The EEG and behavioral continuum of the crocodilian, Caiman sclerops

Electroencephalography and Clinical Neurophysiology, 1973, 34 : 521-538 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

521

THE EEG AND BEHAVIORAL CONTINUUM OF THE CROCODILIAN, CAIMAN

SCLEROPS 1

W. F. FLANIGANJR., R. H. WILCOXAND A.

RECHTSCHAFFEN

Sleep Laboratory, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Accepted for publication: October 17, 1972)

In studies of mammals and birds, limited Mammals vary widely in when, where, and how they sleep. Nevertheless, certain common attention has been given to distinguishing features ordinarily define their behavioral sleep : between sleep defined behaviorally and sleep (a) stereotypic or species-specific posture; (b) defined electrophysiologically. The two generally behavioral quiescence; (c) elevated behavioral correlate well enough so that the electrophysiothreshold; and (d) rapid state reversibility with logical signs are typically used as indicators of relatively intense stimulation. This sleep in behavioral sleep. The gross differences in brain mammals is accompanied by two alternating morphology in non-mammals should alert one electrophysiological patterns: (a) synchronized to dissociations of the familiar mammalian coror slow wave sleep (SWS), characterized by a relations (Broughton 1971). Therefore, clear relatively low frequency, large amplitude electro- distinctions between behavioral and electroencephalogram (EEG) ; and (b) desynchronized physiological measures will be maintained in our or "paradoxical" sleep (PS), differentiated by a study of the caiman, with precedence given to the rapid shift to a relatively high frequency, low behavioral definition of sleep. After all, the elecvoltage EEG 2. Although species differences do trophysiological variables gained their definioccur, PS is typically accompanied by eye move- tional value only from correlations with the ments, lowered neck muscle tone, myoclonic behavior. To then reify the former as the defining twitches of the extremities, and irregularity in variables would invite confusion about what is most universally known as sleep. heart and respiration rates. Sleep has been investigated in three of the four Behavioral sleep and its electrophysiological correlates have also been reported in four orders surviving orders of reptiles. Reported results vary of birds (Klein et al. 1963 ; Ookawa and Gotoh widely and very likely reflect not only species 1964, 1965; Rojas-Ramirez and Tauber 1970; differences, but also variations in methodological Berger and Walker 1972; Van Twyver and rigor. Behavioral sleep (as defined above) has Allison 1972). Although some species differences been described in three species of chelonians are apparent, behavioral sleep has been typically (turtles and tortoises) (Herman et al. 1964; reported to be correlated with an increase in high Vasilescu 1970; Flanigan 1972) during which voltage, slow wave EEG activity. Epochs of EEGs (polymorphous and irregular in form) mammalian-like PS in birds have been described declined in frequency, although mammalian-like prolonged epochs of SWS were not recorded. as extremely short (3-15 sec). Vasilescu (1970) suggested that mammalian-like 1 This research was supported by Grants MH-4151 and PS may be present in the European pond turtle, K5-MH-18,428 from the National Institute of Mental Health, but PS has not been recorded in other chelonians U.S. Public Health Service to Dr. Rechtschaffen. Reprint requests should be sent to W. F. Flanigan, (Herman et al. 1964; Flanigan 1972). Apparently, University of Chicago Sleep Laboratory, 5741 South Drexel lizards and a species of Python also exhibit Avenue, Chicago, I11.60637. behavioral sleep which is accompanied by EEG 2 Monotremes have been reported not to show PS slowing (Tauber et al. 1966, 1968 ; Peyrethon and (Allison and Goff 1968; Allison and Van Twyver 1969).

522 Dusan-Peyrethon 1969), although lowered arousal thresholds have not been specified for chameleons (Tauber et al. 1966), and clear indications of rapid state reversibility are lacking in all lizard and snake studies. Prolonged epochs of mammalian-like SWS have not been recorded in these reptiles; however, two species of lizards reportedly show some of the characteristics o[" mammalian-like PS (Tauber et al. 1966, 1968). Within the order Crocodilia, preliminary, shortterm investigation of juvenile Caiman sclerops has not disclosed the existence of mammalianlike SWS or PS (Rechtschaffen et al. 1968). Behavioral sleep and mammalian-like PS, however. have been reported for a specimen of Caiman latirostris (Peyrethon and DusanPeyrethon 1969). Not all of the above citations should be taken as highly generalized fact. As we shall find in the caiman, the description of sleep behavior and electrophysiology in Reptilia may be highly dependent upon duration and conditions of recording, the monitoring of several parameters simultaneously to aid in the interpretation of single parameters, and the liberty one takes in interpreting suggestive observations. Crocodilians are a logical choice for sleep studies within the class Reptilia. An ancient common ancestry relates birds more closely to crocodilians than to any other major group of vertebrates (cf Romer 1966, p. 166). Moreover, crocodilians and birds may be more closely related to mammals than to any other living vertebrates (Reig 1967, 1970). The present investigation, therefore, was undertaken to determine whether the crocodilian Caiman sclerops exhibits behavioral sleep, and, if so, the electrophysiological correlates of this behavior. MATERIALS AND METHODS

