- Email: [email protected]
Sleep and wakefulness in the green iguanid lizard (Iguana iguana)
Available online at www.sciencedirect.com
Comparative Biochemistry and Physiology, Part A 151 (2008) 305 – 312 www.elsevier.com/locate/cbpa
Sleep and wakefulness in the green iguanid lizard (Iguana iguana) ☆ F. Ayala-Guerrero ⁎, G. Mexicano Facultad de Psicología, Universidad Nacional Autónoma de México and UAEM, Mexico DF 04510, Mexico Received 27 October 2005; received in revised form 23 March 2007; accepted 26 March 2007 Available online 30 March 2007
Abstract The reptile Iguana iguana exhibits four states of vigilance: active wakefulness (AW), quiet wakefulness (QW), quiet sleep (QS) and active sleep (AS). Cerebral activity decreases in amplitude and frequency when passing from wakefulness to QS. Both parameters show a slight increase during AS. Heart rate is at a maximum during AW (43.8 ± 7.9 beats/min), decreases to a minimum in QS (25.3 ± 3.2 beats/min) and increases in AS (36.1 ± 5.7 beats/min). Tonical and phasical muscular activity is present in wakefulness, decreases or disappears in QS and reappears in AS. Single or conjugate ocular movements are observed during wakefulness, then disappear in QS and abruptly reappear in AS. Although these reptiles are polyphasic, their sleep shows a tendency to concentrate between 20:00 and 8:00 h. Quiet sleep occupies the greater percentage of the total sleep time. Active sleep episodes are of very short duration, showing an average of 21.5 ± 4.9 (mean ± SD). Compensatory increment of sleep following its total deprivation was significant only for QS. Reaction to stimuli decreased significantly when passing from wakefulness to sleep. It is suggested that the lizard I. iguana displays two sleep phases behaviorally and somatovegetatively similar to slow wave sleep and paradoxical sleep in birds and mammals. © 2007 Elsevier Inc. All rights reserved. Keywords: Active sleep; Iguana iguana; Quiet sleep; Reptiles
1. Introduction Mammals (Ruckebusch, 1963; Jouvet, 1967; Allison and Van Twyver, 1970; Freemon et al., 1971; Brebbia and Pyne, 1972; Prudom and Klemm, 1973; Mukhametov, 1990; Zepelin, 1994) and birds (Susic and Kovacevic, 1973; Schlehuber et al., 1974; Vasconcelos-Dueñas and Ayala-Guerrero, 1983; Mexicano et al., 1992) present two sleep phases. The first, called slow wave sleep (SWS), is characterized by brain activity of high amplitude slow waves. The second, known as paradoxical sleep (PS) or rapid eye movement sleep (REM), exhibits a low voltage fast frequency electroencephalographic pattern. This ☆
This paper is part of the 5th special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Carolina A. Freire (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of Knut SchmidtNielsen (1915–2007), a great mind in comparative physiology, with books translated into 16 languages, including Spanish and Portuguese, and Cicero Lima (1934–2006), journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. E-mail address: [email protected] (F. Ayala-Guerrero). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.03.027
sleep phase is usually further distinguished by the presence of eye movements, myoclonic twitches of the extremities, reduced muscle tone and autonomic variability. Most sleep studies have been performed in endothermic vertebrates. However, in recent years, the comparative approach towards an understanding of the brain has provided significant new insights. Reptiles appear to be most useful in this approach because of their ancestral relationship to mammals and birds. Sleep in these ectothermic vertebrates remains as a matter of controversy (Walker et al., 1973), since it has been determined almost exclusively by considering the electrical activity of mammalian brain as a prototype for comparative sleep studies. However, the absence of electroencephalographic signs of SWS or PS might imply only the absence of the appropriate neurophysiological generators of such activity but not necessarily of sleep. In contrast, it has been suggested that a number of common behavioral characteristics can be used to delineate sleep for any class of animals. These characteristics include: 1) the spontaneous assumption of a stereotypic specific posture, 2) the maintenance of behavioral immobility, 3) an elevated behavioral response threshold which may be reflected in the intensity of an arousing stimulus and/or in the frequency,
306
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
latency, or duration of a behavioral response, and 4) rapid behavioral state reversibility with stimulation (Flanigan, 1974). It has been reported that several reptile species belonging to chelonians (Vasilescu, 1970; Karmanova and Churnosov, 1972; Flanigan, 1974; Ayala-Guerrero, 1987), crocodilians (Peyrethon and Dusan–Peyrethon, 1969; Flanigan et al., 1973; Warner and Huggings, 1978) and lizards (Tauber et al., 1966, 1968a; Karmanova et al., 1971; Flanigan, 1972; Romo et al., 1978; Huntley, 1987; Ayala-Guerrero and Mexicano, in press) exhibit behavioral sleep. In the particular case of iguanid lizards two phases of sleep have been described in Ctenosaura pectinata (Tauber et al., 1966, 1968a; Ayala-Guerrero and HuitrónReséndiz, 1991), and Ctenosaura similis (Ayala- Guerrero and Vargas-Reyna, 1987; Huitrón-Reséndiz et al., 1992). However in Iguana iguana only one phase has been observed (Flanigan, 1973). Since genetic factors exist in expressing certain sleep characteristics (Liknowski et al., 1991), and I. iguana is, from the phylogenetic point of view, closely related to the other two lizards, it is probable that it also presents two similar sleep phases. 2. Materials and methods 2.1. Surgical and recording procedures Ten adult specimens of the lizard I. iguana of both sexes were used in this study. Stainless steel, electrodes insulated except for the tip were used. Electrodes were implanted, after a period of habituation in our laboratory, under Nembutal anesthesia (35 mg/kg i.p). After removing the superficial scales, the muscle mass overlying the parietal bones on each side was separated through a midline skin incision. Drill holes were made in the skull and the electrodes for chronic bipolar recordings of cerebral activity (EEG) were implanted bilaterally on the anterior and posterior telencephalon. Two electrodes were placed in the skull at the external and internal canthi of each eye for ocular activity recording (EOG). Muscular activity (EMG) and heart rate were obtained from the same pair of electrodes placed in the neck muscles. One screw (ground electrode) was placed in the skull approximately 2.5 cm anterior to the parietal eye. All electrode leads were soldered to a small connector fixed to the skull with acrylic cement. Seven days after postoperative recovery the lizards were placed in a shielded sound attenuated chamber constantly illuminated by a 60 W light bulb, in order to facilitate the observation through a one-way glass window. Chamber temperature during recording sessions varied between 25 and 30 °C. A po1ygraphical recording by animal, under constant visual observations, was taken continuously during 24 h on an 8 channel Grass model III-D electroencephalograph.
calculated. The mean duration and the total number of active sleep episodes over the 24-h period were obtained as well as their nycterohemeral distribution. Muscular activity measurements of each state of vigilance were made in reference to activity exhibited during active wakefulness, which was considered as 100%. Heart rate and frequency of ocular activity were extrapolated to 1 min. Values of states of vigilance obtained during the astronomical day (from 8:00 to 20:00 h) were compared to those of the astronomical night (from 20:00 to 8:00 h) through the Student's t-test. 2.3. Arousal reactions Behavioral response latencies during behavioral waking and behavioral sleep were measured in three additional specimens. Electrical stimulation consisting in dipolar pulses of 50 ms duration at 60 c/s was delivered to pairs of EMG electrodes. The voltage used was 2 V which was randomly applied during AW, QW, QS and AS. Behavioral responses consisted of a head movement accompanied by eyelid, limb and body movements. Experimental trials were conducted during 24 h/day for three consecutive periods or until 30 stimulus presentations during behavioral waking and 50 during behavioral sleep (30 in QS and 20 in AS) had been made. 2.4. Enforced waking These three specimens were also kept behaviorally awake for 48 continuous hours, and baseline recordings were compared with post-stimulation recovery recordings of 24 h. Enforced wakefulness was maintained by lifting, stroking and handling. 3. Results The lizard I. iguana (Fig. 1) displayed the following states of vigilance during the nycterohemeral cycle: active wakefulness (AW), quiet wakefulness (QW), quiet sleep (QS) and active sleep (AS). Each sate showed its own behavioral and electrophysiological features. 3.1. Behavioral and electrophysiological data during AW The iguanid stood on their four legs. The body was raised from the chamber floor, head and neck were elevated, and eyes
2.2. Data analysis Polygraphic recordings were visually analyzed. The total time spent by animals in each state of vigilance during the 24-h period was measured, and the percentage within the period was
Fig. 1. An Iguana iguana during active wakefulness.
