Physiology & Behavior, Vol. 22, pp. 741-745. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Functional Relationship Between Cat Brainstem Neurons During Sleep and Wakefulness TOYOHIKO SATOH, KUNIHIRO EGUCHI AND KAZUSHIGE WATABE
Department of Physiology, School of Dental Medicine, Aichi-Gakuin University Nagoya, 464, Japan ( R e c e i v e d 7 O c t o b e r 1978) S A T O H , T., K. E G U C H I A N D K. W A T A B E . Functional relationship between cat brainstem neurons during sleep and wakefulness. P H Y S I O L . B E H A V . 22(4) 741-745, 1979.--The m o d e of interaction b e t w e e n so-called sleep-wakefulness
centers in the cat lower brainstem was studied on 100 neurons. The magnitude of the spike response of a neuron in one center to electrical stimulation of another center was measured to calculate the index of responsiveness. During REM sleep the index was, in a great majority of cases, significantly smaller as compared with that during wakefulness and slow wave sleep. This reduction in the effectiveness of information transmission between different centers might be the basis of characteristic events occurring during REM sleep. From the behavior of the indexes during sleep-wakefulness cycle, it is suggested that different phases of sleep and wakefulness are realized by a complicated interplay of many sleep-wakefulness centers which would be communicating with one another not only through channels which are activated in a phase-specific manner, but also through a larger number of channels of which activity is modulated differentially during different phases. Sleep-wakefulness mechanism Brainstem neurons Magnitude of evoked unitary discharge relative to spontaneous firing Dynamic relationship between sleep-wakefulness centers
OUR present understanding on the functional organization of the brain structures concerned with the realization of different phases of sleep and wakefulness is confined mostly to its static aspect; that is to say, we know that certain structures are important for a particular phase of sleep and wakefulness, while we are only poorly informed about the role of those structures during other phases. A negative feedback loop between the bulbar reticular formation and the midbrain reticular formation (MRF) has been suggested [1, 2, 11]. The basal forebrain area and the MRF are claimed to be mutually inhibitory [3], and this inhibition is thought to be put into action, at least in part, through the activation of inhibitorysystem in the bulbar reticular formation [11]. Reciporcal relationship has been demonstrated between the MRF and the solitary tract nucleus [4]. The influence of the reticular formation upon the raphe nuclei [13,16], and the interaction between the raphe and the locus coeruleus (LC) [10] have been studied. The onset of REM sleep is characterized by a reciprocal activity between the giganto-cellular tegmental field (FTG) and the LC [7]. The amplitude and configuration of the evoked responses which were elicited by electrical stimulation of one of so-called sleep-wakefulness centers and were recorded from other centers, are modulated during sleep-wakefulness cycle in a manner which is not easily accounted for by a simple, static concept that a particular center is executive only in a particular phase [14]. Present investigation was undertaken to study whether so-called sleep-wakefulness centers are in different relationship with one another during different phases of sleep and wakefulness. Their altered relationship must be somehow expressed in the magnitude of the spike response of indi-
vidual neurons in one center to electrical stimulation of another center. As the background discharge rate (BDR) of the recorded neuron may change from a phase to another, the degree of change in the effectiveness of the stimulated neurons to drive or inhibit the recorded neuron may be correctly expressed by the ratio of the number of evoked spikes to that of background spikes, rather than by the absolute number of the evoked spikes. Phase-dependent modulation, as assessed in this way, may occur at various sites; the stimulated neurons, the recorded neuron and the conduction pathways between the two. Therefore, the altered magnitude of the response is to be understood as representing the sum of the modulation acting on a set of functional units which has a group of stimulated neurons as the input stage and the recorded neuron as the common output element. METHOD
Data Acquisition Under intraperitoneal pentobarbitone anesthesia (40 mg/kg) 12 cats were prepared for chronic experiment. Stainless steel screws were fixed to the skull to monitor the EEG and electrooculogram and to be used as the reference electrode for unit recording. Vinyl-coated wires were embedded in the nuchal muscles for recording the EMG. Parallel, bipolar electrodes for stimulation were made of stainless steel wires of 50/zm in diameter and had tip separation of 0.5 mm along the axis. They were implanted stereotaxically into two sites of the following structures: the MRF, raphe magnus (RM), raphe dorsalis (DRN) and LC. Rectangular pulses of 0.01 msec in duration were delivered every 2.5-5.0 sec at a
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040741-05502.00/0
742
SATOH, EGUCHI AND WATABE
constant intensity of 0.3-0.8 mA. Glass-insulated Pt-Ir microelectrode of 2-7 Mf~ was driven with a hydraulic system which worked at 1 /zm steps and was mounted on a metal cylinder fixed on the trepanned skull. In order to ensure long-time holding of the units, the head of the animal was restrained from moving by tightly clamping the acrylic mound made on the skull. Unit activity was picked up from the MRF, RM, LC and FFG. After the experiment the animal was sacrificed with overdose anesthetics. Recorded and stimulated sites were identified histologically on serial sections stained with Kliiver-Barrera method.
