- Email: [email protected]
pO2 recorded in the amygdaloid complex during the sleep-waking cycle of the cat
EXPERIMENTAL
p0,
KECROLOGY
798405
50,
(1976)
Recorded in the Amygdaloid Sleep-Waking Cycle RICARDO
VELLUTI
ANDJAIME
Nezrrofisiologia, Drpartame+lto de Fisiologia, Farmacologia y Terape’utica, Hospital Rcceiz~ed
Complex during of the Cat M.MOKTI
Facultad de Mrdicina, de Clirticas, Alotztcvideo,
October
the
Dejarfarm~to Uruguay
de
30, 19i5
Oxygen cathodes were implanted chronically in the lateral and basal amygdaloid nuclei and the nucleus reticularis pontis caudalis to record local oxygen availability in the brains of cats. The animals were controlled behaviorally and electrophysiologically to determine their actual state. In every experimental session the oxygen-dependence of the cathodes was tested. During slow-wave sleep, phasic pOa shifts were observed only at the lateral amygdaloid nucleus, characterized by a striking increase in the amplitude of oscillations. The basal amygdaloid nucleus together with the nucleus reticularis pontis caudalis showed p02 oscillating responses only in the REM or paradoxical sleep phase. It is postulated that those variations are due to a local increase of neuronal activity. Hence, the lateral amygdaloid nucleus assumes the role of a subcortical structure closely related to slow sleep and the basal amygdaloid nucleus, found to be activated along with the nucleus reticularis pontis caudalis, is assumed to be related to REM or paradoxical sleep. Changes during quiet and active wakefulness are also described.
INTRODUCTION The amygdaloid complex participates in a variety of brain functions, including behavior. Significant functional differences within the amygdaln have been demonstrated between its phylogenetically older and its more recently developed regions (9). It was established (4, 8) that stimulation of the amygdaloid complex evoked the behavioral responsesof attention, fear, and rage together with an EEG arousal pattern identical to that elicited by stimulation of the brain stem reticular formation. Aioreover, Kreindler and Steriade (IO) obtained desynchronization or slow waves and spindles in the electrocorticogram after stimulation at two different levels of the amygdala. Both arousal and the synchronizing effects persisted even in the cerveau isole preparation. 798 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved
AMYGDALA
PO2
IN
SLEEP
AND
WAKING
799
The description by Fuxe et al. (5) of fibers originating from noradrenergic and serotonergic cell body groups in the lower brain stem, ascending in the medial forebrain bundle and radiating in the amygdaloid complex, gave anatomic support to the existence of connections from subcortical structures which may be related to the sleep-wakefulness cycle. Spontaneous changes in the activity of the amygdaloid complex, closely related to the sleep-waking cycle, were found by Velluti et al. (13) and Garcia Austt et al. (6) using an oxygen cathode, and by Sawa and Delgado ( 11) and Jacobs and McGinty (7) while recording unit discharges. It was our purpose to study the activity of the basal and lateral amygdaloid nuclei in cats with chronically implanted electrodes during the waking and sleep phases using oxygen cathodes. These data were correlated with similar data obtained from the pontine reticular formation (n. reticularis pontis caudalis). Our results show that ~02 behaves differently in each amygdaloid nucleus during slow-wave sleep and REM or paradoxical sleep. MATERIAL
AND
METHODS
Experiments were carried out on eight adult cats (male or female) which were prepared for sleep recordings. Implantation of the electrodes was done under pentobarbital anesthesia and the animals were treated with antibiotics for several days after surgical procedures. Three to four oxygen cathodes were placed in each animal. To control the behavioral state, the electrocorticogram (lateral gyrus), the electrooculogram, and the electromyogram were recorded. Changes in brain ~0s were monitored by means of platinum oxygen cathodes referred to anodes made of AgCl-Ag wires (2, 3, 13). Each cathode was stereotaxically placed in subcortical structures including the nuclei amygdala lateralis (A 11.0, L 12.5, H -6.O), basalis (A 11.0, L 9.0, H -6.0 to -6.5)) and reticularis pontis caudalis (P 2.0, L 3.0, H -4.0)) according to the Snider and Niemer atlas (12). The anodes were implanted in the brain tissue, one for each cathode, and 4 to 5 mm apart. Ten days after surgery, the cats were placed in a dimly lighted, soundproof, isolated box fitted with a one-way mirror, and recordings were made through a multiple-strand cable using a Grass Model 7 Polygraph. A constant voltage of -0.6 V was applied to the platinum electrodes. The oxygen reduction-dependent current was obtained through a voltage drop measured across a 270 a resistance in the polygraph by means of a low level d-c Preamplifier Model 7Pl. The low pass filter of the amplifier was set to a cut-off frequency of 0.1 Hz, so that only the slowest oscillations of the current were monitored. Recordings were also carried out with cut-off frequencies of 0.3 Hz and 15 Hz.
