that we observed. Similarly, there is evidence that PGO waves are more regular during spontaneously occurring REM sleep in chronic decerebrate cats than during REM sleep in intact cats (16). Again, this suggests some alterations in the system that generates the phasic events of REM sleep in decerebrate animals.
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ventilatory response to CO2 is preserved in the car·· bachol-induced state in decerebrate cats. [In inta!;:t cats, the response to CO 2 during the carbachol-induced state is lllaintained; the slope of the CO 2 response curve is not significantly different from waking, but is signifi·· cantly higher than during natural REM sleep (8).] Be· cause the hypoglossal and intercostal nerve activities and respiratory rate remained significantly depressed for the whole period of postural atonia, this also sug· gests that the suppression of phrenic nerve activity may be relatively easily overcome by chemical drive, in contrast to those other respiratory motor outputs and the decrease in respiratory rate. Thus, the absence of a reduction in the motor output to the diaphragm during the steady-state observed in the present study confirms our earlier hypothesis (5) that the large depression of phrenic nerve activity accompanying the postural atonia in the decerebrate, paralyzed and artificially ventilated cats was largely due to the absence of natural chemical feedbacks. In the presence of chemical feedback, the small changes in the diaphragmatic motor output seen in this study are more compatible with minor average changes in the tidal volume seen during natural REM sleep (6) and in the carbachol-induced REM sleep-like state (8) and are in contrast with the significant depression seen in artificially ventilated, decerebrate animals (5). This suggests that, during the carbachol-induced REM sleep-like state and perhaps also during natural REM sleep, the diaphragm is the most important respiratory muscle that can effectively compensate for the reduction in ventilation resulting from cholinergic stimulation within the pons. However, it is noteworthy that, in spite of the increase in phrenic nerve activity following the initial depression, ventilation remained depressed and the end-expiratory CO 2 level substantially increased. The same is also very likely to occur in the carbachol-injected, intact cat (8). The large decreases in respiratory rate, in contrast to the frequently observed periods of accelerated breathing during natural REM sleep (6), may be related to a powerful stimulatory effect of carbachol within the pontine tegmentum that is unlike the presumably les.s intense cholinergic stimulation that occurs during natural REM sleep. A decrease in respiratory rate also occurs during carbachol-induced REM sleep in intact cats (7-9) and it is closely linked to the amount of endogenous acetylcholine released within the pontine tegmentum (9). Thus, the intrinsic cholinoceptive mechanisms in this region of the pons playa role in the control of respiratory timing. Recently it was demonstrated (15) that the respiratory suppression elicited in parallel to postural atonia by electrical stimulation within the pontine tegmentum in decerebrate, spontaneously breathing cats also involves a decrease of the respiratory rate that outlasts the depression of the di-
BREATHING DURING CARBACHOL-INDUCED ATONIA
pression is weaker and can be largely compensated for by chemical feedbacks. Consequently, the carbacholinjected decerebrate animal should provide a suitable model for studying the tonic changes in respiration and upper airway muscle tone that are directly related to the mechanisms underlying the postural atonia of REM sleep and, perhaps, other states that involve a generalized postural atonia such as cataplexy (21) and concussion-induced behavioral suppression (22). The absence of irregular breathing means that additional, thus far undetermined, mechanisms are required for the full expression of the changes in the breathing pattern characteristic of natural REM sleep. Thus, the carbacholinduced state in decerebrate animals may be regarded as a model of selected aspects of the effects of REM sleep on breathing and, as such, may be very useful to study them in isolation from other REM sleep activities. Acknowledgements: We are grateful to Dr. Allan Pack for helpful discussions throughout the course ofthese studies. We thank Rosemarie Cohen for her excellent secretarial support. H.K. was on leave from the Department of Chest Medicine, School of Medicine, Chiba University, Chiba, Japan. This study was supported by National Heart, Lung and Blood Institute Specialized Center for Research Grant HL-42236.
REFERENCES I. Hobson JA, Lydic R, Baghdoyan HA. Evolving concepts of sleep
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cycle generation: from brain centers to neuronal populations. Behav Brain Sci 1986;9:371-448. Siegel JM. Brainstem mechanisms generating REM sleep. In: Kryger MH, Roth T, Dement WC, eds. Principles and practice of sleep medicine. Philadelphia: WB Saunders, 1989; 104-20. Morales FR, Engelhardt JK, Soja PJ, Pereda AE, Chase MH. Motoneuron properties during motor inhibition produced by microinjection of carbachol into the pontine reticular formation of the decerebrate cat. J Neurophysiol 1987;57: 1118-29. Lai YY, Siegel JM. Cardiovascular and muscle tone changes produced by microinjection of cholinergic and glutamatergic agonists in dorsolateral pons and medical medulla. Brain Res 1990;514:27-36. Kimura H, Kubin L, Davies RO, Pack AI. Cholinergic stimulation of the pons depresses respiration in decerebrate cats. J Appl Physiol 1990;69:2280-9. Phillipson EA, Bowes G. Control of breathing during sleep. In: Chemiack NS, Widdicombe JG, eds. Handbook of physiology. The respiratory system. Control of breathing. Bethesda, MD: Am Physiol Soc, 1986, Sect 3, Vol II, Pt 2, Chapt 19,649-89. Lydic R, Baghdoyan HA. Cholinoceptive pontine reticular mechanisms cause state-dependent respiratory changes in the cat. Neurosci Lett 1989;102:211-16. Lydic R, Baghdoyan HA, Wertz R, White DP. Cholinergic reticular mechanisms influence state-dependent ventilatory response to hypercapnia. Am J PhysioI1991;261:R738-R746. Lydic R, Baghdoyan HA, Lorinc Z. Microdialysis of cat pons reveals enhanced acetylcholine release during state-dependent respiratory depression. Am J Physiol1991 ;261:R766-R 770. Orem J. Neuronal mechanisms of respiration in REM sleep. Sleep 1980;3:251-67. Kline LR, Hendricks JC, Davies RO, Pack AI. Control of activity of the diaphragm in rapid-eye-movement sleep. J Appl PhysioI1986;61:1293-1300. Sleep. Vol. 15. No.5, 1992