Data Analysis Spike activity tape-recorded at a frequency response of 5 kHz was fed to the data processor (San-ei Sokki 7T07) to calculate the BDR and to obtain post-stimulus time histograms (PSTH) for each sleep-wakefulness phase. The PSTHs were constructed with 2 msec bin width and 50-100 sweeps. Only the neurons which gave an obvious response in the PSTH in any phase of sleep and wakefulness were submitted to further analysis. The effectiveness for the stimulated site to drive or inhibit the recorded neuron was expressed with an index. The index represents the ratio of the evoked spike number during the response period which may change in duration from one phase to another, to the background spike number during the corresponding period.
stim. FIG. 1. Parameters used to calculate the index of responsiveness from the post-stimulus time histogram. Excitatory (R') and inhibitory (R'~)responses are schematically shown along with the duration of the responses (D' and D"). BG: mean background discharge rate.
55•
R-BG BG
D Dw
where R is the total spike number during response period D in the PSTH schematically shown in Fig. 1. BG is the background spike number during the period corresponding to D. Dw is the duration of the response during quiet wakefulness without gross movement (W). When significant (see below) response was not present during W or during both W and slow wave sleep (S), the duration of the response during S or REM sleep (P) was substituted for Dw, respectively. Unequivocal deflections in the PSTH which were regarded as significant responses had an index greater than + 1.0 in the case of excitation and less than -0.3 in the case of inhibition. As the physiological significance of the output pattern of recorded neuron is totally unknown and the PSTH disregards such an aspect, the above equation contains no term about the discharge pattern. Hence it is possible for two phases with different values in their parameters to have an identical index. When the index and the BDR were calculated in the same phases occurring at a different time, the maximum variance of both values was within 30%. Consequently, the difference in the index and the BDR greater than 30% was regarded as significant. RESULTS
Excitatory responses which gave peaks in the PSTH with index greater than + 1.0 were most often found at about 10 and/or 50 msec. A total of 106 excitatory responses were obtained from 100 neurons. Inhibitory responses with index less t h a n - 0 . 3 had a peak, in general, at 20--30 msec (Fig. 2). Total number of the inhibitory responses was 57. There seemed to be no great difference due to the combination of stimulated and recorded structures. The analysis was done on the responses with latency greater than 2 msec.
IlI~RF
1@-~ RMOMRF
I
5-
Index =
MRFOLC
MRF
40-
I
5-
:: : : : : :
DRN~MRF
20-
MRFOFTG 50 ~-~ 100
20-
0, RM
1SOres
excitatory inhibitory
FIG. 2. Response periods appeared in the PSTHs. Ordinate: number of response. Abscissa: time after stimulation. The arrows point from the stimulated site to the recorded site.
