Reptilian waking EEG: slow waves, spindles and evoked potentials

Electroencephalography and clinical Neurophysiology , 90 (1994) 298-303

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© 1994 Elsevier Science Ireland Ltd. 0013-4694/94/$07.00

EEG93651

Reptilian waking EEG: slow waves, spindles and evoked potentials Luis De Vera a, Julifin Gonzfilez a and Rub6n V. Rial b,, a Departamento de Fisiolog[a, Laboratorio de Biof[sica, Facultad de Medicina, Unit,ersidad de La Laguna, 38320 Tenerife (Spain), and h Departamento de Biologia Fonamental i Cikncies de la Salut, Universidad de les llles Balears, 07071 Palma de Mallorca (Spain)

(Accepted for publication: 26 November 1993)

Summary Signal spectral analysis procedures were used to compute the power spectrum of Gallotia galloti lizards EEG at different (5-35°C) body temperatures. EEG power spectra were mainly characterized by a low frequency peak between 0.5 and 4 Hz which was present at the different body temperatures. A second spectral peak, corresponding to spindles of similar pattern to the sleep spindles of mammals, also appears in the spectra. The peak frequency of the spindles increased with the body temperature. Flash evoked potentials were characterized by a slow triphasic component upon which a spindle was superimposed, adopting a morphology similar to the K complexes of mammalian sleep. The characteristics of this EEG and evoked potentials support the hypothesis of homology between the waking state of the reptiles and the slow wave sleep of mammals. Key words: Slow wave EEG; Slow wave sleep; Spindles; Evoked potentials; Sleep evolution; Spectral analysis

Slow EEG waves (SWs) from 0.5 to 4 Hz with large amplitude (75-400 ~V) are the most prominent characteristic of the quiet, NREM or slow wave sleep in mammals. These SWs disappear after large cortical ablations (Jouvet 1962) but they persist after bilateral thalamic destruction (Villablanca 1974). After these reports, the SWs have been considered as produced in the cortex, and the lack of a well developed forebrain cortex in reptiles is considered as the main cause of failure in demonstrating the SW sleep stage in these animals. However, the cortical origin of the SWs is not yet indisputably established. Typical large amplitude SWs have been recorded in the reticular thalamic nucleus of the cat after disconnection from the cerebral cortex (Steriade et al. 1987). The same type of SW EEG has been demonstrated in the mammalian ventro-lateral thalamic nucleus in close time relation with spike barrages in the neurons of deep cerebellar nuclei, its major source of inputs (Steriade et al. 1971). Moreover, it has been demonstrated that hyperpolarized thalamic neurons show both spindle-like and delta-like oscillations resulting from the interplay of their intrinsic currents (Steriade et al. 1991).These reports suggest

* Corresponding author. Tel.: 34 (9) 71 173000; Fax: 34 (9) 71 173184.

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that the SW origin may actually transcend the cerebral cortex. In the same way, SWs have been reported in the resting EEG of waking, active reptiles (Gonzfilez and Rial 1977) contradicting many studies of reptilian sleep in which the SW EEG seemed to be consistently absent. Some studies from the Russian school (Karmanova 1982) also reported SW EEG. They also stated that, contrarywise to mammals, behavioral activation was correlated with EEG synchronization. On the other hand, reptiles are poikilothermic animals, lacking an advanced physiological temperature control. These animals, however, when active and under natural conditions, are quite able to regulate their body temperature. This is mainly achieved thanks to a well developed behavioral control. Of course, during the low activity phase of the circadian cycle, these mechanisms are put off and the temperature regulation of the body is abandoned. As a consequence, the lack of SW EEG reports in reptiles may not be due to the lack of a well developed cortex but only to a temperature dependent depression of brain activity. This low activity would be present in coincidence with the time of the expected sleep and SW EEG. The failure in recording SW in reptiles would thus be easily explained. There is another important EEG characteristic of the mammalian slow wave sleep. During this stage the

