Sleep-wake states in infant rabbits: Profiles from motility monitoring

Physiology & Behavior, Vol. 22, pp. 1049-1054. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.

Sleep-Wake States in Infant Rabbits: Profiles from Motility Monitoring' E V E L Y N B. T H O M A N A N D L E W I S P. Z E I D N E R

Department of Biobehavioral Sciences, University of Connecticut, Storrs CT 06268 (Received 27 D e c e m b e r 1978) THOMAN, E. B. AND L. P. ZEIDNER. Sleep-wake states in infant rabbits: Profiles from motility monitoring. PHYSIOL. BEHAV. 22(6) 1049-1054, 1979.--Behavioral states in infant rabbits were determined from a single-channel analog recording of motility, including body movements and respiration. Behavioral states were first recorded by direct observation. Two observers simultaneously made 1-hour observations of bunnies at 7, 8, 9, 10, and 19 days of age, recording state every 10 seconds. Inter-observer reliability was assessed from epoch-by-epocb agreement on each observation. The mean percent exact agreement was 85.4. Then the animals were observed for one hour by one observer who recorded from direct observation while motility was recorded from a sensor-platform. Subsequently another person judged state from the analog recordings of motility patterns only. Validity assessment for these observations were also based on exact epoch agreement and the mean percent exact agreement was 84.0. The states recorded from motility patterns included: Waking, Sleep-Wake Transition, Active Sleep, and Quiet Sleep. Although state-related behaviors vary over this age range, the patterns of motility produced by body movements and respiration during waking and sleep states are consistently distinctive. These results suggest that the patterning of motility including motor movements and respiration is a total organismic expression of behavioral state. The procedure of motility recording permits prolonged monitoring periods and makes it possible to study the ontogeny of state organization in freely moving animals. Sleep-wake states Behavioral states Motility and sleep-wake states

Animals: behavioral states

SLEEP-WAKE states in infant animals have been studied by physiological recording and by direct behavioral observation. In altricial animals, including the rabbit, the general finding has been that electrophysiological states cannot be discriminated reliably during the earliest days. of life [3,4]. However, in rabbits, it is possible to discriminate behavioral states from the first day of life, and the configuration of state parameters obtained from behavioral observation converges with those obtained by physiological recording at 7 days of age [8]. In studies of state in infant rabbits we have found that isolation rearing and handling during the In-st 20 days of life have effects on sleep-wake organization at 30 days, when the pups are weaned [1,2]. The predictive potential of behavioral state measures in infant rabbits was also demonstrated: measures of sleep behavior taken from 8 to 15 days of age were related to open-field, exploratory, and social behaviors after weaning [9]. In the studies just cited state was measured by direct observation without instrumentation of the animal. However, direct behavioral observations are extremely tedious and time consuming, and this places limitations on both the number of animals that can be observed and the duration of observation periods for any state study. The present report

Rabbits: sleep-wake states

describes a procedure whereby some states of wakefulness and sleep can be reliably identified in the infant animal by use of a sensor platform which indicates the animal's motility produced by respiration and motor movements. We have found that analog signals from a very sensitive pressure transducer provide patterns that are unique for waking, active sleep, quiet sleep and sleep-wake transition. The frequency and regularity of the signal reflects the frequency and rhythmicity of movements: and the rise time reflects the speed of each movement, These qualities of movement, including those produced by respiration, are the very characteristics which provide the basis for state discriminations when direct behavioral observation of the infant animals are made. This report describes the procedures used for recording motility and presents reliability and validity data of behavioral state judgments from the analog recordings. METHOD

Animals The animals in this study were five Dutch-Belted rabbit littermates, three males and two females, born after a normal

1The research described in this paper was supported by The William T. Grant Foundation, NICHD Grant HD-08195-01A2, and NIMH Predoctoral Fellowship 5268-81-13645.

Copyright © 1979 Brain Research Publications Inc.--0031-9384/79/061049-06502.00/0

1050 gestation of 30-31 days. They all weighed between 40 and 50 g at birth, and were cleaned and fed normally by their mother. The litter was housed in a wooden nest box located within a large stainless steel maternity cage. The mother was locked out of the nest box at all times other than the daily 15-rain feeding period [10]. One of the five animals was observed at each of the following days of age: 7, 8, 9, 11, and 19.

General Procedure State observations of the five rabbits were made starting one hour after the end of the daily feeding period. The bunny was placed on a 9-in. square Plexiglas platform, within a clear Plexiglass cyfinder. A pressure-sensor under the 1/4 in. thick platform was connected through an amplifier to a single channel strip-chart recorder and any movement by the animal, including that produced by respiration during quiescent periods, was recorded as analog signal. A 100 W red light above the observational area maintained the temperature at 30°C, the observed nest temperature. After a 3-min period for the animal to adapt to the change in environment, the observation began. For one hour, two observers recorded the animal's behavioral states. During the next hour one investigator recorded the states of the animal by direct observation while an analog recording was obtained from the platform sensor. The second investigator later judged the animal's states from the chart recording only.