800
VELLUTI
AND
MONT1
In every experimental session, which lasted from 2. to 8 hr, the oxygendependence of the cathodes was tested by changing the oxygen concentration in the cage, replacing air with 100% oxygen. The electrodes responded satisfactorily to systemic ~02 shifts for as long as 150 days, which was the duration of the longest experiment. The oxygen diffusion coefficient at the recording locus was not known, and as a consequence, the ~02 was always uncalibrated. In some instances there was “poisoning” of the cathodes, and they failed to respond to shifts in the enhanced oxygen-breathing concentration. Those electrodes were discarded. Records were obtained at a slow paper speed (9 mm/min), which was optimal to appraise the ~02 changes. Wakefulness, slow-wave sleep, and REM or paradoxica1 sleep were evaluated by observation of the animal and from the polygraphic records of the electrocorticogram, electrooculogram, and electromyogram. Histologic verification of the electrode placements was carried out at the end of the experiments. RESULTS Several basal brain regions along with the lower brain stem and the whole cerebellum have been demonstrated to exhibit a particular ~02 oscillating pattern during the paradoxical phase of sleep (6, 14). Within the amygdala, the lateral amygdaloid nucleus demonstrated a similar ~02 response. This consisted of high-amplitude waves (up to 35 nA) which virtually replaced the previous rhythm. This result consistently appeared in all the animals recorded and, in addition, the ~02 oscillating response has been observed during paradoxical sleep in the nucleus reticularis pontis caudalis. As shown in Fig. 1, there was a close temporal coincidence between the high-amplitude ~02 waves and rapid eye movements or paradoxical sleep. The same figure shows small-amplitude oscillations corresponding to the slow sleep pattern. The insets are the 100% oxygenbreathing test. The increased current recorded demonstrated the oxygen sensitivity of the cathodes. The electrocorticogram and the electromyogram clearly showed the sleep phases. No changes had been demonstrated in previous experiments in neocortex or in specific sensory nuclei such as the medial or lateral geniculate bodies (6). When the cathodes were implanted in the white matter, they did not show any significant shift. The changes observed were present only when the oxygen cathodes were oxygendependent. Moreover, when the electrodes were “poisoned” or not polarized by -0.6 V, the recordings were not altered by any shift in behavioral conditions. The right portion of Fig. 1 presents the ~02 activity during quiet wakefulness. Smaller oscillations were seen, as in other brain structures, in the
AMYGDALA CAT
PO2
IN
SLEEP
AND
801
WAKING
3
POZAL
lmln
100~04
FIG. 1. Oxygen partial pressure changes in amygdaloid and reticular nuclei during slow-wave and rapid-eye-movement sleep. ~0, RPS : oxygen cathode recording from the nucleus reticularis pontis caudalis; ~02 AL: recording of the n. amygdalae lateralis ; pOa AB : recording of the n. amygdalae basalis. ECoG, electrocorticogram ; EMG, electromyogram of the neck muscles. The oxygen cathodes show a similar oscillating pattern during the first slow-wave sleep period at the left of the figure, slightly larger in that located at the n. AL. Subsequently, an 11-min period of paradoxical sleep appeared. The paradoxical sleep oscillating response is clearly observed at the n. RPC and n. AB, as large waves superimposed on the rhythm of slow-wave sleep. Meanwhile, in the n. AL the amplitude of the PO2 oscillations decreased. A sudden awakening stops the oscillating response. The animal again returns to slowwave sleep with slightly larger waves in the n. AL record. A period of quiet wakefulness ends the record. A striking similarity is observed among the recordings of the n. RPC and n. AB. Tonic changes, shown as slow oscillation of the baseline, with parallel behavior, are more evident in the n. RPC and n. AB. The insets show the oxygen dependence of the cathodes tested in every experimental session. The injection of 100% oxygen into the animal box was maintained during one minute (02). Relative calibration of the current is shown in nA.