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Thus, our result may be taken to suggest that breathing irregularities require an intact PGO wave generating system and/or require activation of other, probably suprapontine, brain structures (10). In regard to the influence of vagal input, some reports indicate that vagotomy tends to increase the variability of breathing during REM sleep (17-19). In the present study, bilateral vagotomy did not affect the carbachol-induced prolongations ofTJ and TE or their variability. Phillipson et al. (18) and Foutz et al. (19) show that vagotomy has a smaller effect on the variability of respiratory frequency during REM sleep than during non-REM sleep, which suggests a greater role of the vagus during the latter state. The present finding that the variabilities of breathing were not different in animals with vagi intact or cut during the carbacholinduced REM sleep-like state is consistent with a minor role of vagal afferent control during natural REM sleep (6). In this study, we found that carbachol injections into the dorsal pontine tegmentum of decerebrate, spontaneously breathing cats evoked only transient depressions of the phrenic nerve activity and tidal volume, but long-lasting decreases in respiratory rate, postural muscle tone and the activity of upper airway and rib cage muscles. Although the changes were not significant, there was a trend towards more regular breathing after carbachol, more so in the animals showing vigorous eye movements in response to carbachol injection. This is in contrast to what might be expected based on respiratory changes typically observed during natural REM sleep and raises the question of how useful the acute decerebrate cat with postural atonia produced by pontine carbachol is for the study of the respiratory alterations during REM sleep. On the one hand, we found that the breathing pattern in this preparation differs from that in natural phasic REM sleep in that there is a profound slowing of the respiratory rate and no increase in the irregularities of either the timing or the magnitude of the motor output. On the other hand, as discussed elsewhere (5), the relative depression of activities of different respiratory motor nerves is the same, albeit exaggerated, as in natural REM sleep. In addition, the pontine injection site that gives origin to the respiratory changes that we described is rather small and in a similar location to, if not congruent with, that giving origin to many other signs of REM sleep in intact animals (5,12,20). Moreover, the concentrations of endogenous acetylcholine in this region correlate well with the decreases in respiratory rate induced by carbachol (9). Therefore, the tonic respiratory changes induced by pontine carbachol are likely to represent an exaggerated form of respiratory depression that may be activated during natural REM sleep. In normal conditions, however, this de-
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17. Remmers JE, Bartlett D Jr, Putnam MD. Changes in the respiratory cycle associated with sleep. Respir Physio/ 1976;28: 227-38. 18. Phillipson EA, Murphy E, Kozar LF. Regulation of respiration in sleeping dogs. J Appl Physiol 1976;40:688-93. 19. Foutz AS, Netick A, Dement We. Sleep state effects on breathing after spinal cord transection and vagotomy in the cat. Respir PhysioI1979;37:89-100. 20. Yamamoto K, Mamelak AN, Quattrochi JJ, Hobson JA. A cholinoceptive desynchronized sleep induction zone in the anterodorsal pontine tegmentum: locus of the sensitive region. Neuroscience 1990;39:279-93. 21. Mitler MM, Dement We. Cataplectic-like behavior in cats after micro-injections of carbachol in pontine reticular formation. Brain Res 1974;68:335-43. 22. Hayes RL, Pechura CM, Katayama Y, Povlishock JT, Giebel ML, Becker DP. Activation of pontine cholinergic sites implicated in unconsciousness following cerebral concussion in the cat. Science 1984;223:301-3.
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12. Vanni-Mercier G, Sakai K, Lin JS, Jouvet M. Mapping ofcholinoceptive brain stem structures responsible for the generation of paradoxical sleep in the cat. Arch Ital Bioi 1989; 127: 133-64. 13. Berman AL. The brainstem of the cat: cytoarchitectonic atlas with stereotaxic coordinates. Madison: University of Wisconsin Press, 1968. 14. Lopez-Rodriguez F, Morales FR, Chase MH. Induction of muscle atonia by the microiontophoretic injection of cholinergic drugs into the brainstem reticular formation. Sleep Res 1992; 21:8. 15. Kawahara K, Suzuki M. Descending inhibitory pathway re:sponsible for simultaneously suppression of postural tone and respiration in decerebrate cats. Brain Res 1991;538:303-9. 16. Gadea-Ciria M. Etude sequentielle des pointes ponto-geniculooccipitales (PGO) au cours du sommeil paradoxal chez Ie chait normal, et apres lesions corticales et sous-corticales. These de Doctorat. Lyon, 1972. Cited after: Jones BE. Paradoxical sleep and its chemical/structural substrates in the brain. Neurosci 199 J; 40:637-56.
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Fundamental Research Spontaneous Ventilation and Respiratory Motor Output During Carbachol-Induced Atonia of REM Sleep in the Decerebrate Cat Department of Animal Biology, School of Veterinary Medicine; and Center for Sleep and Respiratory Neurobiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046, U.S.A.