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
307
Fig. 2. Active wakefulness (AW). During this state the EEG is fast (2,3) , the EOG activity is intense (1) and the EMG is tonically high and presents phasical discharges (4). Artifacts produced by animal movements outstand from the EEG background.
were open. Vertical and side-to-side head shaking and attack behavior also occurred. During this state the EEG was polymorphic showing an amplitude of 24.2 ± 2.8 μV (mean ± SD), and a frequency from 9 to 11.4 cycles/s (Fig. 2). EOG activity was intense with a frequency of 46 ± 5.2 movements/min (mean ± SD). The EMG of neck muscles was tonically high with phasical discharges coinciding with head movements. Heart rate was at a maximum reaching 43.8 ± 7.9 beats/min (mean ± SD). 3.2. Behavioral and electrophysiological data during QW Throughout this state of vigilance the limbs were usually flexed, body rested on the cage floor, and head and neck assumed varied degrees of elevation. Eyes were usually open. General activity exhibited by animals was lower than that displayed during AW. Electroencephalographic patterns were similar to those of active wakefulness, with an amp1itude of 22.8 ± 1.9 μV (mean ± SD) and a frequency oscillating between 8 and 9.6 cycles/s (Fig. 3). Electro–
ocular activity was reduced to an average of 9± 1 movements/min (mean ± SD), while muscular tone decreased to 67% of that observed in AW. Heart rate diminished slightly showing an average of 42.1± 6.6 beats/min (mean ± SD). 3.3. Behavioral and electrophysiological data during QS Animals were completely relaxed, motionless showing the four limbs flexed. Head was lowered and rested flat on the cage floor. Their eyes remained closed and no ocular movements were detected. The EEG showed a decrease both in amplitude (9.2 ± 1.7 μV, mean ± SD) and frequency (between 5.4 to 6.6 cycles/s). The most prominent electrophysiological sign of this sleep period was arrhythmic and intermittent high voltage spikes recorded from different places of the brain (Fig. 4). The incidence of spikes showed a marked tendency to disappear during waking. Electromyogram decreased to 5% of the waking level or sometimes disappeared. Heart rate declined to an average of 25.3 ± 3.2 beats/min (mean ± SD).
Fig. 3. Quiet wakefulness (QW). During this state the EEG patterns (2,3) are similar to those of AW, while EOG activity (1) and EMG (4) are slightly reduced.
308
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Fig. 4. Quiet sleep (QS). During this state of sleep some spikes can be observed outstanding from the EEG (2,3). Ocular movements are abolished (1) and EMG (4) declines in comparison that of the previous state.
3.4. Behavioral and electrophysiological data during AS Long periods of QS were intermittently interrupted by short episodes of active sleep. During these episodes there was a generalized motor activity. Although eyes remained closed there were some openings coinciding with blinking and with isolated or bursts of ocular movements. There were also motor automatisms involving head shaking, chewing, or movements of the body and legs. After these automatisms, animals usually reentered to another period of quiet sleep. Active sleep episodes occurred only after several hours of behavioral sleep and they were never observed immediately following wakefulness. Cerebral activity recorded during active sleep was similar to that observed in quiet wakefulness. Its amplitude was 19.2 ± 1.2 μV (mean ± SD) with a frequency predominantly between 8.4 and 9 cycles/s (Fig. 5). Electro–oculogram recordings showed the presence of bursts of eye movements with a frequency of 27.5 ± 4.2 movements/min (mean ± SD). Electro-
myogram exhibited intermittent phasic discharges related to the animal's automatisms. Heart rate was higher than that observed during QS (36.1 ± 5.7 beats/min, mean ± SD). Lizards remained in QS for most of the 24 h period, spending only a small percentage of time in AS (Fig. 6; Table 1). Frequency of active sleep phases was very variable fluctuating between 15 and 30 episodes throughout the whole 24-h period, while their average duration was very short, lasting only some seconds, and showing certain variability from animal to animal (Table 2). The lowest value observed was 15 s. and the highest 29 s. In contrast, the states of sleep were randomly distributed along the nycterohemeral cycle (Fig. 6). Therefore, these animals may be considered as polyphasic. However the time spent in QS between 8:00 and 20:00 h (150 ± 59 min; mean ± SD) was significantly smaller (p b 0.01) than that observed between 20:00 and 8:00 h (570 ± 48 min; mean ± SD). Likewise, the time spent in AS throughout the two mentioned periods varied significantly (p b 0.01), from 1.36 ± 0.61 min (mean ± SD)
Fig. 5. Active sleep (AS). EEG (2,3) shows a slight increase in frequency and amplitude in comparison to QS. Electro–oculographic recordings (1) show bursts of eye movements and EMG exhibits phasic discharges (4).
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
309
Fig. 6. Distribution of vigilance states in a 24-h period of one specimen of Iguana iguana. AW, Active wakefulness; QS, Quiet wakefulness; QS, Quiet sleep; AS, Active sleep.
to 5.84 ± 1.1 min (mean ± SD respectively. These differences were observed in spite of the fact that the animals were studied under constant illumination conditions.
QS and 7.50 for AS). However, this increment was statistically significant only for QS (p b 0.05). 4. Discussion
3.5. Arousal reactions Frequency and latency of behavioral responses to electrical stimulation respectively changed significantly (p b 0.01 ) from 98.4%–100% and 0.53–0.61 sec (variations between animals) for QW to 48.2–52.7% and 1.29–1.92 s for QS, while obtained values for AS were 46.6–50.9% and 1.27–1.89, which did not show significant differences with those of QS. 3.6. Enforced waking Over the course of enforced waking, responses to stimulation varied from threat and attack behavior to attempted escape. Following cessation of stimulation animals assumed a completely relaxed behavior remained continuously immobile with eye closed for about the next 14–20 h. Subsequent epochs of waking were of relatively short duration and periods of behavioral quiescence were of much longer duration than those of baseline conditions over the remainder of the 24–h recovery period. Time spent in wakefulness by these three lizards showed a significant decrement (p b 0.05) from 745.70 min (76.70 for AW and 669.10 min for QW) exhibited under basal conditions to 467.98 min (31.10 for AW and 436.88 for QW) during the recovery period. In contrast, total sleep time increased from 694.30 min (688 for QS and 6.30 for AS) to 972.0 min (964 for
The lizard I. iguana exhibited the type of behavior which is ordinarily classified as sleep when it occurs in endothermic vertebrates (Ayala-Guerrero, 1983; Frank, 1999). When left undisturbed, iguanids entered a state characterized by immobility, long term maintenance of eyelid closure, maximum relaxation of body musculature, lowered respiratory and cardiac activity and substantial and consistent diminished behavioral responsiveness to stimulation. The same type of behavioral sleep has been reported in other reptiles such as the crocodile Caiman sclerops (Flanigan et al., 1973), the turtle Terrapene Table 1 Percentages of vigilance states from 24-h recordings Animal
AW
QW
SWS
REM
1 2 3 4 5 6 7 8 9 10 Average SD
15.30 13.65 30.62 31.99 40.30 25.44 19.96 32.52 28.45 24.76 26.30 8.26
24.38 24.76 29.82 12.36 11.39 16.25 25.73 26.71 23.61 28.13 22.29 6.55
59.81 60.98 35.21 54.94 47.93 57.70 52.80 40.51 47.12 46.66 50.37 8.43
0.50 0.59 0.35 0.69 0.38 0.61 0.51 0.46 0.82 0.45 0.54 0.14
310
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Table 2 Number and mean duration of active sleep episodes from 24-h recordings Animal
Number
Mean duration (s)
1 2 3 4 5 6 7 8 9 10 Average SD
15 26 20 30 19 18 23 16 29 21 21.7 5.2
29 15 17 17 25 23 19 28 24 18 21.5 4.9