basal amygdaloid nucleus and the nucleus reticularis pontis caudalis. On the other hand the lateral amygdaloid nucleus showed a more ample pattern during quiet wakefulness than during paradoxical sleep. It has previously been reported that during very active wakefulness, high-amplitude oscillating patterns were observed in all brain regions from which recordings were taken. The ~02 of the lateral amygdaloid nucleus, which was recorded at the same time and only a few millimeters apart from the basal amygdaloid nucleus, exhibited a different pattern during the sleep-waking cycle. Figure 1 also shows that the ~02 oscillations were diminished during the paradoxical sleep and increased in amplitude during the short slow-sleep periods. This opposite behavior of the two amygdaloid nuclei in the p02
802
VELLUTI
AND
MONT1
CAT 8
EOG
EMG I
I
I
/d 1’”
lmtn
2OOyV
FIG. 2. Oxygen partial pressure changes in two amygdaloid nuclei. EOG, electrooculogram. Same abbreviations as in Fig. 1. The inset shows the position of the electrodes in the amygdaloid complex and the increased current is a consequence of the oxygen delivered into the animal cage (0,). Both cathodes have a similar sensitivity to the inhaled oxygen. During a slow-wave sleep period, at the beginning of the recording shown, large ~02 waves appeared at the n. AL stopping abruptly in the waking state (at arrow). When the animal again goes into slow-wave sleep, the oscillating response reappears. The ~0, of the n. AB, presenting the paradoxical-sleep oscillating response, does not show any significant change during slow-ware sleep or quiet wakefulness.
pattern is more clearly shown in Fig. 2. The paper speed was faster than in Fig. 1 and the high oscillations were better observed. The high-amplitude oscillating pattern was evident, located in the lateral amygdaloid nucleus during the slow-sleep phase (left half of Fig. 2). At the arrow the animal was aroused resulting in an abrupt diminution of the high-amplitude waves. A few minutes later the animal returned to slow-wave sleep and the same phenomenon could be observed again. The figure inset shows the oxpgendependence of the cathodes (which were tested in every experimental session) and the location of the electrodes in the amygdaloid nuclei. DISCUSSION Garcia Austt, Velluti, and Villar (6) supported the hypothesis that the paradoxical sleep 1’02 oscillating response, observed from certain basal brain regions, the reticular formation, and the cerebellum (14), was a consequence of a very highly enhanced local neuronal firing. They postulated that when the local metabolic self-regulatory mechanisms, which include at least two negative feedbacks, failed, such as during very active wakefulness, the system began to oscillate, as occurs in any imperfect feedback circuit.
AMYGDALA
PO-
IN
SLEEP
ASD
WAIi’IbiG
803
Because both neuronal activity and brain blood flow change considerably during sleep, it is presumed that the ~02 changes observed are due primarily to modification in the activity of neuronal aggregates. Variations in brain blood flow are thought to be the consequence of modification in neuronal firing. The basal amygdaloid nucleus showed the oscillating response during paradoxical sleep. This nucleus at the level explored in our experiments can be included as a part of the “~02 paradoxical sleep system” (14). On the other hand, high-amplitude pOz waves were observed during slow-wave sleep periods in the lateral amygdaloid nucleus. If it can be reasoned that enhanced oscillations signify an increased neuronal firing, the lateral amygdaloid nucleus appears to be an active subcortical participant in slow-wave sleep processes. These results appear to demonstrate that this nucleus is another active subcortical structure during slow-wave sleep. By using oxygen cathodes, the amygdaloid complex could be divided into two functionally different parts, one of which was related to paradoxical sleep, and the other to slow-wave sleep. The paradoxical-sleep ~02 oscillating response in the basal amygdaloid nuclei showed a close temporal coincidence with that of the reticular formation (nucleus reticularis pontis caudalis). This link between the amygdala and brain stem structures is also supported by other electrophysiologic and anatomic evidence. It