Summary: Microinjections of carbachol into the pons induce a state that resembles rapid eye movement (REM) sleep in intact cats and, in decerebrate, artificially ventilated cats, produce postural atonia accompanied by a powerful depression of the respiratory motor output. In this study, pontine carbachol was used in decerebrate, spontaneously breathing cats to assess the effects of mechanical and chemical respiratory reflexes on the magnitude and pattern of the carbachol-induced depression of breathing, and tOi determine whether the depression is altered in those animals in which rapid eye movements are present. Phrenic nerve activity and tidal volume were only transiently depressed at the onset of the carbachol-induced postural atonia, whereas the decrease in respiratory rate and the depressions of hypoglossal and intercostal activities persisted until the response was reversed by a pontine microinjection of atropine 15-101 minutes after the onset of carbachol response. Ventilation was reduced to 70% of control during the steady-state conditions. The irregularity of breathing, characterized by the inter-quartile ranges of the distributions of the peak phrenic nerve activity and respiratory timing, did not increase following pontine carbachol. Neither vagotomy nor vigorous eye movements were associated with increased breathing irregularity. This contrasts with the irregular breathing (with minor average chan~:es in ventilation) typical of natural REM sleep. We propose that the carbachol-injected decerebrate cat provides a useful model of the depressant effects that neural events associated with REM sleep may have on breathing. Key Words: Atonia-Carbachol-Control of breathing- PonsREM sleep- Vagus.
Cholinergic mechanisms within the dorsal pontine characterization of the behaviors that can be elicited tegmentum have been repeatedly implicated in the in the decerebrate preparation. In a recent study (5), we showed that cholinergic generation of rapid eye movement (REM) sleep (1,2). Recent demonstrations that these mechanisms can be stimulation within the dorsal pontine tegmentum of pharmacologically activated in an acutely decerebrate decerebrate cats produced a powerful depression ofthe cat by microinjections of carbachol into the pontine respiratory motor output that developed in parallel to tegmentum (3-5) provide a novel animal model to the atonia of postural muscles. A large decrease in study the neural mechanisms related to the postural respiratory rate accompanied a stereotyped suppresatonia of REM sleep and, perhaps, other REM sleep sion of the phrenic, hypoglossal and intercostal nerve phenomena. This new line of investigation of REM activities. Paralyzed, vagotomized and artificially vensleep-related mechanisms may offer significant advan- tilated cats were used in these experiments, which altages by allowing rigid control over the conditions of lowed us to study the changes in various motor outputs the experiment and use ofa large spectrum of invasive in isolation from feedback mechanisms originating in techniques. To understand the relationship between lung mechanoreceptors and central and peripheral chethe effects seen in this acute animal model and thosl~ moreceptors. The strength of the respiratory deprescaused by carbachol in the chronically instrumented, sion observed in these experiments substantially exbehaving cat or in natural REM sleep requires further ceeded that typically found in either natural REM sleep (6,7) or the REM sleep-like state induced in chronically instrumented, intact animals by pontine carbachol inAccepted for publication June 1992. Address correspondence and reprint requests to Dr. Leszek Kubin, jections (7-9). We ascribed this exaggerated depresCenter for Sleep and Respiratory Neurobiology, 972 Maloney Building, Hospital of the University of Pennsylvania, 3600 Spruce Street, sion, in part, to the absence of feedback mechanisms, Philadelphia, Pennsylvania 19104-4283, U.S.A. which allowed the inhibitory mechanisms to appear in
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Hirokazu Tojima, Leszek Kubin, Hiroshi Kimura and Richard O. Davies
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BREATHING DURING CARBACHOL-INDUCED ATONIA
METHODS The results were obtained from 15 adult cats (2.04.0 kg). They were preanesthetized with ketamine (80 mg im) and diazepam (2 mg im) and, after intubation, anesthetized with halothane and decerebrated at a precollicular level. The halothane anesthesia was then discontinued. Recordings were begun - 9 hours after the injection of ketamine-diazepam and -7 hours after the decerebration. In four cats, both vagi were cut in the midcervical region; in the other animals, they were left intact. A detailed description of the surgical preparation and recording techniques was given previously (5).
In all animals, the activity of the C5 branch of one phrenic nerve and either the activity of a dorsal cervical motor nerve branch (C4) or the neck muscle electromyogram (EMG) were recorded (20-2,000 Hz bandwidth). The electrooculogram (EO G) was recorded (0.1-20 Hz bandwidth) with two steel wires inserted bilaterally under the skin overlying the postorbital processes of the zygomatic arches. In addition, in five animals, the activity of the genioglossal branch of the hypoglossal nerve and, in another five animals, the intercostal muscle EMG containing either inspiratory, expiratory or both types of activities were recorded. In an additional three animals, genioglossal and intercostal activities were recorded simultaneously. The animals were placed in a stereotaxic instrument and the anterior cerebellar vermis exposed for microinjections in the pons.
The tracheal CO2 was continuously monitored with an infrared gas analyzer (Godard). Because thes~de cerebrate, spontaneously breathing animals were often hypocapnic in control conditions, O 2 (30-50%) and CO 2 (1-3%) were added in some experiments to the inspired gas mixture to elevate the hypoglossal and/or intercostal activities to a measurable level. Airflow was measured using a pneumotachograph (Fleish 00) and differential pressure transducer. The inspiratory volume was obtained by electronic integration of the airflow signal (CWE PI-830). These variables, the moving averages of the nerve or EMG activities and an event marker were monitored on a chart recorder (Gould T A 2000). The raw nerve and EMG signals and other variables were stored on magnetic tape (H-P 3968A) for subsequent analysis. Solutions of carbachol (Sigma) in saline (10 or 88 mM) with 2% pontamine blue dye were placed in glass micropipettes having tip diameters of 30-40 /-tm. The pipettes were positioned in the dorsal pontine tegmentum using visual landmarks and stereotaxic coordinates, aiming for the coordinates of P3.5, H-4.0 and L2.0 (1,2,12). The injections were made using pressure pulses (NeuroPhore BH-2), and the volume ejected was directly determined by measuring the movement of the meniscus in the pipette with a pocket microscope and reticle. The injected volume of carbachol ranged from 140 to 1,000 nl (mean ± SD: 420 ± 280 n1), corresponding to 0.25-16.0 /-tg. In most animals, after a sufficient period of observation following the carbachol response, atropine S04 (Sigma) (equimolar with carbachol and at a volume of 1-4 times that of the preceding carbachol injection) was injected into the same pontine site. This reversed the carbachol effects. Subsequently, in three animals, a second injection of carbachol was then made in a symmetrical site in the contralateral pons (5) and these three responses were also included in our database. In two of these three animals, the first response to pontine carbachol had to be excluded from analysis for technical reasons. In seven animals, after conclusion of the experiment, the brain stem was removed, fixed in 10% formalin and cryoprotected with 30% sucrose, and the pontine region was cut in 50 /-tm slices in the coronal plane. Every fourth slice from the region containing the blue dye was mounted and counterstained with neutral red, and the center of the injection was subsequently redrawn on a standard cross-section of Berman's atlas (13).