The BDR during W was in most neurons between 20 and 25/see. During S it tended to be slightly lower. During P it was usually between 30 and 40/see and was, in a majority of cases, definitely higher as compared to other phases. Two out of 4 LC neurons had a BDR less than 1/sec during W and S. During P the BDR was distinctly higher. Seven out of 14 RM neurons had a BDR of about 4/see during W and S. During P the BDR was always increased. When the animal entered from W to S, the behavior of the indexes was apparently independent to the change in BDR. During P the index was, in a great majority of cases, smaller than during other phases and was often associated with increased BDR. However, increased or unaltered indexes were also usually associated with increased BDR. According to the mode of change during sleepwakefulness cycle, the indexes were classified into four
BRAINSTEM NEURONS DURING SLEEP
743
TABLE 1 BEHAVIOR DURING SLEEP-WAKEFULNESS PHASES OF THE INDEXES OBTAINED BY VARIOUS COMBINATIONS OF STIMULATION AND RECORDING SITES. X, X' OR X" CORRESPONDS TO AN INDEX OF ANY ONE OF W, S AND P. O REPRESENTS THE LEVEL OF SIGNIFICANCE OF THE INDEX OF EXCITATORY AND INHIBITORY RESPONSES. 1N THE SQUARE BRACKETS, THE NUMBERS OF PHASES WHICH FELL UNDER THE SPECIFIED CATEGORIES OF INDEX ARE SHOWN. Mono-phasic
Bi-phasic
Tri-phasic
No change
X>O>X'-X"
X,>X'-X">O X-X'>X'[>O X-X'>O>X"3
X>X'>X">O X>X'>O>X"
26[X=Wx12, S x l 0 , Px4]
43 [X,=Wx3, S×9, P x l X ' ~ = W x l , Sx2, P x l 6 X":~=Wx 1, Sx2, Px8]
12 [X=Wx2, S x l 0 X"=W×3, P×9]
9
Site of Stim.
Site of Rec.
No. of Response (Neuron)
DRN
MRF
90 (54)
LC
MRF
12 (8)
3 [X=Wx2, P x l ]
4 [X,=0 X'~=P×3 X":~=Px l]
4 [ X = W x l , Sx3 X " = W x 1, P×3]
1
RM
MRF
23 (13)
3 [X=Wxl, Sxl, Pxl]
9 [X,=Sx2, P x l X'~=Px3 X":~=Sx2, Px 1]
9 [X=Wx5, Sx4 X"=Sx 1, Px8]
2
MRF
LC
6 (4)
2 [X=Wxl, Sxl]
2 [X,=Sxl X'[=0 X":~=P x 11
2 [X=Wxl, Sxl X"=P×2]
0
MRF
RM
19 (14)
3 [ X = W x l , S×2]
12 [X,=Wx2 X '~=P×5 X":~=Px5]
4 [X=Wx2, Sx2 X"=Px4]
0
MRF
FTG
13 (7)
3 [X=Wx2, S x l ]
2 [X=Sx2 X"=Px2]
2
163 (100)
6 [X,=Sx2 X'~=P× 1 X":~=Sx 1, P×2]
40 (24.5%)
76 (46.6%)
X-x'-x">O
33 (20.2%)
14 (8.5%)
tory and inhibitory responses are shown in Table 2. During P the inhibitory response o u t n u m b e r e d the excitatory one. All o f the P-specific responses w e r e obtained f r o m the M R F . Bi-phasic group occupied 46.6% o f the total response. During P the index was, most often, u n d e r the level o f significance (X.~ in Table 1) or smallest a m o n g three phases (X~ in Table 1; see also X2~-3 in Table 2). During S the index was, in m a n y cases, greatest a m o n g three phases (Xl in Table 1 and 2). Tri-phasic group occupied 20.2% of the total response. A b o u t 70% of this group were lacking a significant r e s p o n s e in one phase (0>Xq. During P the index was, in most occa-
groups (Table 1): ( 1 ) m o n o - p h a s i c group: significant r e s p o n s e is present only in one phase and absent in o t h e r two phases; (2) bi-phasic group: the r e s p o n s e is present at least in two phases, but the indexes of two phases are not significantly different; (3) tri-phasic group: the indexes during three phases of sleep and wakefulness are significantly different with one another; (4) no change: significant r e s p o n s e is present throughout three phases, but no change in the index. Mono-phasic group occupied 24.5% of the total response. R e s p o n s e s occurring only during W or S w e r e e n c o u n t e r e d at a c o m p a r a b l e f r e q u e n c y , while it was m u c h less f r e q u e n t to h a v e the r e s p o n s e only during P. The numbers of excita-