has been shown that electrical stimulation of the amygdala produces a hypersynchronic discharge which $-opagated to the reticular formation (1). Feindel and Gloor (4) obtained an “arousal” of the amygdaloid EEG during electrical stimulation of the reticular formation. From an anatomic viewpoint, Fuxe et al. (5) have described catecholaminergic and indolaminergic fibers coursing from pontine reticular nuclei (locus coreuleus, nuclei raphe) through the medial forebrain bundle to the amygdaloid complex. Those findings might be regarded as providing additional support to our hypothesis which links the basal amygdaloid nucleus, a basal forebrain structure, with the nucleus reticula& pontine caudalis paradoxical sleep physiology. Unit activity recorded with semimicroelectrodes in the basolateral amygdala by Jacobs and McGinty (7) showed an increased frequency of firing during slow-wave sleep. Our results agree that portions of the amygdaloid complex are particularly related to slow-wave sleep processes. According to our ~02 experiments, the lateral amygdaloid nucleus is apparently the nudear group related to slow-wave sleep,, at least at the level recorded. The differences in our results might be of a technical nature or because of internal differences within the amygdaloid nuclei. Semimicroelectrodes might record activity not only from amygdaloid units, but from the bundles of fibers running through those nuclei. Conversely, it can be
so4
VELLUTI
AND
MONT1
assumed that the oxygen cathodes recorded the pOa of the extracellular compartment. The oxygen availability in this compartment is a function of the local oxygen-consuming metabolic cell body activity and the local oxygen-supplying blood flow. It can be ruled out that the changes were due to modifications in systemic pOa, because these were not found in any regions studied previously and they bore no relationship with either phasic or tonic shifts in arterial pressure (6). Only a greatly increased neuronal firing and its metabolic concomitants could be the source of the oscillating response. It has been reported that during arousal (6, ll), pOs increased tonically and diffusely in all brain regions recorded. During active wakefulness an oscillating response, similar to that seen during paradoxical sleep, was observed and this was interpreted as the result of widespread brain activation. Our method, although not quantifiable, has demonstrated its value in identifying functional relationships between brain structures, even between those which are closely situated and from which classical macroelectroderecorded activity failed to show differences. In summary, it could be established that Iocal metabolic differences, reflecting different functions of the amygdaloid complex, enabled the separation of two nuclear groups belonging to a postulated sleep system: the basal amygdaloid nucleus in connection with the reticular formation (nucleus reticularis pontis caudalis) and paradoxical sleep physiology, and the lateral amygdaloid nucleus which actively participates in slow-wave sleep. REFERENCES KIGUEZ, R., J. D. REIS, R. NAQUET, and H. W. MAGOUN. Propagation of amygdaloid seizures. Ada Neural. Latinoamev. 1: 109-122, 1955. CATER, D. B. Oxygen tension and oxidation reduction in living tissues. Prog. Biophy. Mol. Biol., 10: 154-193, 1960. DAVIES, P. W. The oxygen cathode. Zn “Physical Techniques in Biological Research,” W. L. Nastuk [Ed.]. New York, Academic Press, pp. 137-179. v. 4, 1962. FEINDEL, W., and P. GLOOR. Comparison of electrographic effects of stimulation of the amygdala and brain stem reticular formation in cats. EZertroePzcepltoIngl-. C&a. Newophysiol. 6: 389-402, 1954. FUXE, K., T. HAKFELT, and U. UXGERSTEDT. Distribution of monoamines in the mammalian central nervous system. bz “Metabolism of amines in the brain,” E. Hooper [Ed.]. London : MacMillan, pp. 10-22, 1969. GARCfA Ausrr, E., R. VELLUTI, and J. I. VILLAR. Changes of brain p0, during paradoxical sleep in cats. Phyoiol. Behav. 3: 477-485. 1968. JACOBS, B. L., and D. J. MCGINTY. Amygdala unit activity during sleep and waking. Exper. Neural. 33: l-15, 1971. KAADA, B. R., P. ANDERSEN, and J. JANSEN. Stimulation of the amygd.rloid nuclear complex in unanesthetized cats. Neurology 4: 4864, 1954.
1. ARANA
2. 3. 4. 5. 6. 7. 8.