Data analysis
An effective carbachol injection was characterized by the parallel development ofa depression of postural and hypoglossal or intercostal nerve activities (5). The Sleep, Vol. 15, No.5, 1992
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their full strength (5). Apart from the powerful suppression, no irregularities in either respiratory rate or inspiratory firing of the phrenic nerve were observed. This is, therefore, unlike natural REM sleep, during which respiration is characterized by rapid changes in respiratory rate and fractionations of diaphragmatic activity, the frequency and severity of which have been related to the intensity of the characteristic phasic events of REM sleep such as muscle twitches, rapid eye movements and ponto-geniculo-occipital (PGO) waves (6,10,11). In our previous study (5), the use of paralyzed animals precluded the study of eye movements. We now report the effects of pontine carbachol on ventilation and the respiratory motor output in acutely decerebrated, spontaneously breathing cats in which we also recorded the electrooculogram. Thus, we can now relate the strength and pattern of respiratory depression in these animals to the presence of eye movements and compare these results with those obtained from animals with chemical and mechanical respiratory feedbacks absent.
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are presented as the mean ± standard deviation (SD) throughout this report, except where noted. Changes were considered significant if p < 0.05.
RESULTS Changes in breathing associated with carbachol-induced atonia The responses to 16 carbachol injections were analyzed. Three injections (5b, 6 and 7 in Table 1) were made after reversal of the first response with pontine atropine. They produced effects that were not different from the others (5), and all the responses were pooled together for further analysis. Pontine carbachol produced a stereotyped depression of the recorded respiratory nerve and/or EMG activities and a slowing of the respiratory rate that developed in parallel to the atonia of postural activity, as described previously for artificially ventilated cats (5). The mean latency to the onset of the phrenic response was 275 ± 299 seconds, and that for the postural (neck) motor tone was 313 ± 407 seconds (no significant difference, paired t test). The mean delay from the onset of the response to the time of maximal depression of phrenic activity was 99 ± 148 seconds, and that for neck activity was 93 ± 112 seconds (no significant difference, t test). Unless reversed by an atropine injection, the response could last for a prolonged period of time (> 1 hour) as reported for paralyzed and artificially ventilated cats (5). Figure 1 shows two polygraphic records obtained from a vagotomized (part A-cat Sa in Table 1) and a nonvagotomized (part B-cat 10 in Table 1) animal showing the carbachol-induced suppressions of respiratory nerve activities that developed in parallel to the postural atonia. In the response shown in Fig. lA, pontine carbachol induced vigorous horizontal eye movements, whereas no eye movements were observed in the response shown in Fig. 1B. The peak amplitude of the moving average of the phrenic nerve activity was transiently depressed in the cat shown in A (see arrow) and then returned to the control levels. In the example shown in B, the phrenic activity remained almost unchanged. In both animals, the respiratory rate was characteristically decreased and remained reduced for a prolonged period of time. An initial decrease in the amplitude of the phrenic nerve activity of at least 10% was observed at the onset of the response to carbachol in 11 of the 16 trials, the mean initial reduction for all responses being to 86 ± 11 % of control (p < 0.001, t = 4.84, paired t test, n = 16). However, by the time that steady-state conditions were reached, phrenic nerve activity increased in all but three of the responses to a level equal to or higher
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presence of eye movements as indicated by the EOG signal was confirmed by direct observation. The times to the onset of the effect and to reach the maximum effect were measured from the beginning of the injection. For two injections using 88 mM of carbachol, the response commenced immediately after placement of the pipette in the pons, probably due to leakage from the pipette. In these two cases, the time of insertion of the pipette was regarded as the beginning of the injection. The changes in the peak nerve or EMG activities were determined from the moving averages of the corresponding signals and measured with respect to the baseline (established during periods with no activity). Within each respiratory cycle, inspiration was defined as the period from the onset of the increase in the moving average of phrenic nerve activity to the point of rapid decline and expiration as the period from thl~ rapid decline in the moving average of the phrenic nerve activity to the onset of the next burst. To characterize the changes in respiratory timing, the inspiratory duration (T J) and expiratory duration (T E) were determined for control conditions and various stages of the response. (The total respiratory cycle duration was T J + T E') Measurements taken during the carbachol-induced depression were normalized to the corresponding mean control values (immediately before the carbachol injection) and expressed as percent of the control. In many cases, the response to carbachol consisted of two successive phases-a transient depression, during which phrenic nerve activity was maximally depressed, and a subsequent steady state, with ventilation stabilized at a level still reduced but above that of the initial depression. To characterize these changes, the parameters of ventilation were determined for at least 10 consecutive respiratory cycles in each phase and averaged. For 11 experiments, sufficiently long segments of records were available for off-line statistical analysis of the variability of respiratory timing and phrenic nerve magnitude using an IBM PC-AT computer-based data acquisition and analysis software (EGAA and Spread, RC-Electronics). Statistical distributions (histograms) ofTh TE and peak amplitude of the moving average of the phrenic nerve activity were determined from segments of records containing 50-100 respiratory cycles during control conditions and following the carbachol-induced respiratory depression. The interquartile range (IQR), i.e. the range between the lowest and the highest quartile measured from the histogram of the distribution of the analyzed variable, was used to characterize the variability of each of the studied parameters in each condition. Statistical evaluations of the data were performed using a paired or unpaired Student's t test. The data