Procedure for State Recording by Direct Behavioral Observation Throughout the hour of observation, the bunny's states were code recorded by the observer(s) every 10 seconds, using the behavioral state categories defined by Thoman et al. [8]. These states are: Active Sleep, Active-Quiet Transitional Sleep, Quiet Sleep, Drowse, Indefinite, Freeze Stare, Active Waking, and Quiet Waking (some states are not applicable at all ages). In making a decision about each 10-sec epoch, the frame of reference was the ongoing prevailing state; and unless a state change was clear, the state judgement of the previous 10-sec epoch was maintained. Thus, epoch judgments constitute a "running probe" to determine when a state change occurs. An electronic timer provided a signal to the observer every 10-seconds, with a unique tone at the end of each minute. At the sound of the minute tone, the observer pushed a button which placed a signal On the analog tape so that the recording of the bunny's respiration and motility could be synchronized with the behavioral observations.

Procedure for Judging State from the Analog Tapes A preliminary study indicated that judgements from the respiration-motility analog tapes could not reliably match the complexity of the direct behavioral observations. Therefore, the observers' records were modified to code only the following states: Active Sleep, Quiet Sleep, Sleep-Wake Transition and Waking. Active Sleep. In this state the animal obviously lacks muscle tone and shows generalized twitching of a mild to intense level, especially in the extremities, ears, or face. Other typical behaviors include mouthing, jerks, components of burrowing, scratching, facewashing, or running. Rapid Eye Movements occur sporadically. After eye-

THOMAN .AND ZEIDNER opening (at about 14 days of age), the eyes may be either closed or open: and when the eyes are open the eyelids are usually drooped. Respiration is relatively rapid and irregular. Quiet Sleep. This sleep is marked by a complete lack of motor activity while the animal is in a resting (but not limp/ posture. Although sustained quiescence is the rule, occasional isolated small twitches may appear, and a mild, rhythmic mouthing may occur. Respiration is typically slow and regular. After eye-opening, the eyes may be either closed or open. When open, the eyes are only partly open and have a clouded, staring appearance which is readily visible. Sleep-Wake Transition. This state category includes two states previously defined [8], including drowse and indefinite. In drowse, there is a gradual cessation of motor activity and a corresponding change from an alert posture to a more relaxed one. Prior to eye-opening, drowse is characterized by brief episodes of twitching in close association with brief episodes of head raising, or components of locomotion or grooming. After eye-opening, a salient feature of drowse is the slow closing and opening of the eyes, or drooping of the eyelids, and drooping of the head. Sleep-Wake Transition also applies to indefinite periods when the animal exhibits mixed patterns of behaviors so that neither sleep nor wakefulness dominates the 10-sec epoch. Waking. This state is generally marked by locomotion and gross motor activity. In the pre-eye opening ages, waking may include some mild twitching, but is mainly characterized by an erect posture with components of walking, grooming, and exploratory behaviors. After eye-opening the eyes are wide open and alert, there may also be periods of inactivity during waking. Prior to eye-opening, periods of inactivity can be identified as waking by the characteristic posture and the surrounding epochs of motor activity. The analog tapes were then scored by the judge using these four state categories.

Motility Patterns Associated with States. Although the behavioral expression of states in rabbits varies over age [6,8], the quality of motility expressing state remains consistent. Examples of patterns which are characteristic for the four states are shown in Fig. 1. Waking consists of locomotion or other gross movements, producing an easily recognizable pattern which often satiates the pen recorder. Even when activity during wakefulness is very low, motor behaviors are relatively constant and still identifiable as waking rather than sleep. Transitions between waking and sleep, including drowse, are characterized by intermittent bursts of activity interspersed with relative low level activity. The mixture of large peaks with small ones is a dependable discriminator of these transition periods. Active sleep patterns consist of low level activity with sudden spikes reflecting twitches, jerks and startles. The peaks are very pointed because these movements are very sudden and fast in character. Finally, quiet sleep, seen more often in the older animals, is characterized by little or no movements and, as shown in Fig. 1, creates a very low level constant signal consisting only of respiration which is relatively slow and regular.

Motility Patterns Associated with Behaviors In addition to the major states just described, there are

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RESULTS

Inter-Observer Reliability Five 1-hour observations were made by two observers to assess inter-observer reliability. An observation consisted of 360 ten-second epochs scored by each observer. For each of these observations, comparisons were made between the two observers on each of the 360 epochs. The percent agreement scores were: 82, 82, 83, 86, and 94. The mean

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1053 TABLE 1

P E R C E N T O F O B S E R V A T I O N S P E N T I N E A C H S T A T E A S R E C O R D E D BY O B S E R V E R A N D J U D G E

Waking

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Age in Days

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exact agreement of the five observations by the two observers was 85.4-+2.27 percent.