AMYGDALA
POa IN SLEEP AND WAKING
80.5
B. R. Stimulation and regional ablation of the amygdaloid complex with reference to functional representations. In “The Neurobiology of Amygdala,” B. E. Eleftheriou [Ed.]. Plenum Press, New York, pp. 250-281, 1972. 10. KREINDLER, A., and M. STERIADE. EEG patterns of arousal and sleep induced by stimulating various amygdaloid levels in the cat. Arch. Ital. Biol 102: 576436,1964. 11. SAWA, M., and J. M. R. DELGADO. Amygdala unitary activity in the unrestrained cat. Electroencephologr. Clin. Neurophysiol. 15 : 637-650, 1963. 12. SNIDER, R. S., and W. T. NIEMER. “A Stereotaxic Atlas of the Cat Brain.” The University of Chicago Press. 1961. 13. VISLLUTI, R., J. A. ROIG, L. A. ESCARCENA, J. I. VILLAR, and E. GARCIA AUSTT. Changes of brain pOa during arousal and alertness in unrestrained cats. Acta Neurol. Latinoarner. 11: 368-382, 1965. 14. VELLUTI, R., J. C. VELLUTI, and E. GARCIA Ausrr. Cerebellum pOz and the sleep-waking cycle in cats. Physiol. Behav. (in press). 9. KAADA,
p0,
KECROLOGY
798405
50,
(1976)
Recorded in the Amygdaloid Sleep-Waking Cycle RICARDO
VELLUTI
ANDJAIME
Nezrrofisiologia, Drpartame+lto de Fisiologia, Farmacologia y Terape’utica, Hospital Rcceiz~ed
Complex during of the Cat M.MOKTI
Facultad de Mrdicina, de Clirticas, Alotztcvideo,
October
the
Dejarfarm~to Uruguay
de
30, 19i5
Oxygen cathodes were implanted chronically in the lateral and basal amygdaloid nuclei and the nucleus reticularis pontis caudalis to record local oxygen availability in the brains of cats. The animals were controlled behaviorally and electrophysiologically to determine their actual state. In every experimental session the oxygen-dependence of the cathodes was tested. During slow-wave sleep, phasic pOa shifts were observed only at the lateral amygdaloid nucleus, characterized by a striking increase in the amplitude of oscillations. The basal amygdaloid nucleus together with the nucleus reticularis pontis caudalis showed p02 oscillating responses only in the REM or paradoxical sleep phase. It is postulated that those variations are due to a local increase of neuronal activity. Hence, the lateral amygdaloid nucleus assumes the role of a subcortical structure closely related to slow sleep and the basal amygdaloid nucleus, found to be activated along with the nucleus reticularis pontis caudalis, is assumed to be related to REM or paradoxical sleep. Changes during quiet and active wakefulness are also described.
INTRODUCTION The amygdaloid complex participates in a variety of brain functions, including behavior. Significant functional differences within the amygdaln have been demonstrated between its phylogenetically older and its more recently developed regions (9). It was established (4, 8) that stimulation of the amygdaloid complex evoked the behavioral responsesof attention, fear, and rage together with an EEG arousal pattern identical to that elicited by stimulation of the brain stem reticular formation. Aioreover, Kreindler and Steriade (IO) obtained desynchronization or slow waves and spindles in the electrocorticogram after stimulation at two different levels of the amygdala. Both arousal and the synchronizing effects persisted even in the cerveau isole preparation. 798 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved
AMYGDALA
PO2
IN
SLEEP
AND
WAKING
799
The description by Fuxe et al. (5) of fibers originating from noradrenergic and serotonergic cell body groups in the lower brain stem, ascending in the medial forebrain bundle and radiating in the amygdaloid complex, gave anatomic support to the existence of connections from subcortical structures which may be related to the sleep-wakefulness cycle. Spontaneous changes in the activity of the amygdaloid complex, closely related to the sleep-waking cycle, were found by Velluti et al. (13) and Garcia Austt et al. (6) using an oxygen cathode, and by Sawa and Delgado ( 11) and Jacobs and McGinty (7) while recording unit discharges. It was our purpose to study the activity of the basal and lateral amygdaloid nuclei in cats with chronically implanted electrodes during the waking and sleep phases using oxygen cathodes. These data were correlated with similar data obtained from the pontine reticular formation (n. reticularis pontis caudalis). Our results show that ~02 behaves differently in each amygdaloid nucleus during slow-wave sleep and REM or paradoxical sleep. MATERIAL
AND
METHODS
Experiments were carried out on eight adult cats (male or female) which were prepared for sleep recordings. Implantation of the electrodes was done under pentobarbital anesthesia and the animals were treated with antibiotics for several days after surgical procedures. Three to four oxygen cathodes were placed in each animal. To control the behavioral state, the electrocorticogram (lateral gyrus), the electrooculogram, and the electromyogram were recorded. Changes in brain ~0s were monitored by means of platinum oxygen cathodes referred to anodes made of AgCl-Ag wires (2, 3, 13). Each cathode was stereotaxically placed in subcortical structures including the nuclei amygdala lateralis (A 11.0, L 12.5, H -6.O), basalis (A 11.0, L 9.0, H -6.0 to -6.5)) and reticularis pontis caudalis (P 2.0, L 3.0, H -4.0)) according to the Snider and Niemer atlas (12). The anodes were implanted in the brain tissue, one for each cathode, and 4 to 5 mm apart. Ten days after surgery, the cats were placed in a dimly lighted, soundproof, isolated box fitted with a one-way mirror, and recordings were made through a multiple-strand cable using a Grass Model 7 Polygraph. A constant voltage of -0.6 V was applied to the platinum electrodes. The oxygen reduction-dependent current was obtained through a voltage drop measured across a 270 a resistance in the polygraph by means of a low level d-c Preamplifier Model 7Pl. The low pass filter of the amplifier was set to a cut-off frequency of 0.1 Hz, so that only the slowest oscillations of the current were monitored. Recordings were also carried out with cut-off frequencies of 0.3 Hz and 15 Hz.