TABLE 2 RELATION OF EXCITATORY AND INHIBITORY RESPONSES TO THE BEHAVIOR OF THE INDEX. FOR ABBREVIATIONS REFER TO TABLE 1 Mono-phasic
Bi-phasic
X
X,
Tfi-phasic
X'~+:, excit inhib
excit
inhib
excit
inhib
W
10
9
2
3
1
S
10
5
10
4
P
1
5
1
21
19
13
Tot~
X
No change X"
excit
inhib
excit
inhib
1
9
2
2
2
3
4
19
3
0
1
1
33
13
0
0
26
2
8
37
18
28
5
28
5
excit
inhib
5
9
744
SATOH, EGUCHI AND WATABE
sions, under the level of significance or smallest among three phases (X" in Table 1 and 2). During S the index was frequently greatest among three phases (X in tri-phasic group in Table 1 and 2). Inhibitory responses were relatively small in number (Table 2). Responses with unaltered index throughout three phases were found in 8.5% of the total response. The proportion of the inhibitory response was relatively high in this group (Table 2). From Table 1 it did not seem that the site of stimulation, the site of recording or certain combinations of them were preferentially related with a particular behavior of the index. Therefore it was examined whether the behavior of the indexes is correlated with other factors like the magnitude of the index during W, the BDR during W, the latency of the response, or the polarity of the response, i.e. excitatory or inhibitory. As shown in Table 2, the polarity of the response seemed to be somewhat correlated with sleep phases. Smallest indexes (X2~+X'~ were most often found during P (=74) as compared to W(=6) and S(=8). Among these responses during P with smallest index, 79.7% were excitatory. Monophasic responses during P were, as indicated already, mostly inhibitory. During S there was a tendency that excitatory responses had greatest index among three phases, as seen in Xl of bi-phasic responses and X of mono- and tri-phasic responses. DISCUSSION The latencies of excitatory and inhibitory responses were not greatly different among different combinations of various structures stimulated and recorded, and were similar to the values reported on cat brainstem reticular formation [6, 8,
111. The BDR of the MRF neurons during sleep-wakefulness cycle was consistent with other reports [9,12]. One half of the LC neurons had a very low BDR and, hence, they are presumed to be noradrenaline-containing neurons. There were also neurons with higher discharge rate which were described by Chu and Bloom [5]. One half of the neurons in the RM was slowly discharging, suggesting that they are serotonin-containing neurons. Their discharge rates during sleep-wakefulness cycle were generally in agreement with the report by Sheu et al. [15], excepting slightly lower rate during W in our record which was obtained during quite
wakefulness without gross movement. The behavior of the indexes of the presumably monoamine-containing neurons was not markedly different from that of other neurons in the LC and RM. During P it was very infrequent for the index to be greatest among three phases (X in mono- and tri-phasic responses plus X, in bi-phasic responses in Table 2 = 6+0+2=8), while it was frequent during W(19 +11+5=35) and S(15+22+ 14=51). Smallest index among three phases was most often found during P(X'~+:~in bi-phasic and X" in tri-phasic responses in Table 2=46+28=74), while it was much less often during W(2+4=6) and S(7+ 1=8). It seemed, therefore, that reduction in the effectiveness of information transmission between different centers is one of the outstanding features of P. As the BDR is greatly increased during P in most neurons, the release of the activity of individual neurons from the control by other centers might be the basis of characteristic events occurring phasically or tonically during P; for example, PGO-spikes, REMs, desynchronized EEG, fiat