Changes of brain pO2 during paradoxical sleep in cats

Physiology and Behavior. Vol. 3, pp. 477--485. Pergamon Press 1968. Printed in Great Britain

Changes of Brain P02 During Paradoxical Sleep in Cats' E. G A R C | A A U S T T , R. V E L L U T I A N D J. I. V I L L A R

Laboratorio de Neurofisiologia, Institute de Neurologia, Facultad de Medicina, Hospital de Clinicas, Montevideo, Uruguay (Received 22 J a n u a r y 1968) GAItciA AusYr, E., R. VELLUT!ANDJ. I. VnJ.AR Changesof brainpOt duringparadoxical sleep in cats. PHYSIOL.BEHAV. 3 (3) 477-485, 1968.--Cats were chronically implanted with polarographic cathodes in different regions of the brain to record the local oxygen concentration or pressure. The cortical electrical activity, the electromyogram of the neck muscles and the eye movements were also recorded as indicators of wakefulness and slow paradoxical sleep. Animals were studied in unrestrained conditions during these behavioural states. In every experiment the oxygen-dependance of polarographic electrodes was tested by changing the oxygen concentration in the inhaled gases. During paradoxical sleep phasic pOt changes were observed in some nuclear structures char~terized by a dramatic increase in the amplitude of oscillations. These changes were found in a number of subcortical and brain stem regions (Table 1) and were never observed in the nee and archicortex, in the specific thalamic nuclei or in the white matter. Increased brain pOI oscillations constitute an indicator of the PS just as good or better than the electrucortical activation or the electromyogram. In some cases they heralded the PS. The changes observed in the pOs during the PS differed from those recorded during arousal and alertness because in the two latter situations they extended to all of the brain areas studied. It is postulated that these variations be due to a local increase of neuronal activity. Paradoxical sleep

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CHRONICALLYimplanted polarographic electrodes have been employed to study in animals the cerebral oxygen supply, the influence of carbon dioxide and of a wide variety of drugs [2]. The brain oxygen concentration or pressure (pO.) recorded with polarographic cathodes from the extracellular compartment is fundamentally a function of the local oxygen-consuming metabolic activity and of the local oxygen-supplying blood flow. It has been reported that during alertness and paradoxical sleep, the central nervous system behaves very similarly. The cerebral blood flow [11] and the neuronal activity recorded with microelectrodes from the brain cortex [4] and from the reticular formation [8] vary in both situations along the same lines. Hence it seemed of interest to study the brain pOt changes during PS and to compare them with those observed during alermess in unrestrained cats with chronicallyimplanted polarographic electrodes. Preliminary results in this connection have been reported [5, 6, 12, 13], the pOt changes during arousal and alertness being also described [14].

ends, the wire tips were left exposed 1 mm and 10 mm for the cathode and the anode, respectively. One to four cathodes were stereotaxically implanted in each animal in the cerebral cortex and in deep brain structures. They were anesthetized with sodium pentobarbital intraperitoneally (40 mg/kg) and operated upon under aseptic conditions. Generally, for each cathode one reference silver electrode was implanted in the neighboring area about 5 mm distant. The interelectrode resistance was about 20 K. A constant voltage of -0.7 V was applied to the cathode. Voltage changes were measured across a 270 fi resistance in a Grass Poligraph (Model 5) by means of Low Level DC Preamplifiers (Model 5 Pl). Owing to the resistance ratio the system was actually recording current. The amplifiers had a half-amplitude response at a freqmmcy of 0.1 sec. So only the slowest oscillations of the current were recorded, To control the state of wakefulness, slow sleep (SS) and paradoxical sleep (PS), additional electrodes were implanted in the neck muscles, on both sides of the eyes and in the cerebral cortex, to record simultaneously with the brain pOt changes, the electromyogram(EMG), the eye movements (EM) and the electrocorticogram (ECoG), respectively. In addition, the behavior of the animal was appraised by putting it into a closed plastic transparent cage where the air was renovated at a constant rate. In every experiment the oxygen-dependance of the polarographic electrodes was tested by changing the

MATERIAL AND METHODS

Twenty six cats weighing 2.5-3.5 kg were implanted with polarngraphic electrodes. The anode was made of a AgCI-Ag wire or 0.28 mm dia. coated with epoxy resin. In the first ~ t s the cathode had the same diameter and was isolated in the same way. In the last few experiments the cathode was made of 0. I mm dia. platinum wire coated with

tThis work was carried out with the support of the National Isstitutes of Health of the United States (Grants NB 02306-05--06) 477