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TABLE 1. Characteristics oJrespiratory responses to pontine carbachol injections in individual animals Carbachol Animal # ~,
Dose (pg)
Vagotomy
1,000 500 300 280 280 280 260 250 140
no no no no yes yes yes yes no
100 190 40 200 240 320 110 440 270
Control
Carbachol
Minute ventilation a (% control)
20 61 59 50 14 13 81 53 40
16 33 32 22 11 13 36 52 34
98 70 65 50 81 94 59 107 83
Respiratory rate
Period of observation (minutes) 33 64 34 55 70 65 41 67 27
1,000 800 500 500 340 220 140
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than in the precarbachol state. This secondary increase occurred both in animals that had a transient decrease at the start of the response and in animals in which the transient decrease was absent or less than 10%. On the average, this secondary increase brought the phrenic activity back to a level close to that of the control period (103 ± 20%) and not significantly different from the latter. The average changes in respiratory parameters and blood pressure, determined for the steadystate period after ventilation stabilized following carbachol injection, are shown in Fig. 2 separately for nonvagotomized and vagotomized cats. Separate determinations were made for 11 responses analyzed in
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tively; FC0 2 , end-tidal CO 2 fraction; EOG, electrooculogram. Carbachol (0.5 Itg) was injected into the pons 8 minutes before the beginning of the record. Note a transient depression of the phrenic nerve activity (arrow) and a decrease of the respiratory rate at the onset of the response. The phrenic nerve activity subsequently quickly recovered (to 104% of control), while the reduction of the respiratory rate was maintained (14 minute-I in control, II minute-I during the steady state following carbachol). As a consequence of the decreased rate, ventilation was reduced and the end-expiratory CO 2 elevated by 0.3%. B: Response to pontine carbachol in a cat (lOin Table I) that did not show eye movements. IC, NECK, moving averages of the EMG activities of expiratory intercostal and neck muscles, respectively. The phrenic nerve activity was reduced to 76% of control 12 minutes after the onset of the response and the respiratory rate decreased from 39 minute-I before, to 30 minute-I after carbachol. The small changes seen on the EOG trace after the carbachol injection did not represent eye movements but rather muscle twitches that occurred transiently before the response to carbachol reached its steady state. Note the parallel changes in the neck and intercostal EMGs during the initial stage of the response. Sleep. Vol. 15. No.5. 1992
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11 nonvagotomized, and five responses in four vagotomized, animals. The peak phrenic nerve activity and tidal volume were not significantly different from control in either group of animals, whereas the peak flow and respiratory rate in animals with vagi intact and blood pressure in both animal groups remained significantly reduced. No significant differences in the magnitudes of any aspect of the response were found between the animals with vagi intact and cut (I test), nor did the vagotomy affect the variability of respiration following pontine carbachol (see below). The lack of significance in the carbachol-induced chang1es in the peak flow and respiratory rate in the anima.ls with vagi cut could be due to a lower number of observations in this group. In contrast to the changes in phrenic nerve activity, the hypoglossal and intercostal nerve activities (or corresponding EMGs) remained significantly suppressed for a prolonged period of time. After ventilation stabilized following carbachol injection and phrenic nerve activity was close to or above the control level in most cases, as described above, hypoglossal activity was depressed to 21 ± 15% of control (n = 7), inspiratory intercostal activity to 63 ± 29% (n = 5), and expiratory intercostal activity was completely abolished in all three experiments in which expiratory activity was present in control conditions. The respiratory rate also remained significantly depressed; the mean control rate was 40.0 ± 21.0 minute-I and it fell to 25.5 ± 12:.1 minute-I after carbachol (p < 0.001, t = 4.20, paired t test, n = 16). These observations were made over a Sleep, Vol. 15, No.5, 1992
time period ranging from 15 to 101 minutes following the maximal initial depression (mean: 50 ± 21 minutes). Atropine was then injected into the pons to reverse the carbachol effects or a substantial spontaneous recovery from the carbachol effects became apparent. Subsequent to the microinjections of atropine, the respiratory rate and all nerve activities returned to controllevels, as has been described earlier (5). Ventilation, calculated as the product of the peak phrenic nerve activity (arbitrary units) and respiratory rate, was reduced to 57.1 ± 20.6% of control during the transient maximal depression and then, due to the subsequent increase in peak phrenic activity, recovered to 70.3 ± 19.7% after ventilation stabilized (n = 16). The mean end-tidal CO2 rose by 0.8 ± 0.6% following pontine carbachol, as determined for the nine experiments for which adequate calibrations were available for the whole period of analysis. The decrease in respiratory rate was due to prolongations of both T J and T E' After all but one injection, TI increased (mean control = 0.8 ± 0.5 seconds; carbachol = 1.1 ± 0.6 seconds; p-< 0.001,1 = 8.00, paired t test, n = 16) and after all but two injections, T E increased (mean control = 1.1 ± 0.6 seconds; carbachol = 1.8 ± 1.1 seconds; p < 0.001, 1 = 4.12, paired t test, n = 16). Although the absolute change in T E was larger, the relative change of T J was not different from that of T E. Figure 3A shows the effect of vagotomy on the average peak phrenic nerve activity and respiratory timing before and after carbachol. Figure 3B shows the carbachol-induced changes in the irregularity of the
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respiratory cycle (as assessed by IQRs ofT[ and T E) in vagotomized and nonvagotomized cats. The data used in Fig. 3 are from four responses in vagotomized and seven responses in nonvagotomized animals that were suitable for statistical analysis of breathing irregularities, as explained in Methods. As expected, the control T[ and T E were longer in vagotomized than in nonvagotomized animals, and in both animal groups they were prolonged further during the carbachol-induced depression. Vagotomy had no effect on the IQRs of the respiratory timing in either control or post-carbachol conditions (t test).