EMG and so on. During S the index tended to be greatest among three phases. However, this tendency was far less remarkable as compared to the change during P. Generally speaking, S was more like W in terms of the behavior of the index. There were communication channels between different centers which were activated only during a particular phase and inactivated in other phases. However, there were far more numerous channels of which activity was modulated differentially in two or three phases. These communication channels with phase-specific and multi-phasic properties have been found between all centers studied. The proportion of the channels which were not influenced by sleepwakefulness phases was relatively small. There seemed to be no particular combination of stimulated and recorded sites which was correlated very well with a particular behavior of the indexes. Only the polarity of the response seemed to have some correlation with phase-dependent change in the index. The conclusion which may be reached by the present experiment is that different phases of sleep and wakefulness are realized by a complicated interplay of many sleepwakefulness centers which would be communicating with one another not only through channels which are activated in a phase-specific manner, but also through a larger number of channels of which activity is modulated differentially in different phases of sleep and wakefulness.
REFERENCES
1. Batini, C., F. Magni, M. Palestini, G. F. Rossi and A. Zanchetti. Neural mechanisms underlying the enduring E.E.G. and behavioral activation in the midpontine pretrigeminal cat. Archs ital. Biol. 97: 13-25, 1959. 2. Bonvallet, M. et P. Dell. Contrrle bulbaire du syst/~me activateur. In: Aspects Anatomofonctionnels de la Physiologie du Sommeil, edited by M. Jouvet..Paris: C.N.R.S., 1965, pp. 133148. 3. Bremer, F. Existence of a mutual tonic inhibitory interaction between the preoptic hypnogenic structure and the midbrain reticular formation. Brain Res. 96: 71-75, 1975. 4. Bronzino, J. D. Effect of serotonin and xylocaine upon evoked responses established in neural feedback circuits associated with sleep-waking process. Biol. Psychiat. 3: 217-226, 1971. 5. Chu, N.-S. and F. E. Bloom. Activity patterns of catecholamine-containing pontine neurons in the dorso-lateral tegmentum of unrestrained cats. J. Neurobiol. 5: 527-544, 1974.
6. Fuller, J. H. Brain stem reticular units: synaptic responses to stimulation within the ascending reticular pathways. Brain Res. 112: 299-312, 1976. 7. Hobson, J. A., R. W. McCarley and P. W. Wyzinski. Sleep cycle oscillation: reciprocal discharge by two brainstem neuronal groups. Science 189: 55-58, 1975. 8. Kasamatsu, T. Maintained and evoked unit activity in the mesencephalic reticular formation of the freely behaving cat. Expl Neurol. 28: 450-470, 1970. 9. Kasamatsu, T. Effects of visual deafferentation on mesencephalic reticular activity in freely behaving cats. Expl Neurol. 29: 251-267, 1970. 10. Kostowski, W., R. Samanin, S. R. Bareggi, V. Marc, S. Garattini and L. Valzelli. Biochemical aspects of the interaction between midbrain raphe and locus coeruleus in the rat. Brain Res. 82: 178-182, 1974.
BRAINSTEM NEURONS DURING SLEEP 11. Mancia, M., M. Mariotti and R. Spreafico. Caudo-rostral brain stem reciprocal influences in the cat. Brain Res. 80: 41-51, 1974. 12. Manohar, S., H. Noda and W. R. Adey. Behavior of mesencephalic reticular neurons in sleep and wakefulness. Expl Neurol. 34: 140-157, 1972. 13. Nakamura, S. Two types of inhibitory effects upon brain stem reticular neurons by low frequency stimulation of raphe nucleus in the rat. Brain Res. 93: 140-144, 1975.