478

GARC|A AUSTT, VELLUTI AND VILLAR

oxygen concentration into the cage, replacing air by pure oxygen or nitrogen. The recording of brain pO2 was uncalibrated because the oxygen diffusion coefficient in the implanted region was not known. In some experiments the systemic arterial pressure was recorded by means of an etectromanometer (Statham P23AC) with a catheter chronically implanted in the femoral artery. Animals ~ere studied daily during 3-12 hr periods. Records were obtained at a low paper speed of 9 mm/min. This speed was optimal to appraise the pO2 changes. The electrodes began to respond satisfactorily to systemic pO2 changes within 2-4 days after implantation and in many cases they did so as long as 92 days, the longest period of study. In a few cases there was early poisoning of the electrodes, while in others they never responded to changes in oxygen concentration. These latter animals were discarded. All brains ~ere studied histologically in order to determine the electrode position. RESULTS

During SS animals showed low-frequency and lowamplitude brain pO~ oscillations. This phenomenon was observed in all the studied regions. Frequency was some times higher in sub-cortical structures. As long as the level of SS was kept constant, no changes were observed in the brain pO~. On passing from SS to PS there were important phasic changes of pO2 characterized by a considerable increase in the amplitude and generally a decrease in the frequency. In some instances in which the pO~ recorded during SS failed to exhibit oscillations, these appeared during the period of PS. The constant finding was the increase in the amplitude of oscillations. In some cases an increase in the frequency was observed. These changes in the pattern of pO~ oscillations were constantly found in all of the animals during all the period of PS recorded. However, they were not observed in all the regions studied. In Table 1 the brain regions where pO2 TABLE 1 CHANGES OF pO 2 DURING PARADOXICAL SLEEP

NEOPALLIUM G. suprasylvianus medialis G. lateralis G. coronalis G. ectosylvianus medialis G. suprasylvianus posterior (Visual cortex) PALEOPALLIUM Cortex pyriformis ARCHIPALLIUM Hippocampus dorsalis THALAMUS N. anterior ventralis N. centralis medialis N. corporis geniculatum lateralis HYPOTHALAM US Area preoptica MESENCEPHA LON Formatio reticularis PONS Formatio reticularis

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changes were so far found during PS, are listed. As seen in the pallium, there were no changes during PS, the piriform cortex being the only exception. On the other hand, they were observed in a large number of subcortical and brain stem structures. It is interesting to remark that changes were demonstrated in the thalamus, in the reticular and rhinencephalic nuclei but not in a specific nucleus as the lateral geniculate body. Also, when the cathodes were implanted in the white matter they did not shown any significant change. In Fig. 1 the changes of brain pO, during PS are shown. During a 12 min period of PS, recorded in A between vertical lines 1 and 2, a striking increase in the amplitude of pO2 waves in the mesencephalic reticular formation (MRF) and in the nucleus amigdaloideus (NA), is observed. However, at the same time, the cortical pO2 does not show any significant variations in the pattern of oscillations or in the general level of pressure even though the sensitivity of this cathode was more than twice as high as that of the cathode implanted in the mesencephalic reticular formation, as shown in B. The characteristic changes of the polarographic current during PS were observed only when the cathodes were oxygendependant. When the electrodes were poisoned or when they were not activated by the 0.7 V, the current also evidenced slow oscillations of low amplitude which did not change in any behavioral condition. As seen in Fig. 1 a correlation between the pOs oscillations and the episodes of rapid eye movements was not observed. Figure 2 shows different patterns of changes of mesencephalic pO2 in different animals during PS (between vertical lines). In all these experiments the cortical pO~ did not exhibit any change. In A big oscillations at about the same frequency as during SS are observed; in B oscillations, not present during SS, appeared during PS and a tonic increase of PO2 is seen; in C oscillations of higher amplitude and frequency are shown; in D, between vertical lines 1-2, a pattern similar to C is observed and between 3-4 a slight increase of the pressure is additionally present. In A, B and C the period of PS is followed by a short period of alertness and later on by SS. In D the two phases of PS are interrupted by a short phase of SS evidenced only by the bursting in the ECoG without changes in the EMG. Concomitantly, the pO~ oscillations disappear. As shown in these experiments, the changes of brain pO, were an indicator of PS just as reliable as the EMG, the ECoG or the recording of eye movements. Moreover, in some instances, the pO, changes preceded the changes of these other variables. In Fig. 3 two experiments performed in the same animal on the 8th and on the 37th day after implantation, are shown. In both instances both a long and a short period of PS was selected. The beginning ofpO~ changes during PS is indicated by a vertical line. In A, B and C the increase of pO~ oscillations at the M R F precede the disappearance of the EMG, the electrocortical activation and the appearance of the rapid eye movements. On the 8th day, concomitantly with the phasic changes of pO~ there is also a tonic increase. This latter phenomenon was not present on the 37th day. The same variation in the pattern of changes during PS was observed in other animals. On the other hand cats showed a constant rate of tonic decrease of brain pO~ during PS. This is observed in Fig. 4 where in A the following .sequence was recorded, SS (up to 1), PS (between 1 and 2), a short arousal (immediately after 2) and SS. During PS, great oscillations and a sustained rate of decrease of both pressures in the nucleus centralis medialis