R E P T I L I A N W A K I N G EEG

E E G is currently indented with spindles which are defined as waxing-waning waves at 7-14 Hz grouped in sequences of 1.5-2 sec and recurring each 5-10 sec (Steriade and Desch~nes 1984). These values are the typical ones, but considerable interspecies variation may be found. The spindles are characteristically associated with blockage of information transfer through the thalamus and with unconsciousness (Steriade and McCarley 1990). On occasion the sleep spindles are preceded by large spikes (K complexes) which have been considered as abnormally high evoked potentials (Ujszaszi and Halfisz 1986). Interestingly, very similar spindles have been also described in the E E G of active reptiles (Strejckovfi and Servlt 1973; Gonzfilez and Rial 1977). The reptilian spindles have also been considered as serving thalamo-cortical relations and may easily be evoked by sensory stimulation. Taking together all this information, it appears that the resting E E G of active reptiles has several significant features which are very similar to those observed during the SW sleep of mammals. This coincidence has been used to develop a hypothesis on the evolutionary history of mammalian sleep (Rial et al. 1993). The present work aims at the quantitative characterization of the reptilian waking E E G and its dependence on body temperature. This would confirm the presence of both SW and spindles in the E E G of this animal group and perhaps would serve to explain the failure to recognize these traits in the available reports of reptilian sleep.

Material and methods

Twenty lizards of the species Gallotia galloti of both sexes, 23-31 cm in length, and 15-72 g in body mass from the island of Tenerife (Canary Islands, Spain) have been used. The animals were captured from their natural habitat and kept in terraria under a 12/12 h light-dark schedule, for at least 10-15 days prior to the beginning of any experiments. A temperature of 2024°C was maintained during the light phase, descending to 17-20°C during the dark one. The relative humidity ranged from 50 to 60%. The animals were fed with masticated fruits provided ad libitum and had tap water continuously available. The experiment comprised surgical implantation of electrodes, recording and computerized acquisition of the lizard E E G and flash evoked potentials at 7 monitored temperature levels in the range 5-35°C. Under pentobarbital sodium anesthesia (30 / z g / g body mass intraperitoneally) each animal was implanted with one or two stainless steel, teflon coated electrode(s) of 100 ~ m diameter. The electrode tips were placed on the surface of the right medial cortex. A stainless steel screw was secured to the occipital

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bone outside the brain cavity and served as a reference electrode in monopolar recording. The whole set was glued to the skull with acrylic cement. At least 1 week was allowed for recuperation. No postoperative drugs such as antibiotics were employed. After the postoperative recovery period, the lizards exhibited similar types of general activity to those of the unoperated ones. Core body temperature was monitored by an electrical thermometer (Nihon Kohden M G A III-219) provided with a small thermistor probe that was inserted into the animal's cloaca 2 cm deep; thermistor leads were attached to the tail. The E E G and temperature recording were performed with the animals in a 50 × 28 x 25 cm thermostatic (+0.5°C) chamber. All the experiments were carried out in still air and the animals were free to run in the chamber. Relative humidity in the chamber ranged between 35 and 40%. In all experiments the animal was kept within the chamber for a 24 h habituation period, after which measurements were made. During each recording session, the laboratory was kept dimly lit, and strong noises were avoided. Monopolar and bipolar EEGs were recorded with a Nihon Kohden RM-85 polygraph using a 0.3 sec time constant and a 30 Hz high filter. The E E G was analyzed at body temperatures of 5, 10, 15, 20, 25, 30 and 35°C. The E E G signal from the recording system was fed on-line into an automatic data acquisition and processing system for small animals based on a microcomputer. This system (Gonzfilez and De Vera 1984) has been developed and updated in our laboratory. For Fourier analysis purposes, E E G signal segments of 1 min duration were sampled at 128 Hz. We used the ensemble averaging time series analysis technique (Otnes and Enochson 1978) to compute an average spectrum of the E E G signals recorded at each body temperature. In order to perform this procedure, each of the 1 min series was first divided in segments of 2 sec duration. Then the signal segments were normalized by subtracting the mean value of each segment from each data point, trend removed and cosine tapered over the first and last 10% of the samples (Bendat and Piersol 1971). After the application of these digital preprocessing techniques, the power spectral density functions (PSD) of the different E E G segments were calculated, using a fast Fourier transform (FFT) algorithm. Each PSD was frequency-smoothed by averaging the results for 5 contiguous spectral components. The estimates obtained from all the records at each body temperature were averaged, resulting in one ensemble-averaged PSD per temperature. A Nihon Kohden SLS-2141 photostimulator (constant intensity, 10 ~zsec duration flash), placed 35 cm above the lizard's head, was used to produce flash evoked potentials (FEP). The attitude of the lamp eliminated shadows from the surround. Post-stimulus

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segments. This averaging was not made to extract the FEP from noise or from other signals, but only to characterize its main features.

Results Examples of spontaneous EEGs obtained at the body temperatures of 5, 15, 20 and 35°C are shown in Fig. 1. Samples of analog records are shown in Fig. 5.