745 14. Satoh, T. and N. Kanamori. Reticulo-reticular relationship during sleep and waking. Physiol. Behav. 15: 333-337, 1975. 15. Sheu, Y.-S., J. P. Nelson and F. E. Bloom. Discharge patterns of cat raphe neurons during sleep and waking. Brain Res. 73: 263-276, 1974. 16. Wang, R. Y., D. W. Gallager and G. K. Aghajanian. Stimulation of pontine reticular formation suppresses firing of serotonergic neurones in the dorsal raphe. Nature 264: 365-368, 1976.
Functional Relationship Between Cat Brainstem Neurons During Sleep and Wakefulness TOYOHIKO SATOH, KUNIHIRO EGUCHI AND KAZUSHIGE WATABE
Department of Physiology, School of Dental Medicine, Aichi-Gakuin University Nagoya, 464, Japan ( R e c e i v e d 7 O c t o b e r 1978) S A T O H , T., K. E G U C H I A N D K. W A T A B E . Functional relationship between cat brainstem neurons during sleep and wakefulness. P H Y S I O L . B E H A V . 22(4) 741-745, 1979.--The m o d e of interaction b e t w e e n so-called sleep-wakefulness
centers in the cat lower brainstem was studied on 100 neurons. The magnitude of the spike response of a neuron in one center to electrical stimulation of another center was measured to calculate the index of responsiveness. During REM sleep the index was, in a great majority of cases, significantly smaller as compared with that during wakefulness and slow wave sleep. This reduction in the effectiveness of information transmission between different centers might be the basis of characteristic events occurring during REM sleep. From the behavior of the indexes during sleep-wakefulness cycle, it is suggested that different phases of sleep and wakefulness are realized by a complicated interplay of many sleep-wakefulness centers which would be communicating with one another not only through channels which are activated in a phase-specific manner, but also through a larger number of channels of which activity is modulated differentially during different phases. Sleep-wakefulness mechanism Brainstem neurons Magnitude of evoked unitary discharge relative to spontaneous firing Dynamic relationship between sleep-wakefulness centers
OUR present understanding on the functional organization of the brain structures concerned with the realization of different phases of sleep and wakefulness is confined mostly to its static aspect; that is to say, we know that certain structures are important for a particular phase of sleep and wakefulness, while we are only poorly informed about the role of those structures during other phases. A negative feedback loop between the bulbar reticular formation and the midbrain reticular formation (MRF) has been suggested [1, 2, 11]. The basal forebrain area and the MRF are claimed to be mutually inhibitory [3], and this inhibition is thought to be put into action, at least in part, through the activation of inhibitorysystem in the bulbar reticular formation [11]. Reciporcal relationship has been demonstrated between the MRF and the solitary tract nucleus [4]. The influence of the reticular formation upon the raphe nuclei [13,16], and the interaction between the raphe and the locus coeruleus (LC) [10] have been studied. The onset of REM sleep is characterized by a reciprocal activity between the giganto-cellular tegmental field (FTG) and the LC [7]. The amplitude and configuration of the evoked responses which were elicited by electrical stimulation of one of so-called sleep-wakefulness centers and were recorded from other centers, are modulated during sleep-wakefulness cycle in a manner which is not easily accounted for by a simple, static concept that a particular center is executive only in a particular phase [14]. Present investigation was undertaken to study whether so-called sleep-wakefulness centers are in different relationship with one another during different phases of sleep and wakefulness. Their altered relationship must be somehow expressed in the magnitude of the spike response of indi-
vidual neurons in one center to electrical stimulation of another center. As the background discharge rate (BDR) of the recorded neuron may change from a phase to another, the degree of change in the effectiveness of the stimulated neurons to drive or inhibit the recorded neuron may be correctly expressed by the ratio of the number of evoked spikes to that of background spikes, rather than by the absolute number of the evoked spikes. Phase-dependent modulation, as assessed in this way, may occur at various sites; the stimulated neurons, the recorded neuron and the conduction pathways between the two. Therefore, the altered magnitude of the response is to be understood as representing the sum of the modulation acting on a set of functional units which has a group of stimulated neurons as the input stage and the recorded neuron as the common output element. METHOD