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FIG. 1. p O 2 changes in subeortical structures and in the neocortex during PS. EMG, electromyogram of the neck muscles; ECoG (SSG) electrocorticogram of the suprasylvian gyrus; EM, eye movements; POs, oxygen pressure from the suprasylvian gyrus (SSG), the mesencephalic reticular formation (MRF) and the nucleus amygdaloideus (NA). In A at the beginning of the record, the cat is in SS, the polarographic current exhibits small oscillations with a higher frequency in the subeortical structures. Subsequently, a 12-min period of PS is shown between vertical lines 1 and 2, characterized by the disappearance of the tonic EMG and the appearance of twitches, the activation of the ECoG and the presence of episodic rapid eye movements. Concomitantly with these changes high-amplitude oscillations of slower-frequency in the MRF and in the NA, and no variations in SSG of the brain PO2 are observed. The oscillations axe monophasic with an upward peak and a slight tonic increase of the pressure is observed particularly in the PO2 (NA) recording. After 2 the animal passed from PS to SS and the pOs recording acquires the same characteristics as before 1. In B during a period of wakefulness, the air of the cage was changed by pure oxygen (O2), a sudden increase of all pO~ being provoked. In the recordings from the SSG and the NA the big increase of the currents results in a blockage of the galvanometers (indicated by arrows). The response of the cathode in the SSG is more than twice as intense as that of the cathode in the MRF.

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FIG. 2. pO~ changes in the mesencephalic reticular formation and in the neocortex during PS. A-D, different experiments in four cats. Same abbreviations as in Fig. I. PS is shown between vertical lines 1--2 in A--C and between 1-2 and 3-4 in D with disappearance of tonic EMG and ECoG activation. An increase of the amplitude of pOs oscillations in A, C and O and the appearance of oscillations in B, during PS, is observed in the M R F but not in the cerebral cortex. For more details, see text.

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FIG. 4. Changes in the thalamic and mesencephalic pOs during PS. CM, nucleus c~ntralis medialis. Other abbreviations, as in Fig. I. A, PS between lines 1 and 2. B, PS between lines I and 2 and lines 4 and 5, and arousal after lines 3 and 6. Or, pure oxygen is given to

the animal. The changes in pOt are similar but not identical in both regions during PS. During arousal these are also pO2 oscillations. For ntore details see text.

and in the M R F are observed. On passing from PS to arousal and then to SS the pressure slowly reacquired its previous level and the oscillations decreased. At B, between broken vertical lines 1-2 and 4-5, two short periods of PS are observed with the same changes in both pOl. At vertical line 3 the animal arouses and there is a temporary increase of thalamic pO8 but no changes in the mesencephalic pO~. At the end of the record, pure oxygen (O,) is given to the animal, the increase in both pO, shows that both electrodes are oxygen-dependant. At vertical line 3 an arousal is observed with a transient increase in the thalamic pOs. At 6 again during arousal a similar increase was observed in both pO~, thalamic and mesencephalic. In this animal the pO~ changes in two distant subcortical areas were similar but not identical, a rather constant finding at the subcorticai levels in all the cats studied.