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Effect of eye movements on breathing following pontine carbachol
In nine animals, the carbachol-induced depression of respiratory and postural activity was accompanied by vigorous horizontal eye movements, whereas in the remaining six animals no eye movements were induced. The eye movements, when present, were inSleep, Vol. 15, No.5, 1992
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tense and occurred continuously with a relatively regular amplitude and frequency, rather than in bursts of variable duration and amplitude typical of natural REM sleep in intact cats. Figure 4 shows the distribution of the centers of pontine injection sites for four animals that showed eye movements in response to carbachol injection and three animals that did not. As also noted in another two carbachol studies on intact animals (12,14), there was no clear relationship between the injection site and occurrence of eye movements following carbachol injections. In animals with eye movements, the mean changl~s in phrenic amplitude during either the initial transient depression or the steady-state were not significantly different from those in animals without eye mov,ements. Figures 5A and B show separate histograms of T J and phrenic peak amplitude obtained from one cat (animal 3 in Table 1) in control conditions (open bars) and during a carbachol response with eye movements Sleep, Vol. 15, No.5, 1992
(filled bars). In this cat, carbachol prolonged the mean T J, whereas the IQR ofTJ remained unchanged (40 ms) and increased the peak phrenic nerve activity, whereas the IQR decreased to 82% of control. The mean changes in the peak phrenic nerve activity and respiratory timing in relation to the presence or absence of eye movements are shown in Fig. 5C. The mean data from seven responses in six animals with eye movements and from four responses in four animals without are shown. Neither the peak phrenic nerve activity nor the timing changes induced by carbachol were statistically different between the animals with and without eye movements. Because the intensity of eye movements and phasic REM sleep activity was found to correlate with variability in respiratory drive and timing in natural REM sleep (6,10,11), we determined whether the IQRs of the measured respiratory parameters were larger in those animals that displayed eye movements after the
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carbachol injection than in those without them. The It is based on microinjections of carbachol into the IQRs were determined for those animals for which we pons of decerebrate cats and may be useful for studies had sufficient data for statistical analysis, seven re- of selected aspects of the central neural mechanisms sponses with and four without eye movements. Before of REM sleep. Accordingly, in this study we explored assessing the effect of eye movements or vagotomy on some of the features of the acute model that may be the IQRs, we confirmed (using the Fisher exact test) relevant for studies of REM sleep-related changes in that the two factors, vagotomy and the presence of eye the control of breathing. In contrast to our previous work that described the movements, were independent. For the group with eye movements after the carbachol injection, the IQRs for respiratory suppression by pontine injections of carboth T J and T E tended to decrease slightly compared bachol in decerebrate, paralyzed and artificially vento the control. In contrast, they increased to a variable tilated cats (5), the present report focuses upon the degree in animals without eye movements after car- changes in respiratory motor outputs in spontaneously bachol (see Fig. 5D). The only statistically significant breathing decerebrate cats. Two findings relevant for difference was, however, for the percent change of the the future use of decerebrate cats in studies of REM IQR for T J between the animals with and without eye sleep-related changes in respiration were obtained: 1) movements. In this case, unlike what could be expected The phrenic nerve activity was not depressed in the based on studies during natural REM sleep, the larger steady-state phase of the carbachol response, unlike in variability was seen in animals without eye movements the artificially ventilated cats, whereas the decreases (p < 0.05, t = 2.375, t test). The changes in the IQRs in respiratory rate were similarly profound in the two for the phrenic nerve peak amplitude were evaluated preparations (69% of control in our previous (5) and using the ratio of the peak amplitude before and after 64% in the present study); and 2) the variability ofTJ , carbachol as they were measured in arbitrary units. T E and phrenic peak amplitude did not increase during Following carbachol injection, the IQRs of the phrenic the postural atonia and respiratory depression induced peak amplitude in animals with eye movements re- by pontine carbachol, even in animals in which very mained unchanged (97% of the control), whereas in intense eye movements were induced. There findings the group without eye movements they increased to are in a striking contrast to the pattern of breathing 121 %. These changes were, however, not significant during natural REM sleep (6,10,11). In a recent study of the effects of carbachol on reseither within (paired t test) or between the animal groups (t test). The mean IQRs of both the timing and am- piration in chronically instrumented, intact cats (8), plitude characteristics of breathing, pooled for all 11 the tidal volume, which is closely related to peak diresponses studied, both with and without eye move- aphragmatic EMG activity or phrenic peak amplitude, ments, did not change significantly after carbachol in- was virtually unchanged from the control (waking) levjections (paired t test). Thus, unlike in natural REM el during the REM sleep-like state. This is similar to sleep, the presence of eye movements was not asso- what we found in the decerebrate, spontaneously ciated with increased respiratory variability during the breathing cats of the present study where, in most ancarbachol-induced postural atonia in decerebrate cats. imals, carbachol caused only a transient depression of the peak phrenic nerve activity, which then recovered to the control level. In contrast, in decerebrate,' vaDISCUSSION gotomized and artificially ventilated cats, a suppresNumerous investigators have found that microin- sion of the peak phrenic amplitude to 66% of control jections of cholinergic agonists into the pontine teg- was observed (5). In all three preparations, the reducmentum of cats produce a state that has many char- tion in respiratory rate was maintained throughout the acteristics of natural REM sleep (1,2). This duration of the carbachol effect. Thus, ventilation was pharmacologically-induced state in intact, chronically decreased in these three types of experiments to 72% instrumented animals is characterized by such signs of of control in intact cats (8); 70% in spontaneously natural REM sleep as a desynchronized EEG, postural breathing, acutely decerebrate cats (present study); and muscle atonia, rapid eye movements, a characteristic 46% in artificially ventilated, acutely decerebrate cats hippocampal theta rhythm and PGO waves. There- (calculated as a product of the peak phrenic nerve acfore, it has been regarded as a valid experimental model tivity and respiratory rate) (5). for the physiological state of REM sleep. Recently, it The lack of a maintained suppression of phrenic was also employed to study the REM sleep-related activity in the present experiments may result, in a changes in ventilation and respiratory motor output large part, from a compensatory increase in respiratory (7-9). As an extension of the studies performed on drive due to a rise in PaC0 2 following the initial venchronically instrumented cats, an acute animal model tilatory depression. This, when compared to the results of the postural atonia of REM sleep was proposed (3). from artificially ventilated cats (5), suggests that the