800
VELLUTI
AND
MONT1
In every experimental session, which lasted from 2. to 8 hr, the oxygendependence of the cathodes was tested by changing the oxygen concentration in the cage, replacing air with 100% oxygen. The electrodes responded satisfactorily to systemic ~02 shifts for as long as 150 days, which was the duration of the longest experiment. The oxygen diffusion coefficient at the recording locus was not known, and as a consequence, the ~02 was always uncalibrated. In some instances there was “poisoning” of the cathodes, and they failed to respond to shifts in the enhanced oxygen-breathing concentration. Those electrodes were discarded. Records were obtained at a slow paper speed (9 mm/min), which was optimal to appraise the ~02 changes. Wakefulness, slow-wave sleep, and REM or paradoxica1 sleep were evaluated by observation of the animal and from the polygraphic records of the electrocorticogram, electrooculogram, and electromyogram. Histologic verification of the electrode placements was carried out at the end of the experiments. RESULTS Several basal brain regions along with the lower brain stem and the whole cerebellum have been demonstrated to exhibit a particular ~02 oscillating pattern during the paradoxical phase of sleep (6, 14). Within the amygdala, the lateral amygdaloid nucleus demonstrated a similar ~02 response. This consisted of high-amplitude waves (up to 35 nA) which virtually replaced the previous rhythm. This result consistently appeared in all the animals recorded and, in addition, the ~02 oscillating response has been observed during paradoxical sleep in the nucleus reticularis pontis caudalis. As shown in Fig. 1, there was a close temporal coincidence between the high-amplitude ~02 waves and rapid eye movements or paradoxical sleep. The same figure shows small-amplitude oscillations corresponding to the slow sleep pattern. The insets are the 100% oxygenbreathing test. The increased current recorded demonstrated the oxygen sensitivity of the cathodes. The electrocorticogram and the electromyogram clearly showed the sleep phases. No changes had been demonstrated in previous experiments in neocortex or in specific sensory nuclei such as the medial or lateral geniculate bodies (6). When the cathodes were implanted in the white matter, they did not show any significant shift. The changes observed were present only when the oxygen cathodes were oxygendependent. Moreover, when the electrodes were “poisoned” or not polarized by -0.6 V, the recordings were not altered by any shift in behavioral conditions. The right portion of Fig. 1 presents the ~02 activity during quiet wakefulness. Smaller oscillations were seen, as in other brain structures, in the
AMYGDALA CAT
PO2
IN
SLEEP
AND
801
WAKING
3
POZAL
lmln
100~04
FIG. 1. Oxygen partial pressure changes in amygdaloid and reticular nuclei during slow-wave and rapid-eye-movement sleep. ~0, RPS : oxygen cathode recording from the nucleus reticularis pontis caudalis; ~02 AL: recording of the n. amygdalae lateralis ; pOa AB : recording of the n. amygdalae basalis. ECoG, electrocorticogram ; EMG, electromyogram of the neck muscles. The oxygen cathodes show a similar oscillating pattern during the first slow-wave sleep period at the left of the figure, slightly larger in that located at the n. AL. Subsequently, an 11-min period of paradoxical sleep appeared. The paradoxical sleep oscillating response is clearly observed at the n. RPC and n. AB, as large waves superimposed on the rhythm of slow-wave sleep. Meanwhile, in the n. AL the amplitude of the PO2 oscillations decreased. A sudden awakening stops the oscillating response. The animal again returns to slowwave sleep with slightly larger waves in the n. AL record. A period of quiet wakefulness ends the record. A striking similarity is observed among the recordings of the n. RPC and n. AB. Tonic changes, shown as slow oscillation of the baseline, with parallel behavior, are more evident in the n. RPC and n. AB. The insets show the oxygen dependence of the cathodes tested in every experimental session. The injection of 100% oxygen into the animal box was maintained during one minute (02). Relative calibration of the current is shown in nA.