Ten specimens of Caiman sclerops (C. crocodilus) of indeterminate sex, 49-90 cm in length, approximately 2-5 years of age as estimated by their lengths, and 357-2,570 g in weight at the time of electrode implantation were studied. Animals were maintained on a diet largely of horse heart. Recording electrodes were implanted using continuous hypothermia (12-15°C) alone or in combination with 1-2 mg/kg of Flaxedil prepared

w.F. FI,ANIGANJR. et al. in 1 mg/ml solution. Post-operative behavioral recovery from surgery generally took place within 1-2 h and survival was excellent. Gold plated screw electrodes were placed, following the nomenclature of Crosby (1917), bilaterally in contact with the lateral and medial cortex, dorsal hippocampal cortex, optic tectum, olfactory bulbs and cerebellum. Electrode placement was guided by comparison with specially prepared and preserved specimens with skulls similar in size to those of the experimental animals. When half of the skull was carefully removed at the saggital midline thereby exposing half of the brain in preserved specimens, the three-dimensional coordinates of brain surface electrode placements could be readily determined. A check of electrode positions in 2 sacrificed experimental animals indicated that this technique was very reliable. Bipolar stainless-steel electrodes (1 mm tip separation), insulated except for l mm at the tip, were oriented for areas below the lateral ventricles, and for the ventral pontine tegmentum and ventral bulbar tegmentum of one of the largest specimens. Histological verification was not made, although placements were determined using coordinates derived from preserved specimens. Gold plated screws were placed in the supraorbital bone for electro-oculographic (EOG) recordings. Platinum wires (0.013 cm diameter) were snugly wrapped around extraocular muscles, including the superior recti and superior obliques, in 2 specimens. Stainless-steel wires were inserted in the upper jaw muscles (protractor pterygoideus) and forelimb muscles, and silver plates, braided stainless-steel wire, or Grass gold plated cup electrodes were inserted m nuchal muscles for recording electromyograms (EMGs). Electrode leads were soldered to a miniature connector cemented to the skull. Strain gauges were wrapped around the lower throat and the abdomen to measure palpitation and respiration, respectively. Palpitations appear to correspond with sniffing behavior (Huggins et al. 1968). Good quality recordings could be made for 1 2 months before connector plugs became detached from the skull. Recordings were made on an Offner Model T polygraph at paper speeds varying from 1 to 100 mm/sec. To discourage facing away from the observer and a motion

EEG AND BEHAVIOR OF THE CAIMAN

picture camera, caiman, who rarely locomote if undisturbed, were confined to a 60 cm long channel set just wider than the animal. The recording tank had a glass front and gloss-white sides and back. It was located in a soundattenuated room, electrically shielded, and tilted slightly forward to facilitate both observation through a one-way glass from outside the room and time-lapse photographic recording from inside the room. A 200 watt bulb, positioned for maximum photographic contrast, continuously illuminated the recording tank. Prior to implantation and between studies, the animals also received continuous illumination. Water, maintained at a temperature of 25-28°C, covered the tank floor to a maximal depth of about 3.5 cm. No recordings were used which were obtained prior to 5-7 days of post-operative recovery and habituation to cage and cable. Normative studies were conducted for 6-12 consecutive days following post-operative recovery and/or before the anticipated degradation of recording quality. Animals were fully aroused at the onset of each study, but not disturbed thereafter except to proffer food (at least weekly), add water, or replace strain gauges. The recording tank was not cleaned during a study. Arousal thresholds to visual and/or cutaneous stimulation were evaluated. Visual stimulation consi s ted of an experimenter quietly approaching the recording tank, and lowering a wood rod toward the animal's snout. Undoubtedly, some vibratory stimulation to which waking caiman are extremely sensitive (Rechtschaffen et al. 1968) accompanied this sequence. Cutaneous stimulation involved touching the dorsal surface of the animal with the rod using taps of increasing intensity. Stimuli and responses were also observed by a second experimenter and coded onto the electrographic record. Recordings were procured according to one of two schedules: (a) continuous recordings were made at paper speeds of I 10 mm/sec with higher speed (5-30 mm/sec) 60-see samples once every 10 min; or (b) 60-sec samples were obtained every 10 min at speeds of 15-30 mm/sec. Timelapse photographs were taken at the onset of a sample or 20 sec into the sample period. Photographs recorded both postural behavior and ocular changes. Visual observations were made

523 for 1-2 h intervals or up to 24 consecutive hours with 3X magnification binoculars. Postural and ocular variations were coded onto the electrographic record. To study the effects of prolonged behavioral arousal, 4 animals were kept alert by chronic stimulation for 24 h and 1 animal for 48 h; baseline recordings were compared with post-stimulation sample recordings of 36 h. Periodic 1 h behavioral observations were made during the post-stTmulation periods. Criteria of arousal during chronic stimulation included obligatory movements and/ or locomotion, and the maintenance of both eyes fully open with nictitating membranes retracted. Arousal was induced by stroking, lifting and handling (mouths were taped shut). To minimize stress, stimulation was given only as required to produce the arousal criteria. Heating and cooling studies were conducted on 3 animals for 5 or 24 h with continuous recordings; 24-h baseline and post-treatment records were also obtained. Continuous visual observations were made during each treatment and for a 24 h period thereafter. During heating, body temperatures were kept at 35 36°C for 5 h in caiman M-3 and for 24 h in caiman L-3 by means of a thermostatically controlled room heater. Higher body temperatures were not induced to preclude degrading of caiman body fat (Fromm et al. 1957). Continuous hypothermia was induced in caiman M-5 to the point of immobility (11-12°C) for 5 h. Caiman L-3 was cooled over a 24-h period using the recording room air conditioner. Body temperature was maintained at 14°C, which successfully avoided hypothermic immobility. Pre-treatment body temperature was restored within 2 3 h following heating or cooling. Postural shifts, ocular changes, EEG "spike" activity, respirations, and throat palpitations were tabulated from both continuous recordings with direct observations and record samples with accompanying time-lapsephotographs 1.Samples proved to be very adequate representations of continuous recordings due to infrequent variations in caiman behavior and electrophysiology. Accordingly, most quantitative data were derived Spikes were counted only when their amplitude was at least 3 times that of the background EEG.