Data Acquisition Under intraperitoneal pentobarbitone anesthesia (40 mg/kg) 12 cats were prepared for chronic experiment. Stainless steel screws were fixed to the skull to monitor the EEG and electrooculogram and to be used as the reference electrode for unit recording. Vinyl-coated wires were embedded in the nuchal muscles for recording the EMG. Parallel, bipolar electrodes for stimulation were made of stainless steel wires of 50/zm in diameter and had tip separation of 0.5 mm along the axis. They were implanted stereotaxically into two sites of the following structures: the MRF, raphe magnus (RM), raphe dorsalis (DRN) and LC. Rectangular pulses of 0.01 msec in duration were delivered every 2.5-5.0 sec at a
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040741-05502.00/0
742
SATOH, EGUCHI AND WATABE
constant intensity of 0.3-0.8 mA. Glass-insulated Pt-Ir microelectrode of 2-7 Mf~ was driven with a hydraulic system which worked at 1 /zm steps and was mounted on a metal cylinder fixed on the trepanned skull. In order to ensure long-time holding of the units, the head of the animal was restrained from moving by tightly clamping the acrylic mound made on the skull. Unit activity was picked up from the MRF, RM, LC and FFG. After the experiment the animal was sacrificed with overdose anesthetics. Recorded and stimulated sites were identified histologically on serial sections stained with Kliiver-Barrera method.
Data Analysis Spike activity tape-recorded at a frequency response of 5 kHz was fed to the data processor (San-ei Sokki 7T07) to calculate the BDR and to obtain post-stimulus time histograms (PSTH) for each sleep-wakefulness phase. The PSTHs were constructed with 2 msec bin width and 50-100 sweeps. Only the neurons which gave an obvious response in the PSTH in any phase of sleep and wakefulness were submitted to further analysis. The effectiveness for the stimulated site to drive or inhibit the recorded neuron was expressed with an index. The index represents the ratio of the evoked spike number during the response period which may change in duration from one phase to another, to the background spike number during the corresponding period.
stim. FIG. 1. Parameters used to calculate the index of responsiveness from the post-stimulus time histogram. Excitatory (R') and inhibitory (R'~)responses are schematically shown along with the duration of the responses (D' and D"). BG: mean background discharge rate.
55•
R-BG BG
D Dw
where R is the total spike number during response period D in the PSTH schematically shown in Fig. 1. BG is the background spike number during the period corresponding to D. Dw is the duration of the response during quiet wakefulness without gross movement (W). When significant (see below) response was not present during W or during both W and slow wave sleep (S), the duration of the response during S or REM sleep (P) was substituted for Dw, respectively. Unequivocal deflections in the PSTH which were regarded as significant responses had an index greater than + 1.0 in the case of excitation and less than -0.3 in the case of inhibition. As the physiological significance of the output pattern of recorded neuron is totally unknown and the PSTH disregards such an aspect, the above equation contains no term about the discharge pattern. Hence it is possible for two phases with different values in their parameters to have an identical index. When the index and the BDR were calculated in the same phases occurring at a different time, the maximum variance of both values was within 30%. Consequently, the difference in the index and the BDR greater than 30% was regarded as significant. RESULTS
Excitatory responses which gave peaks in the PSTH with index greater than + 1.0 were most often found at about 10 and/or 50 msec. A total of 106 excitatory responses were obtained from 100 neurons. Inhibitory responses with index less t h a n - 0 . 3 had a peak, in general, at 20--30 msec (Fig. 2). Total number of the inhibitory responses was 57. There seemed to be no great difference due to the combination of stimulated and recorded structures. The analysis was done on the responses with latency greater than 2 msec.