TABLE I Peak frequency of the E E G spindles as a function of the body temperature.

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N: number of E E G signal segments where spindles were present.

REPTILIAN WAKING EEG

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E E G s were polymorphic and mixed in frequency. The main E E G feature was the presence of slow waves at the different body temperatures. It could also be seen that the amplitude of the E E G waves increased with body temperature: at 5°C the amplitude never surpassed 10 /xV (mean + S.D.: 4.5 + 1.8 p,V) while at 35°C components were found reaching up to 60 /xV and more (mean _+ S.D.: 51.6 _+ 5.6/xV). Fig. 2 shows examples of E E G segments at 15°C in which spindles were present. Spindle amplitude was slightly greater than the slow E E G waves. The spindles were uncorrelated with breathing or any other observable behavior. The average power spectra of segments of the E E G in which spindles were present are shown in Fig. 3. The power spectra confirmed the presence of slow waves at all the studied temperatures, the main power fraction being always under 4 Hz. A second spectral peak corresponding to the spindles could also be seen. The peak frequency of the spindles was highly dependent on temperature, shifting from low to high frequencies as temperature increased. A very good linear regression (r = 0.995) between the spindle peak frequencies

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and the t e m p e r a t u r e was obtained (Table I) ranging from less than 5 Hz at 5°C to more than 30 Hz at 35°C. Examples of FEPs are shown in Fig. 4. Each record shows the average of 20 responses. A single F E P consisted of a high voltage (above 200 /xV at 35°C) positive-negative-positive wave which, on many occasions, was prolonged in a short spindle. The amplitude of the FEP and the frequency of the subsequent spindle continued to be highly dependent on body temperature.

Discussion The presence of SW in the resting E E G of the lizard Gallotia galloti has been demonstrated by quantitative analysis. In fact, up to 70% of the E E G power lies in the delta frequencies, between 0.5 and 4 Hz. The amplitude of this SW E E G is quite coincident with the normal one in sleeping mammals. Some reports of reptilian slow waves are known (Gonzfilez and Rial 1977; Karmanova 1982) but there are also many reports which do not show, and even deny, the possibility of delta waves in the reptilian E E G . This may be due to several different causes. (1) Genera and species differences. For instance, Peyrethon and Dusan-Peyrethon (1969), in their classic paper, described important differences between Python, Caiman and Iguana. Present reptiles are a very diversified group which may show extremely different adaptations and lifestyles. These differences must be apparent in the structure and function of their nervous system. (2) Idiosyncratic differences. In our own experience the relative E E G power spectrum varied between different animals. The number of studied specimens in most reports has been quite small to ensure significant results. There are no data on the ontogeny of the reptilian E E G but this factor may also be important in explaining differences between individuals of the same species. In the reported experiments all specimens were adult, aged over 4 - 5 years as estimated from their body size (Castanet and Baez 1988). (3) Temperature effects. This must be a very important effect, as shown in the present results. At low body temperatures the E E G amplitude is quite small: the low frequencies may be present but they show an extremely low voltage. There are many reports failing to measure the temperature of the recorded animals. Other reports only state the ambient temperature but not the body temperature. In addition, most researchers may have been looking for the SW at the wrong time, when the animals were at their lower body temperature and showing their lower brain activity. As a consequence, it must have been quite easy to miss the SW E E G .

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(4) Others. For instance, Tauber et al. (1968) reported a time constant of 0.1 sec in their recording. This is approximately equivalent to a low cut-off frequency of 1.5 Hz. Other reports (Herman et al. 1964; Peyrethon and Dusan-Peyrethon 1969; Flanigan et al. 1973; Flanigan 1974; etc.) did not report the used time constant. The spindles in the reptilian EEG have been correlated with the respiration (Flanigan et al. 1973), but in the presented results this was not the case. The spindles in the forebrain of the turtle (Strejckovfi and ServR 1973) were described as having two components: a rapid one of regular sinusoidal activity at 10-30 Hz and low voltage (15-20 txV) upon which a slow transient, with two or three phases, was superimposed. These authors remarked the similitude between the reptilian spindles and those of the human EEG, as the spindles were easily triggered by sensory stimuli and on occasion (but not always) occurred simultaneously in the thalamus (nucleus rotundus) and in the cortex. It is evident that their description fits fairly well with the spindles described in the present results and with the mammalian sleep spindles.