524 from samples. Qualitative data, however, were based on continuous recordings and observations. RESULTS

Behavior

Although mostly languid, the caiman is capable of a wide continuum of animation which may be described by four characteristic postures. Posture 1. Limbs were flexed, body was raised from the tank floor, head and neck were elevated, and eyes were open (Fig. 1). Animals responded immediately to stimulation with escape locomotion, attack, or pre-attack threat behaviors (loud hissing and snorting with mouth fully open, jaw snapping, and abortive strikes, often while slowly moving backward). Respiratory rate averaged 3.5/rain, but ranged as high as 10/min. Throat palpitations occurred irregularly, averaged 4/min, and could be absent during both threat and attack behaviors. Posture 2. Limbs were flexed but the body rested on the tank floor, and head and neck assumed varying degrees of elevation (Fig. 2).

w.F. FLANIGANJR, et al. Eyes were usually open, although, at times, some lid closure occurred shortly before a more relaxed posture was assumed. Response to stimulation was somewhat less aggressive or defensive, and croaking-like vocalizations sometimes occurred. Usually, an increaseinthroatpalpitation followed stimulation. On occasion, the presentation of food elicited approach behavior. Respiration declined to an average of 2/rain, and palpitations were at maximal levels and ranged from 16/min to 23/min, with rates of 50 70/min not uncommon, particularly when Posture 2 followed Posture 1. Posture 3. Limbs were slightly flexed with one, two, or three legs bent backward approximately parallel to the horizontal body axis and often positioned against the body (Fig. 3). The head was lowered and rested on the tank floor with nostrils above or below the water surface. Eyes might be open, but were more likely to be closed the longer this posture was maintained. Small head and limb movements occurred at irregular

Fig. 3. Caiman in Posture 3, Fig. 1. Caiman in Posture 1.

Fig. 2. Caiman in Posture 2.

Vig. 4. Caiman in Posture 4.

525

EEG AND BEHAVIOR OF THE CAIMAN

intervals when this posture was continuously displayed over several hours or days. Threat or escape responses to cutaneous stimulation were somewhat delayed and very sluggish, especially after both eyes had remained closed for 30-60 min or more. Both respiration and palpitation increased with stimulation. In the absence of stimuli, respiratory rate varied from 0.4/min to 1.3/min, and throat palpitations ranged between 0/min and 7/min. Both respirations and palpitations appeared at irregular intervals and were often shallow; m a x i m u m rates and amplitudes coincided with eyes opening. Two to 3 min intervals between respirations were not unc o m m o n when this posture had been maintained for several days. Posture 4. All limbs were bent backward and usually positioned against the body, the head rested on the tank floor, and eyes were closed (Fig. 4). Body musculature seemed totally relaxed. The threshold for behavioral arousal was substantially elevated after the retention of this posture for about 30 min, often permitting an experimenter not only to enter the recording room, but also to pick up and hold an animal for several seconds before its eyes opened and it attempted to escape and/or exhibited threat behavior. These behaviors were initially poorly coordinated and very sluggish. Respiration and palpitation increased with stimulation. When left undisturbed, respiration was shallow and varied from approximately 0.2/min to 0.4/min; inter-respiratory periods averaged 3 min. Throat palpitations were shallow and ranged from 0 to 3/ min, but were usually absent. All four postures could be seen only when an animal had been left undisturbed for a number of days (Fig. 5 and Table I). Postural changes usually followed sequences such as P1---,P2~ P31 -* P3 z ~ P3 3 -* P4 -~ P3 3 --* P3 2 -* P31 - . P2 ~ P31 --, P3 2 --, P3 3 - . P4... in an irregular, cyclical manner 1. Posture 1 occurred only in response to stimuli ; Posture 2 rarely appeared in the absence of obvious stimulation or spontaneous movement. Posture 4 appeared only after prolonged maintenance of Posture 3, and with increasing frequency and longer durations when animals were undisturbed; it was spontaneously i P31 - 2 Posture 3 with one, two or three limbs extended backward. =

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Fig. 5. Long-term (165 h) variation in behavioral postures (average and range) and cortical spiking (average_ standard deviation) of caiman L-4. Dotted lines denote equipment failures. Abbreviations: 3~-3= Posture 3 with one, two or three limbs bent backward. terminated with eyes opening and then the reassumption of Posture 3. Although posture durations varied between and within caiman, Posture 1 typically gave way to Posture 2 shortly after an experimenter left the recording room. When animals were left undisturbed for 6-12 days, Posture 2 comprised 0.7-6~/o (variations between animals), Posture 3 comprised 6 2 - 9 9 ~ , and Posture 4 comprised 0-32~o of total recording time. In all instances, more than 5 0 ~ of recording time was spent in Postures 3 and 4. Posture 4 appeared in some animals only after a week of non-disturbance and never appeared in others. The presence of personnel even in nearby rooms or on the floors below impeded its adoption. Posture 4 appeared most often when direct observations were discontinued, or over weekends when the laboratory building was for the most part deserted. Seasonality did not appear to affect its appearance.