carolina (Flanigan et al., 1974; Eiland et al., 2001), and the iguanids C. similis (Ayala- Guerrero and Vargas-Reyna, 1987) and C. pectinata (Ayala-Guerrero and Huitrón-Reséndiz, 1991). In contrast, the cerebral activity exhibited by I. iguana, and reptiles in general, throughout their states of vigilance, is different from that of endothermic vertebrates. However, from a behavioral standpoint and according to the tendency followed by other physiological variables, this reptile displays two sleep phases: quiet sleep and active sleep similar to SWS and REM sleep in endothermic vertebrates. The differences of cerebral activity between mammals and I. iguana may be due to the fact that the brain of reptiles is less developed than that of mammals (Butler, 1980; Ebbeson, 1980; Finlay et al., 1991). Slow waves, typical of SWS in mammals, depend on the integrity of the neocortex and on the anatomofunctional relationship between the thalamus and the neocortex (Lindsley et al., 1950; Jouvet, 1967). In reptiles the neocortex and the thalamo–cortical traject are poorly developed (Anderson and Anderson, 1974; Lohman and Smeets, 1991) Therefore, the absence of slow waves during quiet sleep of I. iguana may only imply the absence of the appropriate neurophysiological generators of this EEG activity but not of sleep. This is supported by experimental evidences arising from sleep studies performed in decorticated cats, where slow waves were not present during behavioral sleep (Jouvet, 1962). Moreover, the ontogenetic analysis of sleep in guinea pigs shows the absence of high amplitude slow waves when neocortex has not reached its complete development (Astic et al., 1973). In contrast, the spikes recorded in the EEG of I. iguana during quiet sleep may be considered as an appropriate electrophysiological sign to correlate this sleep phase in reptiles with slow waves sleep in mammals. This is supported by studies performed in normal and decorticated cats where similar spikes have been recorded in the ventral hippocampus during this sleep stage (Jouvet, 1962; Hartse and Rechtschaffen, 1982). The presence of spikes during behavioral sleep in chelonians, (Vasilescu, 1970; Flanigan, 1974; Flanigan et al., 1974; Ayala-Guerrero, 1987) crocodilians (Flanigan et al., 1973) and lizards (Ayala- Guerrero and Vargas-Reyna, 1987; AyalaGuerrero and Huitrón-Reséndiz, 1991), as well as some birds and mammals (Hartse and Rechtschaffen, 1982) provides support for the hypothesis that the spikes may have been a
phylogenetic forerunner of the slow waves which characterize SWS in endothermic vertebrates. These and other physiological signs different from EEG, such as electromyogram, electro– oculogram and electrocardiogram, provide information on the degrees of attention or sleep depth of vertebrates. Thus the decrease of body activity observed in I. iguana when entering into periods of sleep is a common tendency in mammals (Ruckebusch and Morel, 1968; Ruckebusch et al., 1970). Eye movements present during wakefulness disappear when resting, and reappear in bursts during active sleep together with other motor manifestations, as seen in mammals and birds during paradoxical sleep. The decrease in heart rate showed by this reptile when passing from wakefulness to quiet sleep followed by an increase during active sleep has also been described in endotherms (Tauber et al., 1968b; Allison et al., 1972). The previous comparative analysis between quiet sleep of I. iguana and SWS of mammals allows us to consider that both classes of sleep are analogous. On the other hand, active sleep of I. iguana is also similar to paradoxical sleep of birds and mammals, since in the three classes of vertebrates, this type of sleep appears after long periods of SWS or quiet sleep. Moreover, during active or paradoxical sleep of reptiles and mammals motor automatisms are observed. Short duration of active sleep of I. iguana contrasts with that of paradoxical sleep of mammals (Allison and Van Twyver, 1970). However, the other class of endothermic vertebrates (birds) also exhibits paradoxical sleep phases of very short duration. The active sleep phase observed in I. iguana has also been reported in other reptile species such as C. similis (AyalaGuerrero and Vargas-Reyna, 1987), C. pectinata (AyalaGuerrero and Huitrón-Reséndiz, 1991), Emys orbicularis (Vasilescu, 1970), and Dipsosaurus dorsalis (Huntley, 1987). Flanigan (1973) reported the absence of paradoxical sleep in I. iguana. However, he took only recording samples of 60 s every 5 min. In our experience it is very important to take continuous recordings for several hours in order to detect As phases in reptiles, since these phases are of very short duration. Moreover, continuous visual observations throughout the recording period are necessary to distinguish behaviorally the mentioned phases. In this context, visual observations carried out by Flanigan were only done intermittently. Behavioral reactivity to repetitive electrical stimulation decreased significantly when passing from waking to behavioral sleep. Although reactivity was lower during AS than during QS this difference was not statistically significant. The absence of significance in the arousal reaction levels between the two phases of sleep could probably be related to the reduced number of specimens utilized in this experimental manipulation. In contrast, REM sleep is homeostatically regulated (Benington and Heller, 1999), therefore attempts to enter REM sleep increase during selective REM sleep deprivation and extends the time spent in REM sleep during recovery to produce a rebound effect. The concept of REM sleep rebound was initially proposed by Dement (1960) to describe the increase of REM sleep which
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
follows its instrumental suppression. This concept has been extended to encompass any increase of REM sleep above the baseline which follows alterations of the sleep-waking cycle. In this context, following cessation of enforced waking, sleep deprived lizards entered immediately to a sleep behavior and remained immobile with eye closed during a period significantly longer to that of basal conditions. Subsequent epochs of waking were of relatively of shorter duration than those in baseline conditions over the remainder of the 24-h recovery period. Total time spent by animals in QW decreased significantly, while amount of behavioral sleep showed an important increment involving both QS and AS. However, only values of QS reached statistically significant levels. Similar effects have been pharmacologically induced in other species of reptiles (Ayala-Guerrero et al., 1993, 1996) and in several mammalian species (Gottesmann, 1966; Tabushi and Himwhich, 1969) after reserpine or PCPA administration. In contrast, it has been reported that rhythmic oscillation between wakefulness and sleep depends on endogenous mechanisms, but they are also influenced by illumination schedules and light intensity (Aschoff, 1960; Borbely, 1978). Despite of the fact that these reptiles were studied under constant light conditions, the time spent in QS and AS during the astronomical day was significantly smaller than time spent in both sleep phases during astronomical night. This may be the expression of an endogenous mechanism regulating sleep distribution, such as has been described in mammals (Borbely and Neuhaus, 1978). From all previous observations, it seems reasonable to consider that the periods of quiet sleep and active sleep in I. iguana might be ana1ogous to the periods of slow wave sleep and paradoxical sleep respective1y, in mammals and birds. References Allison, T., Van Twyver, H., 1970. Sleep in the moles, Scalopus acuaticus and Condylura cristata. Exp. Neurol. 27, 564–578. Allison, T., Van Twyver, H., Goff, W.R., 1972. Electrophysiological studies of the echidna, Tachyglossus aculeatus.1. Waking and sleeping. Arch. Ital. Biol. 110, 145–184. Anderson, P., Anderson, S.A., 1974. Thalamic origin of cortical rhythmic activity. In: Rémond, A. (Ed.), Handbook of Electroencephalography and Clinical Neurophysiology, Vol 2 part C. Elsevier Publishing Company, Amsterdam, pp. 90–114. Aschoff, J., 1960. Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol. 25, 11–28. Astic, L., Sastre, J.P., Brandon, A.M., 1973. Etude polygraphique des états de vigilance chez le foetus du cobaye. Physiol. Behav. 11, 647–654. Ayala-Guerrero, F., 1983. Filogenia del Sueño: los mamíferos. Bol. Estud. Méd. Biol. Méx. 32, 67–82. Ayala-Guerrero, F., 1987. Sleep in the tortoise Kinosternon sp. Experientia 43, 296–298. Ayala-Guerrero, F., Mexicano, G., in press. Topographical distribution of the locus coeruleus and raphe nuclei in the lizard Ctenosaura pectinata: functional implications on sleep. Comp. Biochem. Physiol., A. Ayala- Guerrero, F., Vargas-Reyna, L., 1987. Sleep and wakefulness in the lizard Ctenosaura similis. Bol. Estud. Méd. Biol. Méx. 35, 25–33. Ayala-Guerrero, F., Huitrón-Reséndiz, S., 1991. Sleep patterns in the lizard Ctenosaura pectinata. Physiol. Behav. 49, 1305–1307. Ayala-Guerrero, F., Huitrón-Reséndiz, S., Mexicano, G., 1993. Effect of reserpine on sleep of a chelonian reptile. Proc. West. Pharmacol. Soc. 36, 227–231.