In other instances the same animal exhibited either a tonic increase or decrease o f the pO, in successive periods of PS. This finding is reported in Fig. 5 in which two periods of PS recorded on different days are shown. At A (between l and 2) a slight increase of the mean level of pOs ( M R F ) is visualized, the opposite changes being seen at B (between 2 and 3). In this animal neither the phasic nor the tonic changes of the systemic arterial pressure (AP) were related to the phasic or tonic variations of pOs at the M R F during PS. When in the same brain area the pOt was responsive both to alertness and PS the pattern of changes was about the same. The only difference found was that the tonic increase in pOs appeared as more constant during alertness than during PS. In Fig. 6 a period of alertness is s h o w n at A (between lines l and 2). After arousal from SS (line I) the animal was seen exploring, moving around the cage. An increase of the

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FIG. 5. PO2 changes in the mesencephalic reticular formation and in the arterial pressure during PS. A and B two experiments performed in the same cat with a 24-hr interval. AP, systemic arterial pressure recorded with a catheter implanted in the femoral artery. Note that the recording at B was obtained with less amplification. Other abbreviations, as in Fig. 1. At A between lines 1 and 2, a period of PS with disappearance of tonic EMG, activation of the ECoG, tonic decrease of mean arterial pressure and tonic increase of POt with greater oscillations, is observed at the same time, but there ar~also slight phasic changes in AP during PS which do not coincide with the phasic POt variations, Between 2 and 3 during a short period of SS the POt oscillations decrease and the AP increases to reach the initial value after 3 during alertness. At this time the POt increases considerably. Arrows indicate blockage of the galvanometers. In B a short period of alertness is observed after 1, accompanied by phasic AP and pOt changes. This episode is followed by another short period of SS with tonic decreases of AP and tonic increase of pOt. Between 2 and 3 during PS, phasic changes and a sustained rate of tonic decrease in POt is observed. AP decreases tonically, the oscillations reach higher amplitude. Between lines 3 and 4 the animal goes into SS, the AP increases and the phasic POt changes disappear. Between 4 and 5 a l-min period of PS is observed with POt oscillations and AP decrease. After 5, during arousal both pressures increase phasically. This experiment shows that neither the phasic nor the tonic changes in AP and brain pO~ are concomitant.

amplitude and a decrease in the frequency of oscillations of the pOt ( M R F ) was observed as long as the animal remained in the same state (up to 2). Subseqnently, it went to SS (after 2) and the pOt reacquired the previous characteristics. At B a period of PS is observed (between 1 and 2) with similar changes in the pOt but with a slightly greater amplitude of the oscillations. The animal passed from PS to alertness (in 2) and maintained this state for about 5 min (up to 3). At the same time the pOt ( M R F ) showed the same pattern of oscillation. A t C a period of PS is observed (between 1 and 4) with a 1 min interruption by SS (between 2 and 3). N o changes arc observed in the E M G during this interruption, but the pOt abruptly stopped the high-amplitude oscillations. In this case again the pOt was a more sensitive indicator of PS than the E M G . DISCUSSION

F r o m the data reported above it can be concluded that the recording of brain pO, in some subeortical structures is a very sensitive indicator of PS. It was found that phasic changes were constant at this stage of sleep, particularly, the clear cut increase of the amplitude of oscillations, as their frequency may increase or diminish. The tonic changes were also inconstant and in either direction, i.e., increase or decrease of p o t according to the animal and even in the same animal, according to the days. It is probable that the first few days after implantation they be due to an artifact, as they disappear within the few subsequent days. This might be ascribed to incomplete cicatrization of the lesion produced by the electrode at implantation as a result of which oxygen diffusion would be disturbed at the zone of the electrode.

In order to clearly appreciate pO~ changes it was necessary to record at a very low speed as the ample oscillations were generally of a very low frequency. Under these conditions the changes of brain pO~ were more or equally effective than the E M G and/or the E C o G in tracing off the PS periods. In the cat the rapid eye movements (REM) were always less useful in determining the PS periods, for they appear after muscular hypotonia and after electrocortical activation. On the other hand, the periods of REM are not present throughout the PS period but in the form of bursts separated by periods o f inactivity. In some instances the periods of PS were heralded by the phasic changes of pOt which appeared before any change observable either in the E M G or in the ECoG. In a previous paper [14] it was reported that during arousal the pOt increased tonically and diffusely in all the brain regions studied. Also during sustained alertness the pOt showed phasic changes characterized by an increase in the amplitude of oscillations similar to the ones described during PS. The changes of brain pOt during PS could be ascribed to increased neuronal activity in some regions of the brain. The same characteristics has been postulated to explain the changes observed during arousal and alertness [14]. Obviously, after complete cicatrization and owing to the size of the polarographic electrodes, it can be assumed that the cathode is recording the pOt of the extracellular compartment. The oxygen concentration in this compartment is a function of the local oxygen-consuming metabolic cellular activity and of the local oxygen-supplying blood flow. One may rule out that the changes be due to modifications of the systemic pOt as they were not found in all of the areas studied