EEG patterns and electrophysiological data With the exception of variations in the inci-

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FABLE I Long-term behavioral and electrophysiological changes in caiman. Posture

Number of epochs

Average epoch duration

Percent total time

Average number spikes

Average respiration

Average throat palpitation

7 t) 11! 5 13 4

1.~ 0.4 0.2

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Percent tune eyes closed

(rain)

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0.7 97.9 1.3

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35.6 21.3 55.0 1550.0

5.8 45.1 17.0 32.0

0 84 94

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Data are typical of all caiman studied, but are derived from animals with (a) clearest long-term electrographic and photographic recordings, and (b) a minimum number of equipment failures (i.e., a minimum amount of disturbance of animals by experimenters). * Continuous electrographic and photographic recordings for 210 h. ** Continuous electrographic and photographic recordings for 165 h.

dence of spikes (to be described later), alternations in caiman EEG patterns were minimal and paralleled postural changes only in the sense that frequencies and amplitudes gradually declined slightly as Postures 3 and 4 became most prevalent (Fig. 6 and Table II). Only with induced or spontaneous arousal was a relatively abrupt change seen. EEGs were polymorphic and mixed in frequency. Olfactory fusiform activity of 17 21 c/sec was reflected in cortical leads. During Posture 1, EEG amplitudes ranged from 2 to 63 llV, bipolar cortical deviations registered predominant frequencies of 4-11 and 19-23 c/sec, and the optic tecti were dominated by 5 13 and 22-26 c/sec activity. When undisturbed for hours or days and while behavioral quiescence (Postures 3-4, eyes closed) prevailed, EEG voltages ranged from 2 to 33 I~V. Bipolar cortical EEGs were dominated by 2 5. 7 10 and 19--23 c/sec activity. Frequencies of 3 9 and 17 25 c/sec predominated in bipolar tectal recordings. Epochs of sustained, high voltage. slow wave activity comparable to mammalian SWS were not seen at all. Nuchal and jaw EMG levels primarily rellected obvious head elevations and jaw positions. Hypotonia, but neither phasic EMG suppressions

nor epochs of atonia, accompanied sustained behavioral quiescence. No potentials indicative of eye movemems (EMs) were recorded from EOG electrodes when both eyes were closed during Posture 3 or 4, Potentials were recorded from EOG electrodes when eyes were open, but they usually did not serve to differentiate EMs unambiguously from nictitating membrane wipes, lid movements, or eyeball retractions. Direct visual observation of the animal, therefore, was the primary determinant of EM activity. Although EMs were rare, two types of conjugate EMs were observed: (a) slow rolling horizontal movements when both eyes were open and Posture 1 or 2 was maintained (recorded only during the first few hours following placement in the recording tank) ; and (b) short epochs (4 7 sec) of rapid horizontal movements accompanied by nictitating men> brane wipes and/or eye blinks upon opening eyes during periods of behavioral quiescence {Fig. 7). On the basis of our electrographic monitoring and visual observation, we conclude that the caiman does not show the electrophysiological characteristics of mammalian-like SWS or PS. The most dramatic feature of the caiman EEG was large amplitude (up to 150 200 /~V),

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Fig. 6. Comparison of electrographic data from caiman L-4 during behavioral arousal and quiescence. EEG recordings are bipolar ; calibrations are for 50 pV. The only dramatic difference in the cortical EEG between the two states is the presence of high voltage spikes during quiescence. Nuchal EMG level is diminished but not abolished during quiescence. Respirations are correlated with strain gauge deflections. TABLE II Predominate cortical EEG frequencies and amplitudes. Caiman

Behavioral arousal*

Behavioral quiescence**

M-2

5 ~ c/sec @ 3.6-13.6/~V 19-22 c/sec @ up to 1.8 #V

3 c/sec @ 4.4 pV 7-8 c/sec @ 3.1-5.6 #V 22-23 c/sec @ up to 1.3 pV

M-5

5-8 c/sec @ 10-53 #V 9-11 c/sec @ 17~3/~V 14 c/sec @ 3.3-10 pV

2-4 c/sec @ 20-23 pV 6-8 c/sec @ 10-33 #V 13-15 c/sec @ 3.3-17 pV

L-1

4-10 c/sec @ 6.8-17 pV 19-22 c/sec @ up to 3.5 ;iV

4-5 c/sec @ 4.4-13.6 ,uV 8-10 c/sec @ 2.3-9.1 pV 21-25 c/sec @ up to 4.2 pV

L-4

5-6 c/sec @ 15.6-28.1 #V 8-11 c/sec @ 9.4-28.1 #V 23 c/sec @ up to 6.3 #V

2 c/sec @ 12.5-21.8 pV 7-10 c/sec @ 6.3-15.6 pV 21-23 c/sec @ up to 4.7 #V

Data are representative of all caiman studied. * Animal in Posture 1. ** Animal in Posture 3 or 4 with eyes closed.

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EEG AND BEHAVIOR OF THE CAIMAN

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arrhythmic "spikes", which occurred monophasically (50 msec duration) or polyphasically, unilaterally or bilaterally, and in or 180° out of phase (Fig. 8). Telencephalic spikes, which were most prevalent, were accompanied by synchronous spikes most frequently in the optic tecti, and inconsistently from dorsal pontine and bulbar tegmentum derivations. Large amplitude cortical spiking was also synchronous with spikes recorded from electrodes implanted in extraocular muscles. In addition, during particularly high rates of cortical spiking, correlated spike activity was seen in olfactory bulb and jaw muscle derivations. Spikes did not appear in neck or forelimb muscles and only very rarely were they obtained from cerebellar placements. Noncortical spiking in the absence of cortical spikes was not found with the exception of tectal spikes which accompanied eye blinks and/or nictitating membrane wipes. Spike incidence decreased or transiently

vanished with induced or spontaneous arousal (Fig. 9), during periods of respiration (Fig. 10), and immediately following rhythmic discharges and "spike bursts" (see below). Overall, average spike rate increased as a function of the duration of non-disturbance and as an animal traversed the behavioral continuum from Posture 1 to Posture 4, although wide variation accompanied a continuation of Posture 3 (Fig. 5 and Table I). Spiking increased when eyes were closed in Posture 2; no consistent relationship was found between spike rate and eye closure for Posture 3 or Posture 4. During quiescence, all animals exhibited similar arrhythmic patterns of spiking: spike incidence tended to increase gradually to a relative maximum over several minutes only to be followed by a span of about 10-30 sec marked by very infrequent spiking before the cycle was repeated. Six animals, however, also displayed brief periods of rhythmic spiking (1-3 spikes/sec)