311
Ayala-Guerrero, F., Huitrón-Reséndiz, S., Mexicano, G., 1996. Effect of a depleter of cerebral monoamines on sleep patterns of a Chelonian reptile. Drug Dev. Res. 39, 115–120. Benington, J.H., Heller, C., 1999. Implications of sleep deprivation experiments for our understanding of sleep homeostasis. Sleep 22, 1033–1037. Borbely, A.A., 1978. Effects of light on sleep and activity rhythms. Prog. Neurobiol. 10, 1–31. Borbely, A.A., Neuhaus, H.V., 1978. Circadian rhythm of sleep and motor activity in the rat during skeleton photoperiod, continuous darkness and continuous light. J. Comp. Physiol. 128, 37–46. Brebbia, R.D., Pyne, T.E., 1972. Environmental hypothermia and cerebral electrical activity of the little brown bat Myotis lucifugus. Psychophysiology 9, 122–123. Butler, A.B., 1980. Cytoarchitectonic and connectional organization of the lacertilian telencephalon with comments on vertebrates forebrain evolution. In: Ebbesson, S.O.E. (Ed.), Comparative Neurology of the Telencephalon. Plenum press, New York, pp. 297–325. Dement, W.C., 1960. The effect of dream deprivation. Science 131, 1705–1707. Ebbeson, S.O.E., 1980. Comparative Neurology of the Telencephalon. Plenum Press, New York. Eiland, M.M., Lyamin, O.I., Siegel, J.M., 2001. State-related discharge of neurons in the brain stem of freely moving box turtles, Terrapene carolina major. Arch. Ital. Biol. 139, 23–34. Finlay, B.L., Innocenti, G., Scheich, H., 1991. The neocortex. Ontogeny and phylogeny. Plenum Press, New York. Flanigan, W.F., 1972. Behavioral states and electroencephalograms of reptiles. In: Chase, M.H. (Ed.), Perspectives in the Brain Sciences. 1. The sleeping brain. Brain Information Service, Los Angeles, pp. 14–18. Flanigan, W.F., 1973. Sleep and wakefulness in iguanid lizards, Ctenosaura pectinata and Iguana iguana. Brain Behav. Evol. 8, 401–436. Flanigan, W.F., 1974. Sleep and wakefulness in chelonian reptiles.II. The redfooted tortoise, Geochelone carbonaria. Arch. ltal. Biol. 112, 253–277. Flanigan, W.F., Wilcox, R.H., Rechtschaffen, A., 1973. The EEG and behavioral continuum of the crocodilian, Caiman sclerops. Electroencephalogr. Clin. Neurophysiol. 34, 521–538. Flanigan, W.F., Knight, C.P., Hartse, K.M., Rechtschaffen, A., 1974. Sleep and wakefulness in chelonian reptiles. 1. The box turtle, Terrapene carolina. Arch. Ital. Biol. 112, 227–252. Frank, M.G., 1999. Phylogeny and evolution of rapid eye movement (REM) sleep. In: Mallick, B.N., Inoue, S. (Eds.), Rapid Eye Movement Sleep. Narosa Publishing House, New Delhi, India, pp. 17–38. Freemon, F.R., Mc New, J.J., Adey, W.R., 1971. Chimpanzee sleep stages. Electroencephalogr. Clin. Neurophysiol. 31, 485–489. Gottesmann, C., 1966. Reserpine et vigilance chez le rat. C. R. Soc. Biol. 160, 2056–2061. Hartse, K.M., Rechtschaffen, A., 1982. The effect of amphetamine, nembutal, alpha-methyl-tyrosine, and parachlorophenylalanine on sleep-related spike activity of the tortoise, Geochelone carbonaria, and on the cat ventral hippocampus spike. Brain Behav. Evol. 21, 199–222. Huitrón-Reséndiz, S., Mexicano, G., Ayala-Guerrero, F., 1992. Effect of atropine sulphate on sleep of the iguanid lizard Ctenosaura similis. Proc. West. Pharmacol. Soc. 35, 157–160. Huntley, A.C., 1987. Electrophysiological and behavioral correlates of sleep in the desert iguana Dipsosaurus dorsalis. Comp. Biochem. Physiol. A 86, 325–330. Jouvet, M., 1962. Recherchees sur les structures nerveuses et les mécanismes responsables des différentes phases du sommeil physiologique. Arch. Ital. Biol. 100, 125–206. Jouvet, M., 1967. Neurophysio1oy of the states of sleep. Physiol. Rev. 47, 117–177. Karmanova, J.G., Churnosov, E.V., 1972. Electrophysiological investigation of natural sleep and waking in turtles and hens. J. Evol. Biochem. Physiol. 8, 47–53. Karmanova, J.G., Belekhova, M.G., Churnosov, E.V., 1971. Behavioral and e1ectrographic patterns of natural sleep and wakefulness in reptiles. Fiziol. Zh. SSSR 57, 504–511. Liknowski, P., Kerkhofs, M., Hauspie, R., Mendlewicz, J., 1991. A genetic study of sleep in twins living apart. Sleep Res. 20 A, 41.
312
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Lindsley, D.B., Schreiner, L.H., Knowles, W.B., Magoun, H.W., 1950. Behavioral and EEG changes following chronic brain stem lesions in the cat. Electroencephalogr. Clin. Neurophysiol. 2, 483–498. Lohman, A.H., Smeets, W.J., 1991. The dorsal ventricular ridge and cortex of reptiles in historical and phylogenetic perspective. In: Finlay, B.L., Innocenti, G., Scheich, H. (Eds.), The Neocortex: Ontogeny and phylogeny, vol. 200. Plenum Press, New York, pp. 59–74. Mexicano, G., Huitrón-Reséndiz, S., Ayala-Guerrero, F., 1992. Effect of 5hydroxytryptophan (5-HTP) on sleep in parachlorophenylalanine (PCPA) pretreated birds. Proc. West. Pharmacol. Soc. 35, 17–20. Mukhametov, L., 1990. Le sommeil des mammiféres marins. La Recherche 21 (217), 41–47. Peyrethon, J., Dusan-Peyrethon, D., 1969. Etude polygraphique du cycle veillesommeil chez trois generes de reptiles. C. R. Soc. Biol (Paris) 163, 181–186. Prudom, A.E., Klemm, W.R., 1973. Electromyographic correlates of sleep behavior in a mammal, the armadillo Dasypus novemcinctus. Physiol. Behav. 10, 275–282. Romo, R., Cepeda, C., Velasco, M., 1978. Behavioral and electrophysiological patterns of wakefulness–sleep in the lizard (Phrynosoma regali). Bol. Estud. Méd. Biol. Méx. 30, 13–18. Ruckebusch, Y., 1963. Etude EEG et comportamentale des alternances veillesommeil chez l´ane. C. R. Soc. Biol. (Paris) 157, 840–844. Ruckebusch, Y., Morel, M.T., 1968. Etude polygraphique du sommeil chez le porc. C. R. Soc. Biol. (Paris) 162, 1346–1354. Ruckebusch, Y., Barbey, P., Guillemont, P., 1970. Les étates de sommeil chez le cheval (Equus caballus). C. R. Séances Soc. Biol. (Paris) 164, 658–666.
Schlehuber, C.I., Flanigan, D., Laage, D.G., Spooner, E., 1974. Paradoxical sleep in the chick (Gallus domesticus). Behav. Biol. 11, 537–546. Susic, V.T., Kovacevic, R.M., 1973. Sleep patterns in the owl Strix aluco. Physiol. Behav. 11, 313–317. Tabushi, K., Himwhich, H.E., 1969. The acute effects of reserpine on the sleep– wakefulness cycle in rabbits. Psychopharmacology 16, 240–252. Tauber, E.S., Roffwarg, H.P., Weitzman, E.D., 1966. Eye movements and electroencephalographic activity during sleep in diurnal lizard. Nature 212, 1612–1613. Tauber, E.S., Rojas-Ramírez, J.A., Hernández-Peón, R., 1968a. Electrophysio1ogica1 and behavioral correlates of wakefulness and sleep in the lizard Ctenosaura pectinata. Electroencephalogr. Clin.Neurophysiol. 24, 424–433. Tauber, E.S., Michel, F., Roffwarg, H.P., 1968b. Preliminary note on the sleep and waking cycle in the desert hedgehog (Paracchinus hypome1as). Psychophysio1ogy 5, 201. Vasconcelos-Dueñas, 1., Ayala-Guerrero, F., 1983. Filogenia del sueño en las aves. Bol. Estud. Méd. Biol. Méx. 32, 83–90. Vasilescu, E., 1970. Sleep and wakefulness in the tortoise (Emys orbicularis). Rev. Roum. Biol., Ser. Zool. 15, 177–179. Walker, J.M., Berger, R.J., Scott, T.D., 1973. Absence of electrophysiological signs of sleep in the tortoise (Testudo denticulata). Sleep Res. 2, 88. Warner, B.F., Huggings, S.E.A., 1978. An electroencephalographic study of sleep in young caiman in colony. Comp. Biochem. Physiol. A 59, 139–144. Zepelin, H., 1994. Mammalian sleep. In: Kryger, M.H., Roth, T., Dement, W.C. (Eds.), Principles and Practice of Sleep Medicine. W.B. Saunders Company, Philadelphia, pp. 69–80.