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aphragmatic activity. This phenomenon may also be explained by stimulation-induced release of acetylcholine in amounts exceeding those occurring during natural REM sleep. Taken together, these data point to the dorsolateral pons as the site where po~tural atonia, cholinergic receptors and mechanisms controlling respiratory rate are closely interrelated. . A characteristic feature of breathing during natural phasic REM sleep in animals and man is profound irregularity of both frequency and tidal volume, with an average increase of the former and a minor change of the latter when compared to non-REM sleep or wakefulness (6). In contrast to breathing during natural REM sleep, we did not find an increased variability in respiratory timing or peak phrenic nerve activity during the carbachol-induced postural atonia in decerebrate cats. In several studies, the irregularity of breathing during natural phasic REM sleep was found to increase roughly proportionally to the intensity of phasic phenomena (6,10,11). In this study, we took advantage of the fact that, following carbachol injections, the postural atonia was not always accompanied by rapid eye movements. This has been reported earlier in chronic animal studies (12,14). Moreover, the presence of eye movements is not clearly related to the injection site (12), and it has been proposed that factors other than the site and the dose determine whether eye movements will occur in association with carbacholinduced atonia (14). In this study, we found also that in decerebrate cats eye movements did not occur in every response and their presence was not clearly related to the site of the injection (Fig. 4). To assess whether breathing irregularities can be related to the presence of such phasic events as eye movements following carbachol injections, we analyzed the variability of breathing separately in cats with and without eye movements. Surprisingly, the variabilities of breathing were slightly smaller in those animals in which vigorous eye movements were evoked than in the animals without eye movements. It must be noted, however, that the pattern of eye movements evoked by carbachol in these decerebrate cats was more regular than that typically observed during both natural REM sleep and carbachol-induced REM sleep in intact cats (1,12). Thus, we cannot exclude the fact that the mechanism of generation of eye movements in our preparation is different from that of natural REM sleep. Such a difference may account for the absence of any increase in the irregularity of breathing coincident with eye movements that we observed. Similarly, there is evidence that PGO waves are more regular during spontaneously occurring REM sleep in chronic decerebrate cats than during REM sleep in intact cats (16). Again, this suggests some alterations in the system that generates the phasic events of REM sleep in decerebrate animals.
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ventilatory response to CO2 is preserved in the car·· bachol-induced state in decerebrate cats. [In inta!;:t cats, the response to CO 2 during the carbachol-induced state is lllaintained; the slope of the CO 2 response curve is not significantly different from waking, but is signifi·· cantly higher than during natural REM sleep (8).] Be· cause the hypoglossal and intercostal nerve activities and respiratory rate remained significantly depressed for the whole period of postural atonia, this also sug· gests that the suppression of phrenic nerve activity may be relatively easily overcome by chemical drive, in contrast to those other respiratory motor outputs and the decrease in respiratory rate. Thus, the absence of a reduction in the motor output to the diaphragm during the steady-state observed in the present study confirms our earlier hypothesis (5) that the large depression of phrenic nerve activity accompanying the postural atonia in the decerebrate, paralyzed and artificially ventilated cats was largely due to the absence of natural chemical feedbacks. In the presence of chemical feedback, the small changes in the diaphragmatic motor output seen in this study are more compatible with minor average changes in the tidal volume seen during natural REM sleep (6) and in the carbachol-induced REM sleep-like state (8) and are in contrast with the significant depression seen in artificially ventilated, decerebrate animals (5). This suggests that, during the carbachol-induced REM sleep-like state and perhaps also during natural REM sleep, the diaphragm is the most important respiratory muscle that can effectively compensate for the reduction in ventilation resulting from cholinergic stimulation within the pons. However, it is noteworthy that, in spite of the increase in phrenic nerve activity following the initial depression, ventilation remained depressed and the end-expiratory CO 2 level substantially increased. The same is also very likely to occur in the carbachol-injected, intact cat (8). The large decreases in respiratory rate, in contrast to the frequently observed periods of accelerated breathing during natural REM sleep (6), may be related to a powerful stimulatory effect of carbachol within the pontine tegmentum that is unlike the presumably les.s intense cholinergic stimulation that occurs during natural REM sleep. A decrease in respiratory rate also occurs during carbachol-induced REM sleep in intact cats (7-9) and it is closely linked to the amount of endogenous acetylcholine released within the pontine tegmentum (9). Thus, the intrinsic cholinoceptive mechanisms in this region of the pons playa role in the control of respiratory timing. Recently it was demonstrated (15) that the respiratory suppression elicited in parallel to postural atonia by electrical stimulation within the pontine tegmentum in decerebrate, spontaneously breathing cats also involves a decrease of the respiratory rate that outlasts the depression of the di-
BREATHING DURING CARBACHOL-INDUCED ATONIA
pression is weaker and can be largely compensated for by chemical feedbacks. Consequently, the carbacholinjected decerebrate animal should provide a suitable model for studying the tonic changes in respiration and upper airway muscle tone that are directly related to the mechanisms underlying the postural atonia of REM sleep and, perhaps, other states that involve a generalized postural atonia such as cataplexy (21) and concussion-induced behavioral suppression (22). The absence of irregular breathing means that additional, thus far undetermined, mechanisms are required for the full expression of the changes in the breathing pattern characteristic of natural REM sleep. Thus, the carbacholinduced state in decerebrate animals may be regarded as a model of selected aspects of the effects of REM sleep on breathing and, as such, may be very useful to study them in isolation from other REM sleep activities. Acknowledgements: We are grateful to Dr. Allan Pack for helpful discussions throughout the course ofthese studies. We thank Rosemarie Cohen for her excellent secretarial support. H.K. was on leave from the Department of Chest Medicine, School of Medicine, Chiba University, Chiba, Japan. This study was supported by National Heart, Lung and Blood Institute Specialized Center for Research Grant HL-42236.