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(Fig. ll) and intensive spiking or spike bursts (5-10/sec) (Fig. 12) with epoch durations of about 25-100 and 1-3 sec, respectively. Both patterns were followed by intervals all but devoid of spikes. Rhythmic spiking and spike bursts occurred only during Posture 3 with two or three legs bent backward and were extremely rare during a normative study. Their appearance could be limited to a single epoch (Fig. 11) or they might continue in an irregular fashion for up to an hour or more at a time. On occasion, they were recorded when both eyes were closed, but normally appeared in conjunction with periods of eye openings. Although respirations and/or palpitations (ordinarily correlates of heightened arousal) accompanied both patterns of spiking, responses to visual and cutaneous stimulation were extremely sluggish. Cutaneous stimulation did not eliminate subsequent epochs of intensive spiking or spike bursts unless an animal was induced to assume Posture t or 2.

Prolonyed behavioral wakelulness During periods when wakefulness was enforced by stimulation, spontaneous activity varied irregularly. At times, constant handling was required to keep both eyes open and to induce locomotion; at other times there was spontaneous locomotion which was invariably preceded by several rapid respirations. Respiratory rates averaged about 2/min, but occasionally rose as high as lO/min. Urination and vocalization were particularly frequent during the initial 12 h of stimulation. Following cessation of stimulation and cable reconnection, animals usually assumed Posture 3 (with two or three legs bent backward) immediately or within 2 5 h. and remained virtually immobile with their eyes closed for about the next 24 36 h. Episodic eye openings appeared during the remainder of the 36 h recovery period. Spike bursts appeared in 3 animals with initial latencies between 5 and 23 h. None of these

EEG AND BEHAVIOROF THE CAIMAN

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cortical spiking above baseline levels (Table III) during subsequent recovery. Recordings for caiman M-3 were of rather poor quality initially and became much degraded during recovery from prolonged arousal; accordingly, spike counts may be unreliable for this animal. The overall results, of course, could be a chance effect stemming from the small sample size of 5 animals. However, in view of similar data for the turtle and the tortoise (Flanigan, in preparation), we feel that spike rebound following prolonged behavioral arousal is a reliable result.

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F i g . 11. Rhythmic spiking epoch in caiman M-4 during Posture 3. Tracings are continuous. EEG recordings are bipolar; calibrations are for 25/~V. When spiking becomes very intense, throat palpitations (indicated by strain gauge deflection) may occur without a correlated reduction of spike activity. Note that spikes appear in the olfactorybulb tracing when cortical spike activity becomes very intense.

animals had exhibited spike bursts in their preceding baseline recordings. Four of 5 animals who experienced enforced behavioral arousal for 24 or 48 h exhibited a substantial increase in

During the heating and the post-heating periods, caiman usually maintained Posture 3 with two or three legs bent backward. The animal heated for 24 h adopted Posture 4 (eyes closed) for 90 rain in the immediate post-treatment period. This same animal also displayed yawning behavior toward the end of heating and during the post-treatment period just after emerging from Posture 41. Although only a trend toward increased spiking was seen during and after shortterm heating, heating for 24 h produced a 227 increase in spike rate above baseline. Spike bursts appeared both during heating and in the immediate post-treatment period (none were observed in the preceding baseline recordings). During 24 h of cooling, the animal remained primarily in Posture 2 or with both head and body resting on the tank floor and limbs slightly flexed. Both eyes were usually open. Cooling 1 This was the only incidence of yawning observed in cable-connected animals.

TABLE III Spike activity before and after prolonged behavioral arousal. Caiman

Duration of arousal

A Pre-stimulation average spikes/min

B Post-stimulation average spikes/min

Percent (B-A)/tA)

M-2 M-3 L-l** L-3 L-4

24 h 24 h 24 h 24 h 48 h

1.0 4.0 9.2 6.0 7.5

8.1" 1.9 12.7" 8.7* 11.7

+710.0

* Spike clusters were recorded during these periods. ** Duration of pre- and post-stimulation recordings was 34 h.

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Fig. 12. Spike burst in caiman L-3 with both eyes open while in Posture 3. EEG and EOG recordings are bipolar. Calibrations are for 25/~V,

diminished spike incidence to 71% below baseline. DISCUSSION

It is always precarious to compare the behaviors of species phylogenetically distant from one another ; similarly appearing behaviors may actually serve different functions in different species. As we do not yet understand the functional significance of sleep, we remain fixed at essentially a behavioral level. Proceeding at this level, there is no question that the behavior we have observed in the Caiman sclerops bears a definite resemblance to what is called sleep in mammals and birds. Posture 4 and Posture 3 with two or three limbs bent backward (see below) meet the criteria for behavioral sleep outlined earlier. Over the course of several days and in the absence of obvious stimulation, caiman behavior incrementally approached and culminated in a state typified by total immobility, a characteristic posture, continuous eyelid closure,

maximum relaxation of body musculature, low metabolic activity and substantially diminished behavioral responsiveness to stimulation. State reversibility was readily obtained by stimulation. Chronic stimulation for 24 or 48 h facilitated the early appearance of caiman sleep compared to the several days of quiescence normally required for its appearance in the untreated animal--~an effect similar to sleep rebounds in sleep-deprived mammals. During prolonged application of heat, the rapid adoption and maintenance of Postures 3 and 4 suggest that sublethal elevation of body temperature was soporific for the caiman. A similar phenomenon may occur during sun basking in a natural environment. Some animals exhibited behavioral sleep bul were never observed in Posture 4. In these caiman, behavioral sleep was unambiguously present after Posture 3 with two or three limbs bent backward (Posture 32 3) and eyelid closure had been maintained for several hours; the appearance of Posture 3 2 - 3 closely paralleled the temporal pattern of Posture 4 seen in other caiman during