Comparative Biochemistry and Physiology, Part A 151 (2008) 305 – 312 www.elsevier.com/locate/cbpa
Sleep and wakefulness in the green iguanid lizard (Iguana iguana) ☆ F. Ayala-Guerrero ⁎, G. Mexicano Facultad de Psicología, Universidad Nacional Autónoma de México and UAEM, Mexico DF 04510, Mexico Received 27 October 2005; received in revised form 23 March 2007; accepted 26 March 2007 Available online 30 March 2007
Abstract The reptile Iguana iguana exhibits four states of vigilance: active wakefulness (AW), quiet wakefulness (QW), quiet sleep (QS) and active sleep (AS). Cerebral activity decreases in amplitude and frequency when passing from wakefulness to QS. Both parameters show a slight increase during AS. Heart rate is at a maximum during AW (43.8 ± 7.9 beats/min), decreases to a minimum in QS (25.3 ± 3.2 beats/min) and increases in AS (36.1 ± 5.7 beats/min). Tonical and phasical muscular activity is present in wakefulness, decreases or disappears in QS and reappears in AS. Single or conjugate ocular movements are observed during wakefulness, then disappear in QS and abruptly reappear in AS. Although these reptiles are polyphasic, their sleep shows a tendency to concentrate between 20:00 and 8:00 h. Quiet sleep occupies the greater percentage of the total sleep time. Active sleep episodes are of very short duration, showing an average of 21.5 ± 4.9 (mean ± SD). Compensatory increment of sleep following its total deprivation was significant only for QS. Reaction to stimuli decreased significantly when passing from wakefulness to sleep. It is suggested that the lizard I. iguana displays two sleep phases behaviorally and somatovegetatively similar to slow wave sleep and paradoxical sleep in birds and mammals. © 2007 Elsevier Inc. All rights reserved. Keywords: Active sleep; Iguana iguana; Quiet sleep; Reptiles
1. Introduction Mammals (Ruckebusch, 1963; Jouvet, 1967; Allison and Van Twyver, 1970; Freemon et al., 1971; Brebbia and Pyne, 1972; Prudom and Klemm, 1973; Mukhametov, 1990; Zepelin, 1994) and birds (Susic and Kovacevic, 1973; Schlehuber et al., 1974; Vasconcelos-Dueñas and Ayala-Guerrero, 1983; Mexicano et al., 1992) present two sleep phases. The first, called slow wave sleep (SWS), is characterized by brain activity of high amplitude slow waves. The second, known as paradoxical sleep (PS) or rapid eye movement sleep (REM), exhibits a low voltage fast frequency electroencephalographic pattern. This ☆
This paper is part of the 5th special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Carolina A. Freire (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of Knut SchmidtNielsen (1915–2007), a great mind in comparative physiology, with books translated into 16 languages, including Spanish and Portuguese, and Cicero Lima (1934–2006), journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. E-mail address: [email protected] (F. Ayala-Guerrero). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.03.027
sleep phase is usually further distinguished by the presence of eye movements, myoclonic twitches of the extremities, reduced muscle tone and autonomic variability. Most sleep studies have been performed in endothermic vertebrates. However, in recent years, the comparative approach towards an understanding of the brain has provided significant new insights. Reptiles appear to be most useful in this approach because of their ancestral relationship to mammals and birds. Sleep in these ectothermic vertebrates remains as a matter of controversy (Walker et al., 1973), since it has been determined almost exclusively by considering the electrical activity of mammalian brain as a prototype for comparative sleep studies. However, the absence of electroencephalographic signs of SWS or PS might imply only the absence of the appropriate neurophysiological generators of such activity but not necessarily of sleep. In contrast, it has been suggested that a number of common behavioral characteristics can be used to delineate sleep for any class of animals. These characteristics include: 1) the spontaneous assumption of a stereotypic specific posture, 2) the maintenance of behavioral immobility, 3) an elevated behavioral response threshold which may be reflected in the intensity of an arousing stimulus and/or in the frequency,
306
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
latency, or duration of a behavioral response, and 4) rapid behavioral state reversibility with stimulation (Flanigan, 1974). It has been reported that several reptile species belonging to chelonians (Vasilescu, 1970; Karmanova and Churnosov, 1972; Flanigan, 1974; Ayala-Guerrero, 1987), crocodilians (Peyrethon and Dusan–Peyrethon, 1969; Flanigan et al., 1973; Warner and Huggings, 1978) and lizards (Tauber et al., 1966, 1968a; Karmanova et al., 1971; Flanigan, 1972; Romo et al., 1978; Huntley, 1987; Ayala-Guerrero and Mexicano, in press) exhibit behavioral sleep. In the particular case of iguanid lizards two phases of sleep have been described in Ctenosaura pectinata (Tauber et al., 1966, 1968a; Ayala-Guerrero and HuitrónReséndiz, 1991), and Ctenosaura similis (Ayala- Guerrero and Vargas-Reyna, 1987; Huitrón-Reséndiz et al., 1992). However in Iguana iguana only one phase has been observed (Flanigan, 1973). Since genetic factors exist in expressing certain sleep characteristics (Liknowski et al., 1991), and I. iguana is, from the phylogenetic point of view, closely related to the other two lizards, it is probable that it also presents two similar sleep phases. 2. Materials and methods 2.1. Surgical and recording procedures Ten adult specimens of the lizard I. iguana of both sexes were used in this study. Stainless steel, electrodes insulated except for the tip were used. Electrodes were implanted, after a period of habituation in our laboratory, under Nembutal anesthesia (35 mg/kg i.p). After removing the superficial scales, the muscle mass overlying the parietal bones on each side was separated through a midline skin incision. Drill holes were made in the skull and the electrodes for chronic bipolar recordings of cerebral activity (EEG) were implanted bilaterally on the anterior and posterior telencephalon. Two electrodes were placed in the skull at the external and internal canthi of each eye for ocular activity recording (EOG). Muscular activity (EMG) and heart rate were obtained from the same pair of electrodes placed in the neck muscles. One screw (ground electrode) was placed in the skull approximately 2.5 cm anterior to the parietal eye. All electrode leads were soldered to a small connector fixed to the skull with acrylic cement. Seven days after postoperative recovery the lizards were placed in a shielded sound attenuated chamber constantly illuminated by a 60 W light bulb, in order to facilitate the observation through a one-way glass window. Chamber temperature during recording sessions varied between 25 and 30 °C. A po1ygraphical recording by animal, under constant visual observations, was taken continuously during 24 h on an 8 channel Grass model III-D electroencephalograph.
calculated. The mean duration and the total number of active sleep episodes over the 24-h period were obtained as well as their nycterohemeral distribution. Muscular activity measurements of each state of vigilance were made in reference to activity exhibited during active wakefulness, which was considered as 100%. Heart rate and frequency of ocular activity were extrapolated to 1 min. Values of states of vigilance obtained during the astronomical day (from 8:00 to 20:00 h) were compared to those of the astronomical night (from 20:00 to 8:00 h) through the Student's t-test. 2.3. Arousal reactions Behavioral response latencies during behavioral waking and behavioral sleep were measured in three additional specimens. Electrical stimulation consisting in dipolar pulses of 50 ms duration at 60 c/s was delivered to pairs of EMG electrodes. The voltage used was 2 V which was randomly applied during AW, QW, QS and AS. Behavioral responses consisted of a head movement accompanied by eyelid, limb and body movements. Experimental trials were conducted during 24 h/day for three consecutive periods or until 30 stimulus presentations during behavioral waking and 50 during behavioral sleep (30 in QS and 20 in AS) had been made. 2.4. Enforced waking These three specimens were also kept behaviorally awake for 48 continuous hours, and baseline recordings were compared with post-stimulation recovery recordings of 24 h. Enforced wakefulness was maintained by lifting, stroking and handling. 3. Results The lizard I. iguana (Fig. 1) displayed the following states of vigilance during the nycterohemeral cycle: active wakefulness (AW), quiet wakefulness (QW), quiet sleep (QS) and active sleep (AS). Each sate showed its own behavioral and electrophysiological features. 3.1. Behavioral and electrophysiological data during AW The iguanid stood on their four legs. The body was raised from the chamber floor, head and neck were elevated, and eyes
2.2. Data analysis Polygraphic recordings were visually analyzed. The total time spent by animals in each state of vigilance during the 24-h period was measured, and the percentage within the period was
Fig. 1. An Iguana iguana during active wakefulness.