REFERENCES I. Hobson JA, Lydic R, Baghdoyan HA. Evolving concepts of sleep
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5. 6.
7. 8. 9. 10. II.
cycle generation: from brain centers to neuronal populations. Behav Brain Sci 1986;9:371-448. Siegel JM. Brainstem mechanisms generating REM sleep. In: Kryger MH, Roth T, Dement WC, eds. Principles and practice of sleep medicine. Philadelphia: WB Saunders, 1989; 104-20. Morales FR, Engelhardt JK, Soja PJ, Pereda AE, Chase MH. Motoneuron properties during motor inhibition produced by microinjection of carbachol into the pontine reticular formation of the decerebrate cat. J Neurophysiol 1987;57: 1118-29. Lai YY, Siegel JM. Cardiovascular and muscle tone changes produced by microinjection of cholinergic and glutamatergic agonists in dorsolateral pons and medical medulla. Brain Res 1990;514:27-36. Kimura H, Kubin L, Davies RO, Pack AI. Cholinergic stimulation of the pons depresses respiration in decerebrate cats. J Appl Physiol 1990;69:2280-9. Phillipson EA, Bowes G. Control of breathing during sleep. In: Chemiack NS, Widdicombe JG, eds. Handbook of physiology. The respiratory system. Control of breathing. Bethesda, MD: Am Physiol Soc, 1986, Sect 3, Vol II, Pt 2, Chapt 19,649-89. Lydic R, Baghdoyan HA. Cholinoceptive pontine reticular mechanisms cause state-dependent respiratory changes in the cat. Neurosci Lett 1989;102:211-16. Lydic R, Baghdoyan HA, Wertz R, White DP. Cholinergic reticular mechanisms influence state-dependent ventilatory response to hypercapnia. Am J PhysioI1991;261:R738-R746. Lydic R, Baghdoyan HA, Lorinc Z. Microdialysis of cat pons reveals enhanced acetylcholine release during state-dependent respiratory depression. Am J Physiol1991 ;261:R766-R 770. Orem J. Neuronal mechanisms of respiration in REM sleep. Sleep 1980;3:251-67. Kline LR, Hendricks JC, Davies RO, Pack AI. Control of activity of the diaphragm in rapid-eye-movement sleep. J Appl PhysioI1986;61:1293-1300. Sleep. Vol. 15. No.5, 1992
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Thus, our result may be taken to suggest that breathing irregularities require an intact PGO wave generating system and/or require activation of other, probably suprapontine, brain structures (10). In regard to the influence of vagal input, some reports indicate that vagotomy tends to increase the variability of breathing during REM sleep (17-19). In the present study, bilateral vagotomy did not affect the carbachol-induced prolongations ofTJ and TE or their variability. Phillipson et al. (18) and Foutz et al. (19) show that vagotomy has a smaller effect on the variability of respiratory frequency during REM sleep than during non-REM sleep, which suggests a greater role of the vagus during the latter state. The present finding that the variabilities of breathing were not different in animals with vagi intact or cut during the carbacholinduced REM sleep-like state is consistent with a minor role of vagal afferent control during natural REM sleep (6). In this study, we found that carbachol injections into the dorsal pontine tegmentum of decerebrate, spontaneously breathing cats evoked only transient depressions of the phrenic nerve activity and tidal volume, but long-lasting decreases in respiratory rate, postural muscle tone and the activity of upper airway and rib cage muscles. Although the changes were not significant, there was a trend towards more regular breathing after carbachol, more so in the animals showing vigorous eye movements in response to carbachol injection. This is in contrast to what might be expected based on respiratory changes typically observed during natural REM sleep and raises the question of how useful the acute decerebrate cat with postural atonia produced by pontine carbachol is for the study of the respiratory alterations during REM sleep. On the one hand, we found that the breathing pattern in this preparation differs from that in natural phasic REM sleep in that there is a profound slowing of the respiratory rate and no increase in the irregularities of either the timing or the magnitude of the motor output. On the other hand, as discussed elsewhere (5), the relative depression of activities of different respiratory motor nerves is the same, albeit exaggerated, as in natural REM sleep. In addition, the pontine injection site that gives origin to the respiratory changes that we described is rather small and in a similar location to, if not congruent with, that giving origin to many other signs of REM sleep in intact animals (5,12,20). Moreover, the concentrations of endogenous acetylcholine in this region correlate well with the decreases in respiratory rate induced by carbachol (9). Therefore, the tonic respiratory changes induced by pontine carbachol are likely to represent an exaggerated form of respiratory depression that may be activated during natural REM sleep. In normal conditions, however, this de-
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Sleep, Vol. 15, No.5, 1992
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