EEG AND BEHAVIOR OF THE CAIMAN

normative recordings, for it was initially displayed only after an animal had been undisturbed for several days, and then reappeared in an irregular, cyclical fashion. Behavioral sleep also coincided with Posture 32- 3 following prolonged behavioral wakefulness; all animals rapidly assumed and maintained Posture 32-3 during recovery, but none exhibited Posture 4. On occasion, physical confinement precluded the adoption of Posture 4 during behavioral sleep if a caiman entered a state of behavioral quiescence while remaining partially squeezed into a comer of the recording tank or positioned against a tank side. When this occurred, only two or three limbs could be extended backward without gross body movement (which did not take place so long as Posture 3 was maintained). Formal study of thresholds for behavioral responsiveness to stimulation by varying the intensity of a single stimulus parameter was difficult with the Caiman sclerops. Animals rapidly adapted to the presentation of stimuli such as light, sound, somatic electric shock, or even pulling the cable connected to the head, so that graded responses to stimuli of different intensities soon disappeared. This resulted in caiman exhibiting behavioral and/or electrophysiological changes only at maximum levels of stimulation irrespective of the posture displayed at the time of stimulus presentation (Posture 1 was an exception). In view of this "all or none" reaction to unidimensional stimuli, variation in behavioral responsiveness to stimulation was limited to qualitative observations° Such observations, however, left no equivocacy as to the substantial decline in arousability which accompanied behavioral sleep. For example, caiman responded at once if stimulated during Posture 1, but when Posture 4 was adopted, animals could be picked up and handled for several seconds before any observable response to stimulation appeared. The caiman differs from mammalian and avian forms not in the range of its active-quiet behaviors, but in its speed of transition from the active to quiet portions of the range. In contrast to therapid shifts from wakefulness to sleep which can occur within a matter of minutes in the mammal or bird, it normally takes a caiman at least several days in a quiet, undisturbed environ-

533 ment to progress from Posture 1 to Posture 41, These reptiles seem to lack a "switching mechanism" to take them quickly from a state of unambiguous wakefulness to one of unambiguous sleep. In view of the common heritage shared by modem crocodilians and birds, and, perhaps, by crocodilians, birds and mammals, it could be speculated that the abrupt transitions from wakefulness to sleep found in mammals and birds may represent independently evolved temporal compressions of a behavioral continuum not unlike the one now seen in the caiman. However, the apparent lack of a fast switching mechanism has not been reported for noncrocodilian reptiles (Herman et al. 1964; Tauber et al. 1966, 1968; Peyrethon and Dusan-Peyrethon 1969; Vasilescu 1970; Flanigan 1972) which suggests that the absence of a switching mechanism in the caiman may represent a relatively recent adaptation to environmental demands. Many previous investigations of sleep and wakefulness in reptiles, though, appear to have been concerned more with whether or not an animal exhibited electrophysical events which resembled the electrophysiological correlates of mammalian behavioral sleep than with a thorough delineation and description of behavioral states and associated electrophysical changes. As caiman require a number of days and, at times, up to a week before displaying behavioral sleep, we can not over-emphasize the importance of conducting studies which employ continuous and protracted observations, and which utilize an exceptionally quiet, undisturbed environment before statements are made regarding sleep in a particular species. Caiman EEGs in the present investigation generally resembled those described by Parsons and Huggins (Parsons and Huggins 1965a, b; Huggins et al. 1968). The EEGs paralleled those of mammals and birds only in the sense that a small gradual overall decline in frequency accompanied behavioral quiescence and sleep. The amplitudes of dominant frequencies declined slightly in contrast to the marked elevation of voltage levels which accompany SWS in mam1 Similar behavior has been observed in this laboratory in 2 specimens of the American alligator (A!ligator mississipiensis) of size and weight similar to the caiman used in these studies.)

534 mals and birds. Although brief epochs of rapid EMs were recorded during behavioral quiescence, they were correlated with a transient increase in EEG frequency and amplitude, throat palpitation, and eye openings, i.e., apparently transient arousals. We were unable to confirm the report of PS in a single specimen of Caiman latirostris (Peyrethon and Dusan-Peyrethon 1969). Although the caiman lacks the typical mammalian EEG correlates of behavioral sleep, this does not necessarily mean that the sleep of this reptile is not analogous to that of mammals and birds. The mammalian sleep EEG serves as a phenomenological not a functional definition of sleep (Koella 1967), which is perhaps most apparent when behavior and EEG are dissociated following treatment with such pharmacological agents as atropine sulfate or physostigmine (Wickler 1952 ; Bradley 1968). Mammalian electrophysiological indices of sleep occur when the appropriate cytoarchitecture is present, and it is likely that caiman fail to exhibit mammalianlike sleep EEGs due to the absence of appropriate physiological substrates. In the domestic cat, the slow wave EEG activity of behavioral sleep disappears following decortication (Jouvet 1961) although behavioral sleep continues to occur, whereas both behavioral and electrophysiological signs of sleep are drastically reduced when raphe nuclei in the brain stem are destroyed (Jouvet 1967). Crocodilians do not possess a mammalian-like neocortex, but distinct raphe nuclei may exist in the brain stem (Broughton 1971) and, accordingly, the caiman might be expected to display behavioral sleep and not SWS. A deficiency in particular electrophysical sleep correlates, therefore, does not necessarily imply the absence of the functional sleep state itself, nor does it imply that behavioral sleep in the caiman (or any other non-mammalian form) is not analogous (or, perhaps, homologous) to similarly appearing behaviors in mammals. The presence and often dramatic elevation o1" spike activity in caiman EEGs was the major electrophysiological change found associated with behavioral quiescence and sleep in this reptile. Intensification and short latency of EEG spiking following enforced wakefulness also imply that sleep deprivation is possible in the