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
307
Fig. 2. Active wakefulness (AW). During this state the EEG is fast (2,3) , the EOG activity is intense (1) and the EMG is tonically high and presents phasical discharges (4). Artifacts produced by animal movements outstand from the EEG background.
were open. Vertical and side-to-side head shaking and attack behavior also occurred. During this state the EEG was polymorphic showing an amplitude of 24.2 ± 2.8 μV (mean ± SD), and a frequency from 9 to 11.4 cycles/s (Fig. 2). EOG activity was intense with a frequency of 46 ± 5.2 movements/min (mean ± SD). The EMG of neck muscles was tonically high with phasical discharges coinciding with head movements. Heart rate was at a maximum reaching 43.8 ± 7.9 beats/min (mean ± SD). 3.2. Behavioral and electrophysiological data during QW Throughout this state of vigilance the limbs were usually flexed, body rested on the cage floor, and head and neck assumed varied degrees of elevation. Eyes were usually open. General activity exhibited by animals was lower than that displayed during AW. Electroencephalographic patterns were similar to those of active wakefulness, with an amp1itude of 22.8 ± 1.9 μV (mean ± SD) and a frequency oscillating between 8 and 9.6 cycles/s (Fig. 3). Electro–
ocular activity was reduced to an average of 9± 1 movements/min (mean ± SD), while muscular tone decreased to 67% of that observed in AW. Heart rate diminished slightly showing an average of 42.1± 6.6 beats/min (mean ± SD). 3.3. Behavioral and electrophysiological data during QS Animals were completely relaxed, motionless showing the four limbs flexed. Head was lowered and rested flat on the cage floor. Their eyes remained closed and no ocular movements were detected. The EEG showed a decrease both in amplitude (9.2 ± 1.7 μV, mean ± SD) and frequency (between 5.4 to 6.6 cycles/s). The most prominent electrophysiological sign of this sleep period was arrhythmic and intermittent high voltage spikes recorded from different places of the brain (Fig. 4). The incidence of spikes showed a marked tendency to disappear during waking. Electromyogram decreased to 5% of the waking level or sometimes disappeared. Heart rate declined to an average of 25.3 ± 3.2 beats/min (mean ± SD).
Fig. 3. Quiet wakefulness (QW). During this state the EEG patterns (2,3) are similar to those of AW, while EOG activity (1) and EMG (4) are slightly reduced.
308
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Fig. 4. Quiet sleep (QS). During this state of sleep some spikes can be observed outstanding from the EEG (2,3). Ocular movements are abolished (1) and EMG (4) declines in comparison that of the previous state.
3.4. Behavioral and electrophysiological data during AS Long periods of QS were intermittently interrupted by short episodes of active sleep. During these episodes there was a generalized motor activity. Although eyes remained closed there were some openings coinciding with blinking and with isolated or bursts of ocular movements. There were also motor automatisms involving head shaking, chewing, or movements of the body and legs. After these automatisms, animals usually reentered to another period of quiet sleep. Active sleep episodes occurred only after several hours of behavioral sleep and they were never observed immediately following wakefulness. Cerebral activity recorded during active sleep was similar to that observed in quiet wakefulness. Its amplitude was 19.2 ± 1.2 μV (mean ± SD) with a frequency predominantly between 8.4 and 9 cycles/s (Fig. 5). Electro–oculogram recordings showed the presence of bursts of eye movements with a frequency of 27.5 ± 4.2 movements/min (mean ± SD). Electro-
myogram exhibited intermittent phasic discharges related to the animal's automatisms. Heart rate was higher than that observed during QS (36.1 ± 5.7 beats/min, mean ± SD). Lizards remained in QS for most of the 24 h period, spending only a small percentage of time in AS (Fig. 6; Table 1). Frequency of active sleep phases was very variable fluctuating between 15 and 30 episodes throughout the whole 24-h period, while their average duration was very short, lasting only some seconds, and showing certain variability from animal to animal (Table 2). The lowest value observed was 15 s. and the highest 29 s. In contrast, the states of sleep were randomly distributed along the nycterohemeral cycle (Fig. 6). Therefore, these animals may be considered as polyphasic. However the time spent in QS between 8:00 and 20:00 h (150 ± 59 min; mean ± SD) was significantly smaller (p b 0.01) than that observed between 20:00 and 8:00 h (570 ± 48 min; mean ± SD). Likewise, the time spent in AS throughout the two mentioned periods varied significantly (p b 0.01), from 1.36 ± 0.61 min (mean ± SD)
Fig. 5. Active sleep (AS). EEG (2,3) shows a slight increase in frequency and amplitude in comparison to QS. Electro–oculographic recordings (1) show bursts of eye movements and EMG exhibits phasic discharges (4).
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
309
Fig. 6. Distribution of vigilance states in a 24-h period of one specimen of Iguana iguana. AW, Active wakefulness; QS, Quiet wakefulness; QS, Quiet sleep; AS, Active sleep.
to 5.84 ± 1.1 min (mean ± SD respectively. These differences were observed in spite of the fact that the animals were studied under constant illumination conditions.
QS and 7.50 for AS). However, this increment was statistically significant only for QS (p b 0.05). 4. Discussion
3.5. Arousal reactions Frequency and latency of behavioral responses to electrical stimulation respectively changed significantly (p b 0.01 ) from 98.4%–100% and 0.53–0.61 sec (variations between animals) for QW to 48.2–52.7% and 1.29–1.92 s for QS, while obtained values for AS were 46.6–50.9% and 1.27–1.89, which did not show significant differences with those of QS. 3.6. Enforced waking Over the course of enforced waking, responses to stimulation varied from threat and attack behavior to attempted escape. Following cessation of stimulation animals assumed a completely relaxed behavior remained continuously immobile with eye closed for about the next 14–20 h. Subsequent epochs of waking were of relatively short duration and periods of behavioral quiescence were of much longer duration than those of baseline conditions over the remainder of the 24–h recovery period. Time spent in wakefulness by these three lizards showed a significant decrement (p b 0.05) from 745.70 min (76.70 for AW and 669.10 min for QW) exhibited under basal conditions to 467.98 min (31.10 for AW and 436.88 for QW) during the recovery period. In contrast, total sleep time increased from 694.30 min (688 for QS and 6.30 for AS) to 972.0 min (964 for
The lizard I. iguana exhibited the type of behavior which is ordinarily classified as sleep when it occurs in endothermic vertebrates (Ayala-Guerrero, 1983; Frank, 1999). When left undisturbed, iguanids entered a state characterized by immobility, long term maintenance of eyelid closure, maximum relaxation of body musculature, lowered respiratory and cardiac activity and substantial and consistent diminished behavioral responsiveness to stimulation. The same type of behavioral sleep has been reported in other reptiles such as the crocodile Caiman sclerops (Flanigan et al., 1973), the turtle Terrapene Table 1 Percentages of vigilance states from 24-h recordings Animal
AW
QW
SWS
REM
1 2 3 4 5 6 7 8 9 10 Average SD
15.30 13.65 30.62 31.99 40.30 25.44 19.96 32.52 28.45 24.76 26.30 8.26
24.38 24.76 29.82 12.36 11.39 16.25 25.73 26.71 23.61 28.13 22.29 6.55
59.81 60.98 35.21 54.94 47.93 57.70 52.80 40.51 47.12 46.66 50.37 8.43
0.50 0.59 0.35 0.69 0.38 0.61 0.51 0.46 0.82 0.45 0.54 0.14
310
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Table 2 Number and mean duration of active sleep episodes from 24-h recordings Animal
Number
Mean duration (s)
1 2 3 4 5 6 7 8 9 10 Average SD
15 26 20 30 19 18 23 16 29 21 21.7 5.2
29 15 17 17 25 23 19 28 24 18 21.5 4.9