basal amygdaloid nucleus and the nucleus reticularis pontis caudalis. On the other hand the lateral amygdaloid nucleus showed a more ample pattern during quiet wakefulness than during paradoxical sleep. It has previously been reported that during very active wakefulness, high-amplitude oscillating patterns were observed in all brain regions from which recordings were taken. The ~02 of the lateral amygdaloid nucleus, which was recorded at the same time and only a few millimeters apart from the basal amygdaloid nucleus, exhibited a different pattern during the sleep-waking cycle. Figure 1 also shows that the ~02 oscillations were diminished during the paradoxical sleep and increased in amplitude during the short slow-sleep periods. This opposite behavior of the two amygdaloid nuclei in the p02
802
VELLUTI
AND
MONT1
CAT 8
EOG
EMG I
I
I
/d 1’”
lmtn
2OOyV
FIG. 2. Oxygen partial pressure changes in two amygdaloid nuclei. EOG, electrooculogram. Same abbreviations as in Fig. 1. The inset shows the position of the electrodes in the amygdaloid complex and the increased current is a consequence of the oxygen delivered into the animal cage (0,). Both cathodes have a similar sensitivity to the inhaled oxygen. During a slow-wave sleep period, at the beginning of the recording shown, large ~02 waves appeared at the n. AL stopping abruptly in the waking state (at arrow). When the animal again goes into slow-wave sleep, the oscillating response reappears. The ~0, of the n. AB, presenting the paradoxical-sleep oscillating response, does not show any significant change during slow-ware sleep or quiet wakefulness.
pattern is more clearly shown in Fig. 2. The paper speed was faster than in Fig. 1 and the high oscillations were better observed. The high-amplitude oscillating pattern was evident, located in the lateral amygdaloid nucleus during the slow-sleep phase (left half of Fig. 2). At the arrow the animal was aroused resulting in an abrupt diminution of the high-amplitude waves. A few minutes later the animal returned to slow-wave sleep and the same phenomenon could be observed again. The figure inset shows the oxpgendependence of the cathodes (which were tested in every experimental session) and the location of the electrodes in the amygdaloid nuclei. DISCUSSION Garcia Austt, Velluti, and Villar (6) supported the hypothesis that the paradoxical sleep 1’02 oscillating response, observed from certain basal brain regions, the reticular formation, and the cerebellum (14), was a consequence of a very highly enhanced local neuronal firing. They postulated that when the local metabolic self-regulatory mechanisms, which include at least two negative feedbacks, failed, such as during very active wakefulness, the system began to oscillate, as occurs in any imperfect feedback circuit.
AMYGDALA
PO-
IN
SLEEP
ASD
WAIi’IbiG
803
Because both neuronal activity and brain blood flow change considerably during sleep, it is presumed that the ~02 changes observed are due primarily to modification in the activity of neuronal aggregates. Variations in brain blood flow are thought to be the consequence of modification in neuronal firing. The basal amygdaloid nucleus showed the oscillating response during paradoxical sleep. This nucleus at the level explored in our experiments can be included as a part of the “~02 paradoxical sleep system” (14). On the other hand, high-amplitude pOz waves were observed during slow-wave sleep periods in the lateral amygdaloid nucleus. If it can be reasoned that enhanced oscillations signify an increased neuronal firing, the lateral amygdaloid nucleus appears to be an active subcortical participant in slow-wave sleep processes. These results appear to demonstrate that this nucleus is another active subcortical structure during slow-wave sleep. By using oxygen cathodes, the amygdaloid complex could be divided into two functionally different parts, one of which was related to paradoxical sleep, and the other to slow-wave sleep. The paradoxical-sleep ~02 oscillating response in the basal amygdaloid nuclei showed a close temporal coincidence with that of the reticular formation (nucleus reticularis pontis caudalis). This link between the amygdala and brain stem structures is also supported by other electrophysiologic and anatomic evidence. It has been shown that electrical stimulation of the amygdala produces a hypersynchronic discharge which $-opagated to the reticular formation (1). Feindel and Gloor (4) obtained an “arousal” of the amygdaloid EEG during electrical stimulation of the reticular formation. From an anatomic viewpoint, Fuxe et al. (5) have described catecholaminergic and indolaminergic fibers coursing from pontine reticular nuclei (locus coreuleus, nuclei raphe) through the medial forebrain bundle to the amygdaloid complex. Those