w. t~. FLANIGANJR. el al. caiman. The absence or diminution of spiking which accompanied respiration during behavioral quiescence may represent a transient or incomplete arousal sufficient to elevate the nostrils above the water surface when the animal sleeps in normal surroundings. Large amplitude, arrhythmic spikes seem to be a common attribute of the EEG of reptiles and at least some amphibians. Generally, they have been described in association with behavioral quiescence or sleep. 'roads of the genus Buff) exhibit spike activity during behavioral sleep (Segura 1966). Spikes have also been reported during quiescence in the Python and a species of chameleon (Tauber et al. 1966: Peyrethon and Dusan-Peyrethon 1969): they are continually present but greatest when eyes are closed in the 19uana (Peyrethon and Dusan- Peyrethon 1969). However, in another genus of iguanid lizard, the Ctenosaura, spike rate has been reported to increase with stimulation (Tauber et al. 1968), and in a Caiman latirostris they have been noted to rise with stimulation and to decline when eyes are closed (Peyrethon and Dusan-Peyrethon 1969). In preliminary studies of the eastern box turtle, Terrapene carolina, tetencephalic EEG spikes have been found to accompany behavioral quiescence (neck and limbs relaxed, eyes closed) and to disappear with spontaneous or induced behavioral arousal (Flanigan 1972). The presence of spiking during behavioral quiescence in many reptilian forms (including crocodilians and turtles which belong to probably the most widely separated orders of reptiles) provides strong inferential support for hypothesizing that ancient reptiles including those ancestral to birds and mammals exhibited a similar elect rophysiological phenomenon which may have been retained in some manner by modern birds and mammals. Cortical as well as subcortical spike activity has been reported during sleep in a number of mammals and in birds. The most widely noted pattern is the so-called ponto-geniculate-occipital (PGO) spike activity which appears throughout the visual system and is coincident with the rapid EMs of PS in many mammals. PS in the cat is marked by spiking at the level of the pons and through most of the visual system (Mikiten et al. 1961 ; Bizzi and Brooks 1963: Brooks and Bizzi 1963: Mouret et al. 1963: Michel et al. 1964a)

535

EEG AND BEHAVIOR OF THE CAIMAN

including the extraocular muscles (Michel et al. 1964b; Rechtschaffen et al. 1971). Bursts of spiking in the lateral geniculate nucleus and striate cortex are concomitant with EM activity during PS in capuchin and squirrel monkeys (Perachio 1967). Among Old World primates, pontine spiking (Weitzman et al. 1965) and geniculate spiking (Berger 1967) are associated with PS epochs in rhesus monkeys, and geniculate and, occasionally, occipital spikes can be seen in the baboon during PS (Bert et al. 1970). Spiking has also been recorded from periorbital electrodes during PS in humans (Rechtschaffen et al, 1970). PS spiking which may or may not be related to PGO activity has been reported in the lateral geniculate and striate cortex of the owl monkey (Perachio and Linnstaedter 1970), in the posterior colliculus and, on occasion, the cortex of bats (Brebbia and Paul 1969), and in the cerebellum of the guinea pig (Pellet 1966). SWS is also marked by spike activity in a number of species. Perhaps the most intriguing reports are those of high voltage spikes from the hippocampus and other rhinencephalic structures which characterize SWS in both normal and decorticate cats (Jouvet 1962; Jouvet et al. 1959). Geniculate and cortical spike bursts have also been seen during SWS in the owl monkey (Perachio and Linnstaedter 1970), and bats have been noted to display collicular and cortical spiking during SWS (Brebbia and Paul 1969). In birds, EEG spike-like activity has been described in juvenile domestic chickens shortly after the onset of or during behavioral sleep (Ookawa and Gotoh 1965 ; Ookawa and Takagi 1968). If caiman spike activity is analogous to mammalian spike activity such as PGO spikes, it represents the only mammalian electrophysiological sleep correlate that we have found in this reptile. That the caiman spike may be analogous to the PGO spike is suggested by the association of telencephalic and extraocular spikes. It should be noted, however, that volume-conducted potentials from the brain may have contributed to the phasic electrographic activity recorded from caiman extraocular muscles, since only a thin membrane separates their eyes from brain tissue. Although most PGO activity normally occurs in PS, continuous spiking appears in cats given

parachlorophenylalanine or reserpine (Jouvet 1967 ; Dement 1969) which can be interpreted as the breakdown of some unknown spike-containing mechanism. Possibly the caiman has not evolved a mechanism which restricts PGO spikes to discrete periods of sleep. Preliminary drug studies, however, argue against an analogy with the PGO spike. Reserpine enhances PGO activity (Delorme et al. 1965; Jouvet 1967) in cats, but when given to a caiman (2 mg/kg, i.p.) it eliminated all spiking within a day. MAO inhibitors impede PS and PGO activity in mammals (Jouvet et al. 1965; Jouvet 1967). Pargyline (20 mg/kg, i.p.) injected into 3 caiman, however, elicited rhythmic spiking, spike bursts, and an overall increase in spike incidence within 24 h. Another possibility is that caiman spike activity may be analogous to SWS spike activity such as the limbic system spikes reported for the cat. In this connection it should be noted that the cortex overlying the lateral ventricles in the caiman is extremely thin, and that spikes recorded from the dorsal telencephalon may in fact stem completely or at least partially from volume-conducted potentials which originate in structures below the ventricles. At present, though, there is simply not enough" information to decide whether or not the caiman spike bears any definite relationship (topological or functional) to the spike activities correlated with mammalian and avian behavioral sleep. SUMMARY