carolina (Flanigan et al., 1974; Eiland et al., 2001), and the iguanids C. similis (Ayala- Guerrero and Vargas-Reyna, 1987) and C. pectinata (Ayala-Guerrero and Huitrón-Reséndiz, 1991). In contrast, the cerebral activity exhibited by I. iguana, and reptiles in general, throughout their states of vigilance, is different from that of endothermic vertebrates. However, from a behavioral standpoint and according to the tendency followed by other physiological variables, this reptile displays two sleep phases: quiet sleep and active sleep similar to SWS and REM sleep in endothermic vertebrates. The differences of cerebral activity between mammals and I. iguana may be due to the fact that the brain of reptiles is less developed than that of mammals (Butler, 1980; Ebbeson, 1980; Finlay et al., 1991). Slow waves, typical of SWS in mammals, depend on the integrity of the neocortex and on the anatomofunctional relationship between the thalamus and the neocortex (Lindsley et al., 1950; Jouvet, 1967). In reptiles the neocortex and the thalamo–cortical traject are poorly developed (Anderson and Anderson, 1974; Lohman and Smeets, 1991) Therefore, the absence of slow waves during quiet sleep of I. iguana may only imply the absence of the appropriate neurophysiological generators of this EEG activity but not of sleep. This is supported by experimental evidences arising from sleep studies performed in decorticated cats, where slow waves were not present during behavioral sleep (Jouvet, 1962). Moreover, the ontogenetic analysis of sleep in guinea pigs shows the absence of high amplitude slow waves when neocortex has not reached its complete development (Astic et al., 1973). In contrast, the spikes recorded in the EEG of I. iguana during quiet sleep may be considered as an appropriate electrophysiological sign to correlate this sleep phase in reptiles with slow waves sleep in mammals. This is supported by studies performed in normal and decorticated cats where similar spikes have been recorded in the ventral hippocampus during this sleep stage (Jouvet, 1962; Hartse and Rechtschaffen, 1982). The presence of spikes during behavioral sleep in chelonians, (Vasilescu, 1970; Flanigan, 1974; Flanigan et al., 1974; Ayala-Guerrero, 1987) crocodilians (Flanigan et al., 1973) and lizards (Ayala- Guerrero and Vargas-Reyna, 1987; AyalaGuerrero and Huitrón-Reséndiz, 1991), as well as some birds and mammals (Hartse and Rechtschaffen, 1982) provides support for the hypothesis that the spikes may have been a
phylogenetic forerunner of the slow waves which characterize SWS in endothermic vertebrates. These and other physiological signs different from EEG, such as electromyogram, electro– oculogram and electrocardiogram, provide information on the degrees of attention or sleep depth of vertebrates. Thus the decrease of body activity observed in I. iguana when entering into periods of sleep is a common tendency in mammals (Ruckebusch and Morel, 1968; Ruckebusch et al., 1970). Eye movements present during wakefulness disappear when resting, and reappear in bursts during active sleep together with other motor manifestations, as seen in mammals and birds during paradoxical sleep. The decrease in heart rate showed by this reptile when passing from wakefulness to quiet sleep followed by an increase during active sleep has also been described in endotherms (Tauber et al., 1968b; Allison et al., 1972). The previous comparative analysis between quiet sleep of I. iguana and SWS of mammals allows us to consider that both classes of sleep are analogous. On the other hand, active sleep of I. iguana is also similar to paradoxical sleep of birds and mammals, since in the three classes of vertebrates, this type of sleep appears after long periods of SWS or quiet sleep. Moreover, during active or paradoxical sleep of reptiles and mammals motor automatisms are observed. Short duration of active sleep of I. iguana contrasts with that of paradoxical sleep of mammals (Allison and Van Twyver, 1970). However, the other class of endothermic vertebrates (birds) also exhibits paradoxical sleep phases of very short duration. The active sleep phase observed in I. iguana has also been reported in other reptile species such as C. similis (AyalaGuerrero and Vargas-Reyna, 1987), C. pectinata (AyalaGuerrero and Huitrón-Reséndiz, 1991), Emys orbicularis (Vasilescu, 1970), and Dipsosaurus dorsalis (Huntley, 1987). Flanigan (1973) reported the absence of paradoxical sleep in I. iguana. However, he took only recording samples of 60 s every 5 min. In our experience it is very important to take continuous recordings for several hours in order to detect As phases in reptiles, since these phases are of very short duration. Moreover, continuous visual observations throughout the recording period are necessary to distinguish behaviorally the mentioned phases. In this context, visual observations carried out by Flanigan were only done intermittently. Behavioral reactivity to repetitive electrical stimulation decreased significantly when passing from waking to behavioral sleep. Although reactivity was lower during AS than during QS this difference was not statistically significant. The absence of significance in the arousal reaction levels between the two phases of sleep could probably be related to the reduced number of specimens utilized in this experimental manipulation. In contrast, REM sleep is homeostatically regulated (Benington and Heller, 1999), therefore attempts to enter REM sleep increase during selective REM sleep deprivation and extends the time spent in REM sleep during recovery to produce a rebound effect. The concept of REM sleep rebound was initially proposed by Dement (1960) to describe the increase of REM sleep which
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
follows its instrumental suppression. This concept has been extended to encompass any increase of REM sleep above the baseline which follows alterations of the sleep-waking cycle. In this context, following cessation of enforced waking, sleep deprived lizards entered immediately to a sleep behavior and remained immobile with eye closed during a period significantly longer to that of basal conditions. Subsequent epochs of waking were of relatively of shorter duration than those in baseline conditions over the remainder of the 24-h recovery period. Total time spent by animals in QW decreased significantly, while amount of behavioral sleep showed an important increment involving both QS and AS. However, only values of QS reached statistically significant levels. Similar effects have been pharmacologically induced in other species of reptiles (Ayala-Guerrero et al., 1993, 1996) and in several mammalian species (Gottesmann, 1966; Tabushi and Himwhich, 1969) after reserpine or PCPA administration. In contrast, it has been reported that rhythmic oscillation between wakefulness and sleep depends on endogenous mechanisms, but they are also influenced by illumination schedules and light intensity (Aschoff, 1960; Borbely, 1978). Despite of the fact that these reptiles were studied under constant light conditions, the time spent in QS and AS during the astronomical day was significantly smaller than time spent in both sleep phases during astronomical night. This may be the expression of an endogenous mechanism regulating sleep distribution, such as has been described in mammals (Borbely and Neuhaus, 1978). From all previous observations, it seems reasonable to consider that the periods of quiet sleep and active sleep in I. iguana might be ana1ogous to the periods of slow wave sleep and paradoxical sleep respective1y, in mammals and birds. References Allison, T., Van Twyver, H., 1970. Sleep in the moles, Scalopus acuaticus and Condylura cristata. Exp. Neurol. 27, 564–578. Allison, T., Van Twyver, H., Goff, W.R., 1972. Electrophysiological studies of the echidna, Tachyglossus aculeatus.1. Waking and sleeping. Arch. Ital. Biol. 110, 145–184. Anderson, P., Anderson, S.A., 1974. Thalamic origin of cortical rhythmic activity. In: Rémond, A. (Ed.), Handbook of Electroencephalography and Clinical Neurophysiology, Vol 2 part C. Elsevier Publishing Company, Amsterdam, pp. 90–114. Aschoff, J., 1960. Exogenous and endogenous components in circadian rhythms. Cold Spring Harbor Symp. Quant. Biol. 25, 11–28. Astic, L., Sastre, J.P., Brandon, A.M., 1973. Etude polygraphique des états de vigilance chez le foetus du cobaye. Physiol. Behav. 11, 647–654. Ayala-Guerrero, F., 1983. Filogenia del Sueño: los mamíferos. Bol. Estud. Méd. Biol. Méx. 32, 67–82. Ayala-Guerrero, F., 1987. Sleep in the tortoise Kinosternon sp. Experientia 43, 296–298. Ayala-Guerrero, F., Mexicano, G., in press. Topographical distribution of the locus coeruleus and raphe nuclei in the lizard Ctenosaura pectinata: functional implications on sleep. Comp. Biochem. Physiol., A. Ayala- Guerrero, F., Vargas-Reyna, L., 1987. Sleep and wakefulness in the lizard Ctenosaura similis. Bol. Estud. Méd. Biol. Méx. 35, 25–33. Ayala-Guerrero, F., Huitrón-Reséndiz, S., 1991. Sleep patterns in the lizard Ctenosaura pectinata. Physiol. Behav. 49, 1305–1307. Ayala-Guerrero, F., Huitrón-Reséndiz, S., Mexicano, G., 1993. Effect of reserpine on sleep of a chelonian reptile. Proc. West. Pharmacol. Soc. 36, 227–231.