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Judge-Observer Validity Five 1-hour observations were made by an observer simultaneously with the respiration-motility recording, and state was later independently coded from the analog recording by a judge. Table 1 presents the distribution of states as recorded by the observer and the judge for each animal. F o r each observation, comparisons were made between the observer and judge for agreement on each of the 360 epochs. The percent agreement scores were: 75, 80, 83, 91, and 91. Over the total observation, the mean agreement was 84.0-+3.13 percent. This is virtually the same as the interobserver agreement.

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The respiration-motility recording procedure permits continuous monitoring of state. The typical presentation of data from continuous recording is a profile of states over time. Figures 3 and 4 present one-hour state profiles of animals at 9 and 11 days of age including the profile derived from the behavioral o b s e r v e r ' s record and that derived from the judge's record, which was based on the chart record only. The data for these figures were smoothed by excluding any state changes lasting less than six 10-second epochs. Judge-observer agreement was not changed by this smoothing procedure, but the resulting profiles are more appropriate for presenting data from long-term continuous monitoring. The similarity between state patterning recorded by the observer and judgements made from the analog records is apparent from these figures. DISCUSSION

The results of this study demonstrate that states of wakefulness, active sleep, quiet sleep and sleep-wake transition can be discriminated from analog recordings of motility in infant rabbits. The validity of the procedure of judging state from motility patterns was assessed by determining agreement between the observer and judge on an epoch-by-epoch basis within every observation. Inter-observer reliability also used the epoch as the unit for analysis. If one were only interested in the amount of time an animal spent in a particular state over an extended observation, then the unit for analysis of reliability and validity would be the mean, rather than the individual epoch. However, state researchers are also interested in the sequencing of states (e.g., from quiet to active sleep), and thus it is necessary to establish that the

FIG. 3. One-hour state profile of 9-day-old bunny from recording of state by direct observation and from judgement of analog tape. W i

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state by direct observation and from judgment of analog tape. epoch itself is reliable, since a state change is the definition of a sequence [5,7]. It is for this reason that we have focused upon the epoch in our reliability and validity evaluations. The motility recording procedure clearly does not permit all of the state discriminations that are possible from direct

1054

THOMAN AND Z E I I ) N E R

behavioral observation. In this study we were able to discriminate: waking, sleep-wake transition, active sleep, and quiet sleep. From behavioral observations it is possible to separate the waking state into active waking and quiet alertness, to separate the sleep-wake transition periods into drowse and indefinite, and to identify reliably active sleepquiet sleep transition periods. Additionally, from direct observation it is possible to identify specific state related behaviors [8].However, burrowing, rocking, and hiccupping movements produce unique rhythmic patterns which are readily apparent in the motility recordings. The choice of

recording methodology is a function of the question of interest. The motility recording procedure permits prolonged recordings of the major states of sleep and waking in young rabbits in contrast to one- or two-hour observation periods that are the usual feasible time limit for direct observations. Developmental changes in state profiles can be studied in freely moving animals. Variations in the developmental course can be related to prenatal and postnatal factors, and the predictive potential of early measures for later development can be explored with minimal interference from observational procedures.

REFERENCES

1. Denenberg, V. H., D. DeSantis, S. Waite and E. B. Thoman. The effects of handling in infancy on behavioral states in the rabbit. Physiol. Behav. 18: 553-557, 1977. 2. DeSantis, D., S. Waite, E. B. Thoman and V. H. Denenberg. Effects of isolation rearing upon behavioral state organization and growth in the rabbit. Behav. Biol. 21: 273-285, 1977. 3. Ellingson, R. J. Development of wakefulness-sleep cycles and associated EEG paterns in mammals. In: Sleep and the Maturing Nervous System, edited by C. D. Clemente, D. P. Purpura and F. E. Mayer. New York: Academic Press, 1972, pp. 165174. 4. Jouvet-Mounier, D., L. Astic and D. Lacote. Ontogenesis of the states of sleep in rat, cat, and guinea pig during the first postnatal month. Devl. Psychobiol., 2: 216--239, 1970. 5. Monroe, L. J. Inter-rater reliability and the role of experience in scoring sleep EEG records I. Psychophysiology 5: 376--384, 1969.

6. Shimizu, A. and H. E. Himwich. The ontogeny of sleep in kittens and young rabbits. EEG Clin. Neurophysiol. 24: 307, 1968. 7. Thoman, E. B. and W. D. Tynan. Sleep-wake states in human infants: Profiles from motility monitoring. Physiol. Behm'.. in press, 1979. 8. Thoman, E. B., S. P. Waite, D. T. DeSantis and V. H. Denenberg. Ontogeny of sleep and wake states in the rabbit. Anita. Behav. 27: 95--106, 1979. 9. Waite, S. P., D. DeSantis, E. B. Thoman and V. H. Denenberg. The predictive validity of neonatal state variables in the rabbit. Biol. Behav. 2: 249-261, 1977. 10. Zarrow, M. X., V. H. Denenberg and C. O. Anderson. Rabbit: Frequency of suckling in the pup. Science 150: 1835, 1965.