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The similitude of the reptilian FEP and the K complexes to those of mammalian sleep is also quite clear. It should be stressed, as a special feature, that the reptilian FEP has shown a very large amplitude, which makes the use of averaging techniques unnecessary. This marks an important difference from the mammalian evoked potentials. At the same time, the FEP shows a close similitude with the K complexes of mammalian sleep. In this respect, the K complexes of human sleep are considered as extremely large evoked potentials (Ujszaszi and Halfisz 1986). The only difference between the reptilian FEP and the K complexes lies in the broad frequency range of the following spindle in reptiles, that clearly exceeds the range of sigma activity (14 Hz). However, the high frequency variability of the reptilian spindles is clearly due to the effects of temperature. Mammals having a fixed temperature, it is not surprising to find a more constant frequency in their responses. Summarizing, the resting EEG of the lizard Gallotia galloti shows SW and other remarkable similitudes with that of SW sleeping mammals. This supports the hypothesis that the reptilian waking state and the

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REPTILIAN WAKING EEG

mammalian SW sleep are homologous states and that the true evolutionary acquisition of mammals is not sleep, but advanced wakefulness (Rial et al. 1993). The correlation found between EEG synchronization and behavioral activation (Karmanova 1982) further supports this idea. This research was in part supported by the "Fondo de lnvestigaciones Sanitarias (FIS) Ministerio de Sanidady Consumo," Grant 91/0609, from the "Consejerla de Educaci6n del Gobierno de Canarias," Grant 09/08.03.90, and from the "Direcci6n General de lnvestigaci6n Cientlfica y T6cnica (DGICYT), Ministerio de Educaci6n y Ciencia," Grant SM90-0007.

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303 Jouvet, M. Recherches sur les structures nerveuses et les m6chanismes responsables des diff6rentes phases du sommeil physiologique. Arch. Ital. Biol., 1962, 100: 125-206. Karmanova, I.G. Evolution of Sleep: Stages of the Formation of the Wakefulness-Sleep Cycle in Vertebrates. Karger, Basel, 1982. Otnes, R.K. and Enochson, L. Applied Time Series Analysis. Vol. 1. Basic Techniques. Wiley-Interscience, New York, 1978. Peyrethon, J. and Dusan-Peyrethon, D. Etude polygraphique du cycle veille-sommeil chez trois genres de reptiles. C.R. Soc. Biol. (Paris), 1969, 163: 181-186. Rial, R.V., Nicolau, M.C., L6pez-Garcia, J.A. and Almirall, H. On the evolution of waking and sleeping. Comp. Biochem. Physiol., 1993, 104A: 189-193. Steriade, M. and Desch~nes, M. The thalamus as as neuronal oscillator. Brain Res. Rev., 1984, 8: 1-63. Steriade, M. and McCarley, R.W. Brainstem Control of Wakefulness and Sleep. Plenum Press, New York, 1990: 309-324. Steriade, M., Ap6stol, V. and Oakson, G. Control of unitary activities in cerebello-thalamic pathways during wakefulness and synchronized sleep. J. Neurophysiol., 1971, 34: 384-413. Steriade, M., Domich, L., Oakson, G. and Desch~nes, M. The deafferented reticular thalamic nucleus generates spindle rhythmicity. J. Neurophysiol., 1987, 57: 260-273. Steriade, M., Curr6 Dossi, R. and Nufiez, A. Network modulation of a slow intrinsic oscillation of cat thalamocortical neurons implicated in sleep delta waves: cortically induced synchronization of brainstem cholinergic suppression. J. Neurosci., 1991, 11: 32003217. Strejckovfi, A. and Servlt, Z. Isolated head of the turtle. A useful experimental model in the physiology and pathophysiology of the brain. Physiol. Bohemoslov., 1973, 22: 37-41. Tauber, E.S., Rojas-Ramlrez, J. and Hernandez-Pe6n, R. Electrophysiological and behavioral correlates of wakefulness and sleep in the lizard, Ctenosaura pectinata. Electroenceph. clin. Neurophysiol., 1968, 24: 424-433. Ujszaszi, J. and Halfisz, P. Late component variants of single auditory evoked responses during non-REM sleep stage 2 in man. Electroenceph. clin. Neurophysiol., 1986, 64: 260-268. Villablanca, J. Role of the thalamus in sleep control: sleep-wakefulness studies in chronic diencephalic and athalamic cats. In: O. Petre-Quadens and J.D. Schlag (Eds.), Basic Sleep Mechanisms. Academic Press, New York, 1974: 51-78.