Caiman were implanted with chronic electrodes in forebrain, midbrain, hindbrain, orbital cavities, and in jaw, nuchal and forelimb muscles. Strain gauges measured throat palpitations ("sniffing") and respiration. Recordings and photographs were obtained for 6-12 consecutive days. Four characteristic postures were associated with levels of behavioral arousal: (1) limbs flexed, body raised from floor, head and neck elevated, eyes open; animals responded immediately and aggressively to stimulation; (2) posture similar to above, but with trunk resting on floor; animals responded as above, but less aggressively; (3) limbs slightly flexed with one to three legs relaxed and extended backward against body, head

536

w.F. FLANIGANJR. eta/.

rested on floor, eyes were mostly closed ; responses to stimulation were sluggish, especially when eyes were closed ;(4) all limbs extended backward against body, head on floor, eyes closed ; animals could be handled for several seconds prior to eyes opening and movement (the latter was poorly coordinated and very sluggish). Posture 4 and+ in certain instances, Posture 3 denote behavioral sleep, i.e., motor quiescence, stereotypic posture, increased response threshold and rapid state reversibility. EEGs (polymorphic and irregular in form) were somewhat higher in frequencies and larger in amplitudes during behavioral arousal, but changes across the posture-arousal continuum were minimal. Neither epochs of slow wave nor paradoxical sleep were recorded. Large amplitude (up to 200 #V), arrhythmic spikes (50 msec duration) characterized caiman EEG and were most prevalent in the telencephalon. Spiking increased with behavioral quiescence and decreased or transiently vanished with induced or spontaneous arousal and respiration. Spike incidence substantially increased during recovery from 24 or 48 h of induced behavioral arousal. The association of spikes with quiescence and their apparent compensatory rebound after enforced arousal encourage the hypothesis that they are analogs of EEG spike activity reported to accompany mammalian and avian behavioral sleep.

RESUME LE CONTINUUM

EEG ET

COMPORTEMENTAL

DU

CROCODILE CAIMAN SCLEROPS

Des caimans ont 6t6 implant6s avec des 616ctrodes chroniques au niveau du dienc6phale, du m6senc6phale, du tronc c6r6bral post6rieur, des cavit6s orbitaires et au niveau des muscles de lajoue, de la nuque et des membres ant6rieurs. Des jauges de tension mesurent les palpitations de la gorge ("reniflements") et la respiration. Des enregistrements et des photographies ont 6t6 obtenus pendant 6 ~ 12 jours cons6cutifs. Quatrepostures caract6ristiques sont associ6es fi des niveaux comportementaux de vigilance:

(1) les membres flechis, le corps soulev6 du plancher, la t~te et le c o u e n 616vation, les yeux ouverts; les animaux r6pondent imm6diatement et de fation agressive fi la stimulation: (2) posture similaire 'h ce qui a 6t6 d6crit ci-dessus, mais le tronc reposant sur le plancher: les animaux r6pondent comme ci-dessus, mais de fa~on moins agressive; (3) les membres 16g6rement fl6chis avec une fi trois jambes d6tendues et 6tendues vers l'arri6re contre le corps, la t6te reposant sur le plancher, les yeux 6tant principalement ferm6s; les r6ponses a la stimulation sont paresseuses, sp6cialement lorsque les yeux sont ferm6s; (4) tousles membres sont 6tendus vers l'arri6re contre le corps, la t~te sur le plancher, les yeux ferm6s; les animaux pouvaient atre manipul6s pendant plusieurs secondes avant d'ouvrir les yeux et avant qu'un mouvement ne survienne (ce dernier 6tant real coordonn6 et tr6s paresseux). La posture 4 et dans certains cas la posture 3 d6notent le sommeil comportemental+ c'est-~-direla tranqui llit6 motrice+ la posture st6r6otyp6e, l'616vation du seuil de r6ponse, et la r6versibilit6 rapide de cet 6tat. Les EEGs polymorphes et irr6guliers ont des fr6quences et des amplitudes quelque peu sup6rieures au cours de l'6veil comportemental. mais les modifications tout au long du continuum posture-vigilance sont minimes. Aucune 6poque d'ondes lentes ni de sommeil paradoxal n'a 6t~ enregistr6e. Des pointes de grande amplitude (jusqu'fi 200 ltV) et arythmiques caract6risent I'EEG des caimans et pr6dominent principalement au niveau du t61enc6phale. Ces d6charges de pointes augmentent avec la tranquillit~ comportementale et diminuent ou disparaissent de fa~on transitoire avec l'6veil induit ou spontan6 et la respiration. La survenue des pointes augmente de fa~on importante au cours de la r6cup6ration de 24 ou 48 h d'6veil comportemental induit. L'association de pointes et de tranquillite et le rebound compensatoire apparent de pointes apr6s vigilance renforc6e sont en faveur de l'hypoth6se suivant laquelle ces pointes sont analogues ~ l'activit6 de pointes EEG d6critc comme concomitante du sommeil comportemental des mammif6res et des oiseaux.

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