findings might be regarded as providing additional support to our hypothesis which links the basal amygdaloid nucleus, a basal forebrain structure, with the nucleus reticula& pontine caudalis paradoxical sleep physiology. Unit activity recorded with semimicroelectrodes in the basolateral amygdala by Jacobs and McGinty (7) showed an increased frequency of firing during slow-wave sleep. Our results agree that portions of the amygdaloid complex are particularly related to slow-wave sleep processes. According to our ~02 experiments, the lateral amygdaloid nucleus is apparently the nudear group related to slow-wave sleep,, at least at the level recorded. The differences in our results might be of a technical nature or because of internal differences within the amygdaloid nuclei. Semimicroelectrodes might record activity not only from amygdaloid units, but from the bundles of fibers running through those nuclei. Conversely, it can be
so4
VELLUTI
AND
MONT1
assumed that the oxygen cathodes recorded the pOa of the extracellular compartment. The oxygen availability in this compartment is a function of the local oxygen-consuming metabolic cell body activity and the local oxygen-supplying blood flow. It can be ruled out that the changes were due to modifications in systemic pOa, because these were not found in any regions studied previously and they bore no relationship with either phasic or tonic shifts in arterial pressure (6). Only a greatly increased neuronal firing and its metabolic concomitants could be the source of the oscillating response. It has been reported that during arousal (6, ll), pOs increased tonically and diffusely in all brain regions recorded. During active wakefulness an oscillating response, similar to that seen during paradoxical sleep, was observed and this was interpreted as the result of widespread brain activation. Our method, although not quantifiable, has demonstrated its value in identifying functional relationships between brain structures, even between those which are closely situated and from which classical macroelectroderecorded activity failed to show differences. In summary, it could be established that Iocal metabolic differences, reflecting different functions of the amygdaloid complex, enabled the separation of two nuclear groups belonging to a postulated sleep system: the basal amygdaloid nucleus in connection with the reticular formation (nucleus reticularis pontis caudalis) and paradoxical sleep physiology, and the lateral amygdaloid nucleus which actively participates in slow-wave sleep. REFERENCES KIGUEZ, R., J. D. REIS, R. NAQUET, and H. W. MAGOUN. Propagation of amygdaloid seizures. Ada Neural. Latinoamev. 1: 109-122, 1955. CATER, D. B. Oxygen tension and oxidation reduction in living tissues. Prog. Biophy. Mol. Biol., 10: 154-193, 1960. DAVIES, P. W. The oxygen cathode. Zn “Physical Techniques in Biological Research,” W. L. Nastuk [Ed.]. New York, Academic Press, pp. 137-179. v. 4, 1962. FEINDEL, W., and P. GLOOR. Comparison of electrographic effects of stimulation of the amygdala and brain stem reticular formation in cats. EZertroePzcepltoIngl-. C&a. Newophysiol. 6: 389-402, 1954. FUXE, K., T. HAKFELT, and U. UXGERSTEDT. Distribution of monoamines in the mammalian central nervous system. bz “Metabolism of amines in the brain,” E. Hooper [Ed.]. London : MacMillan, pp. 10-22, 1969. GARCfA Ausrr, E., R. VELLUTI, and J. I. VILLAR. Changes of brain p0, during paradoxical sleep in cats. Phyoiol. Behav. 3: 477-485. 1968. JACOBS, B. L., and D. J. MCGINTY. Amygdala unit activity during sleep and waking. Exper. Neural. 33: l-15, 1971. KAADA, B. R., P. ANDERSEN, and J. JANSEN. Stimulation of the amygd.rloid nuclear complex in unanesthetized cats. Neurology 4: 4864, 1954.
1. ARANA
2. 3. 4. 5. 6. 7. 8.
AMYGDALA
POa IN SLEEP AND WAKING
80.5
B. R. Stimulation and regional ablation of the amygdaloid complex with reference to functional representations. In “The Neurobiology of Amygdala,” B. E. Eleftheriou [Ed.]. Plenum Press, New York, pp. 250-281, 1972. 10. KREINDLER, A., and M. STERIADE. EEG patterns of arousal and sleep induced by stimulating various amygdaloid levels in the cat. Arch. Ital. Biol 102: 576436,1964. 11. SAWA, M., and J. M. R. DELGADO. Amygdala unitary activity in the unrestrained cat. Electroencephologr. Clin. Neurophysiol. 15 : 637-650, 1963. 12. SNIDER, R. S., and W. T. NIEMER. “A Stereotaxic Atlas of the Cat Brain.” The University of Chicago Press. 1961. 13. VISLLUTI, R., J. A. ROIG, L. A. ESCARCENA, J. I. VILLAR, and E. GARCIA AUSTT. Changes of brain pOa during arousal and alertness in unrestrained cats. Acta Neurol. Latinoarner. 11: 368-382, 1965. 14. VELLUTI, R., J. C. VELLUTI, and E. GARCIA Ausrr. Cerebellum pOz and the sleep-waking cycle in cats. Physiol. Behav. (in press). 9. KAADA,