311
Ayala-Guerrero, F., Huitrón-Reséndiz, S., Mexicano, G., 1996. Effect of a depleter of cerebral monoamines on sleep patterns of a Chelonian reptile. Drug Dev. Res. 39, 115–120. Benington, J.H., Heller, C., 1999. Implications of sleep deprivation experiments for our understanding of sleep homeostasis. Sleep 22, 1033–1037. Borbely, A.A., 1978. Effects of light on sleep and activity rhythms. Prog. Neurobiol. 10, 1–31. Borbely, A.A., Neuhaus, H.V., 1978. Circadian rhythm of sleep and motor activity in the rat during skeleton photoperiod, continuous darkness and continuous light. J. Comp. Physiol. 128, 37–46. Brebbia, R.D., Pyne, T.E., 1972. Environmental hypothermia and cerebral electrical activity of the little brown bat Myotis lucifugus. Psychophysiology 9, 122–123. Butler, A.B., 1980. Cytoarchitectonic and connectional organization of the lacertilian telencephalon with comments on vertebrates forebrain evolution. In: Ebbesson, S.O.E. (Ed.), Comparative Neurology of the Telencephalon. Plenum press, New York, pp. 297–325. Dement, W.C., 1960. The effect of dream deprivation. Science 131, 1705–1707. Ebbeson, S.O.E., 1980. Comparative Neurology of the Telencephalon. Plenum Press, New York. Eiland, M.M., Lyamin, O.I., Siegel, J.M., 2001. State-related discharge of neurons in the brain stem of freely moving box turtles, Terrapene carolina major. Arch. Ital. Biol. 139, 23–34. Finlay, B.L., Innocenti, G., Scheich, H., 1991. The neocortex. Ontogeny and phylogeny. Plenum Press, New York. Flanigan, W.F., 1972. Behavioral states and electroencephalograms of reptiles. In: Chase, M.H. (Ed.), Perspectives in the Brain Sciences. 1. The sleeping brain. Brain Information Service, Los Angeles, pp. 14–18. Flanigan, W.F., 1973. Sleep and wakefulness in iguanid lizards, Ctenosaura pectinata and Iguana iguana. Brain Behav. Evol. 8, 401–436. Flanigan, W.F., 1974. Sleep and wakefulness in chelonian reptiles.II. The redfooted tortoise, Geochelone carbonaria. Arch. ltal. Biol. 112, 253–277. Flanigan, W.F., Wilcox, R.H., Rechtschaffen, A., 1973. The EEG and behavioral continuum of the crocodilian, Caiman sclerops. Electroencephalogr. Clin. Neurophysiol. 34, 521–538. Flanigan, W.F., Knight, C.P., Hartse, K.M., Rechtschaffen, A., 1974. Sleep and wakefulness in chelonian reptiles. 1. The box turtle, Terrapene carolina. Arch. Ital. Biol. 112, 227–252. Frank, M.G., 1999. Phylogeny and evolution of rapid eye movement (REM) sleep. In: Mallick, B.N., Inoue, S. (Eds.), Rapid Eye Movement Sleep. Narosa Publishing House, New Delhi, India, pp. 17–38. Freemon, F.R., Mc New, J.J., Adey, W.R., 1971. Chimpanzee sleep stages. Electroencephalogr. Clin. Neurophysiol. 31, 485–489. Gottesmann, C., 1966. Reserpine et vigilance chez le rat. C. R. Soc. Biol. 160, 2056–2061. Hartse, K.M., Rechtschaffen, A., 1982. The effect of amphetamine, nembutal, alpha-methyl-tyrosine, and parachlorophenylalanine on sleep-related spike activity of the tortoise, Geochelone carbonaria, and on the cat ventral hippocampus spike. Brain Behav. Evol. 21, 199–222. Huitrón-Reséndiz, S., Mexicano, G., Ayala-Guerrero, F., 1992. Effect of atropine sulphate on sleep of the iguanid lizard Ctenosaura similis. Proc. West. Pharmacol. Soc. 35, 157–160. Huntley, A.C., 1987. Electrophysiological and behavioral correlates of sleep in the desert iguana Dipsosaurus dorsalis. Comp. Biochem. Physiol. A 86, 325–330. Jouvet, M., 1962. Recherchees sur les structures nerveuses et les mécanismes responsables des différentes phases du sommeil physiologique. Arch. Ital. Biol. 100, 125–206. Jouvet, M., 1967. Neurophysio1oy of the states of sleep. Physiol. Rev. 47, 117–177. Karmanova, J.G., Churnosov, E.V., 1972. Electrophysiological investigation of natural sleep and waking in turtles and hens. J. Evol. Biochem. Physiol. 8, 47–53. Karmanova, J.G., Belekhova, M.G., Churnosov, E.V., 1971. Behavioral and e1ectrographic patterns of natural sleep and wakefulness in reptiles. Fiziol. Zh. SSSR 57, 504–511. Liknowski, P., Kerkhofs, M., Hauspie, R., Mendlewicz, J., 1991. A genetic study of sleep in twins living apart. Sleep Res. 20 A, 41.
312
F. Ayala-Guerrero, G. Mexicano / Comparative Biochemistry and Physiology, Part A 151 (2008) 305–312
Lindsley, D.B., Schreiner, L.H., Knowles, W.B., Magoun, H.W., 1950. Behavioral and EEG changes following chronic brain stem lesions in the cat. Electroencephalogr. Clin. Neurophysiol. 2, 483–498. Lohman, A.H., Smeets, W.J., 1991. The dorsal ventricular ridge and cortex of reptiles in historical and phylogenetic perspective. In: Finlay, B.L., Innocenti, G., Scheich, H. (Eds.), The Neocortex: Ontogeny and phylogeny, vol. 200. Plenum Press, New York, pp. 59–74. Mexicano, G., Huitrón-Reséndiz, S., Ayala-Guerrero, F., 1992. Effect of 5hydroxytryptophan (5-HTP) on sleep in parachlorophenylalanine (PCPA) pretreated birds. Proc. West. Pharmacol. Soc. 35, 17–20. Mukhametov, L., 1990. Le sommeil des mammiféres marins. La Recherche 21 (217), 41–47. Peyrethon, J., Dusan-Peyrethon, D., 1969. Etude polygraphique du cycle veillesommeil chez trois generes de reptiles. C. R. Soc. Biol (Paris) 163, 181–186. Prudom, A.E., Klemm, W.R., 1973. Electromyographic correlates of sleep behavior in a mammal, the armadillo Dasypus novemcinctus. Physiol. Behav. 10, 275–282. Romo, R., Cepeda, C., Velasco, M., 1978. Behavioral and electrophysiological patterns of wakefulness–sleep in the lizard (Phrynosoma regali). Bol. Estud. Méd. Biol. Méx. 30, 13–18. Ruckebusch, Y., 1963. Etude EEG et comportamentale des alternances veillesommeil chez l´ane. C. R. Soc. Biol. (Paris) 157, 840–844. Ruckebusch, Y., Morel, M.T., 1968. Etude polygraphique du sommeil chez le porc. C. R. Soc. Biol. (Paris) 162, 1346–1354. Ruckebusch, Y., Barbey, P., Guillemont, P., 1970. Les étates de sommeil chez le cheval (Equus caballus). C. R. Séances Soc. Biol. (Paris) 164, 658–666.
Schlehuber, C.I., Flanigan, D., Laage, D.G., Spooner, E., 1974. Paradoxical sleep in the chick (Gallus domesticus). Behav. Biol. 11, 537–546. Susic, V.T., Kovacevic, R.M., 1973. Sleep patterns in the owl Strix aluco. Physiol. Behav. 11, 313–317. Tabushi, K., Himwhich, H.E., 1969. The acute effects of reserpine on the sleep– wakefulness cycle in rabbits. Psychopharmacology 16, 240–252. Tauber, E.S., Roffwarg, H.P., Weitzman, E.D., 1966. Eye movements and electroencephalographic activity during sleep in diurnal lizard. Nature 212, 1612–1613. Tauber, E.S., Rojas-Ramírez, J.A., Hernández-Peón, R., 1968a. Electrophysio1ogica1 and behavioral correlates of wakefulness and sleep in the lizard Ctenosaura pectinata. Electroencephalogr. Clin.Neurophysiol. 24, 424–433. Tauber, E.S., Michel, F., Roffwarg, H.P., 1968b. Preliminary note on the sleep and waking cycle in the desert hedgehog (Paracchinus hypome1as). Psychophysio1ogy 5, 201. Vasconcelos-Dueñas, 1., Ayala-Guerrero, F., 1983. Filogenia del sueño en las aves. Bol. Estud. Méd. Biol. Méx. 32, 83–90. Vasilescu, E., 1970. Sleep and wakefulness in the tortoise (Emys orbicularis). Rev. Roum. Biol., Ser. Zool. 15, 177–179. Walker, J.M., Berger, R.J., Scott, T.D., 1973. Absence of electrophysiological signs of sleep in the tortoise (Testudo denticulata). Sleep Res. 2, 88. Warner, B.F., Huggings, S.E.A., 1978. An electroencephalographic study of sleep in young caiman in colony. Comp. Biochem. Physiol. A 59, 139–144. Zepelin, H., 1994. Mammalian sleep. In: Kryger, M.H., Roth, T., Dement, W.C. (Eds.), Principles and Practice of Sleep Medicine. W.B. Saunders Company, Philadelphia, pp. 69–80.