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FIG. 6. pO~ changes in the mesencephalic reticular formation during alertness and paradoxical sleep. Same abbreviations as in Fig. 1. A, between 1 and 2, during a period of alertness when the animal is exploring, moving around the cage, the pO= shows phasic changes. The high amplitude episodes in the ECoG are movement artifacts. B, between 1 and 2 during PS, similar changes are observed. This period is followed by an episode of alertness (between lines 2 and 3) and the oscillations persist but with a lower amplitude. In C, another period of PS is showed between 2 and 3, as evidenced by the bursting in the ECoG but without changes in the EMG. Concomitantly, the pO, oscillations are interrupted. For more details see text.

and because they bear no relationstup to either the phasical or tonic changes in arterial pressure. Still it might be speculated that these brain pO, changes be the result of respiratory modifications concomitant to the PS [9, 10] and that the changes be seen only or preponderantly in some regions of the brain owing to their peculiar characteristics, for instance, capillary vascularization. However, this interpretation may also be fully discarded inasmuch as when the systemic pO= is raised by changing the inhaled air for pure oxygen, the increase in brain pO, is comparable in all the areas, even in those where no changes were observed during the PS (Figs. 1 and 4). The local homeostasis in the CNS is ensured by a well known chernical regulating mechanism. When the neuronal activity increases, the local carbon dioxide concentration or pressure (pCO=) also increases in the extracellular compartment as a result of the diffusional flow from the intracellular compartment. The increased pCO= causes a rise of local blood flow by diminishing the vascular resistance. This rise in blood flow provokes an increase of the local pOz which has previously decreased as a result of the cellular intake. This mechanism of control is adjusted by two negative feedbacks because the changes in local blood flow determine

opposite changes in local pCO= and the changes in pO= have an effect opposite to that of pCO= upon vascular resistance. We postulated [14] that these systems of control are surely effective under conditions of moderate neuronal activity, the local extracellular pO= remaining at relatively constant levels. This would be the situation of the animal in relaxed wakefulness and SS. On the other hand, under conditions of very increased neuronal activity, the system presents phasic and tonic changes indicative of a less effective control of the homeostasis. If this be actually the case, these local pO, changes might be regarded as reflecting a great local increase of neuronal activity. We would thus have at our disposal, a method of determining [1] which regions of the nervous system do actually participate actively in IS, [2] their relative importance and [3] the differences with wakeful activity. To support the first point is the fact that the brain structures where modifications were present during paradoxical sleep, have been described as integrating the system of PS [7, 9]. As for the second point, a larger number of experiments is still required prior to a quantitative evabaation. Finally, the differences of PS, with wakefulness and SS have been clearly established. Our results substantiate the dualistic concept of Jouvet [10] in that both types of sleep involve

CHANGES OF BRAIN pO2 DURING PARADOXICAL SLEEP different mechanisms. It becomes evident that, with regard to SS, during PS, something very different may be going on in some brain structures, to judge by the conspicuous modifications in the 100,. A number of studies have attempted to demonstrate that during wakefulness and PS, the nervous system behaves very similarly. The slow cortical potentials, [3, 15], the cortical impedance [11, the cerebral blood flow [1 I] and the neuronal activity recorded with microelectrodes from the brain cortex [4] and from the reticular formation [8], vary in both situations along the same lines. Our own findings demonstrate that pO~ changes at arousal and alertness and during PS, although often similar, are essentially different owing to their different distribution. At arousal and alertness generalized changes in the pO~ were o b ~ r v c d while during PS they were confined to only some paleocortical, subcortical and brain stem structures. Finally, it is interesting to emphasize that the desynchronized electrical activity is not always a reliable indicator of

485 the functional state of the underlying structure because where this activity is very similar, as in the neocortex during PS and alertness, pOs recording in both situations demonstrates a very different physiological condition. Indeed, in the lateral gcniculate, where high voltage sharp waves are recorded during PS [9, 10], it was never possible to observe changes in the pO, and conversely, in the pontine reticular formation, where the same electrographic changes are recorded, the pO, simultaneously show important modifications. Similar considerations hold good for other variables which change in the cerebral cortex in the same way during alertness and PS, the steady potentials [3, 5] and the impedance [1]. This difference between the behavior of the pO, and of these electrical variables warrants the assumption that at least during PS changes of the cathodic current are not provoked by variations in the electromotive forces or of the impedance and, conversely, that variations in the pOs are not, in turn, responsible for changes in the cortical impedance.

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