IlI~RF
1@-~ RMOMRF
I
5-
Index =
MRFOLC
MRF
40-
I
5-
:: : : : : :
DRN~MRF
20-
MRFOFTG 50 ~-~ 100
20-
0, RM
1SOres
excitatory inhibitory
FIG. 2. Response periods appeared in the PSTHs. Ordinate: number of response. Abscissa: time after stimulation. The arrows point from the stimulated site to the recorded site.
The BDR during W was in most neurons between 20 and 25/see. During S it tended to be slightly lower. During P it was usually between 30 and 40/see and was, in a majority of cases, definitely higher as compared to other phases. Two out of 4 LC neurons had a BDR less than 1/sec during W and S. During P the BDR was distinctly higher. Seven out of 14 RM neurons had a BDR of about 4/see during W and S. During P the BDR was always increased. When the animal entered from W to S, the behavior of the indexes was apparently independent to the change in BDR. During P the index was, in a great majority of cases, smaller than during other phases and was often associated with increased BDR. However, increased or unaltered indexes were also usually associated with increased BDR. According to the mode of change during sleepwakefulness cycle, the indexes were classified into four
BRAINSTEM NEURONS DURING SLEEP
743
TABLE 1 BEHAVIOR DURING SLEEP-WAKEFULNESS PHASES OF THE INDEXES OBTAINED BY VARIOUS COMBINATIONS OF STIMULATION AND RECORDING SITES. X, X' OR X" CORRESPONDS TO AN INDEX OF ANY ONE OF W, S AND P. O REPRESENTS THE LEVEL OF SIGNIFICANCE OF THE INDEX OF EXCITATORY AND INHIBITORY RESPONSES. 1N THE SQUARE BRACKETS, THE NUMBERS OF PHASES WHICH FELL UNDER THE SPECIFIED CATEGORIES OF INDEX ARE SHOWN. Mono-phasic
Bi-phasic
Tri-phasic
No change
X>O>X'-X"
X,>X'-X">O X-X'>X'[>O X-X'>O>X"3
X>X'>X">O X>X'>O>X"
26[X=Wx12, S x l 0 , Px4]
43 [X,=Wx3, S×9, P x l X ' ~ = W x l , Sx2, P x l 6 X":~=Wx 1, Sx2, Px8]
12 [X=Wx2, S x l 0 X"=W×3, P×9]
9
Site of Stim.
Site of Rec.
No. of Response (Neuron)
DRN
MRF
90 (54)
LC
MRF
12 (8)
3 [X=Wx2, P x l ]
4 [X,=0 X'~=P×3 X":~=Px l]
4 [ X = W x l , Sx3 X " = W x 1, P×3]
1
RM
MRF
23 (13)
3 [X=Wxl, Sxl, Pxl]
9 [X,=Sx2, P x l X'~=Px3 X":~=Sx2, Px 1]
9 [X=Wx5, Sx4 X"=Sx 1, Px8]
2
MRF
LC
6 (4)
2 [X=Wxl, Sxl]
2 [X,=Sxl X'[=0 X":~=P x 11
2 [X=Wxl, Sxl X"=P×2]
0
MRF
RM
19 (14)
3 [ X = W x l , S×2]
12 [X,=Wx2 X '~=P×5 X":~=Px5]
4 [X=Wx2, Sx2 X"=Px4]
0
MRF
FTG
13 (7)
3 [X=Wx2, S x l ]
2 [X=Sx2 X"=Px2]
2
163 (100)
6 [X,=Sx2 X'~=P× 1 X":~=Sx 1, P×2]
40 (24.5%)
76 (46.6%)
X-x'-x">O
33 (20.2%)
14 (8.5%)
tory and inhibitory responses are shown in Table 2. During P the inhibitory response o u t n u m b e r e d the excitatory one. All o f the P-specific responses w e r e obtained f r o m the M R F . Bi-phasic group occupied 46.6% o f the total response. During P the index was, most often, u n d e r the level o f significance (X.~ in Table 1) or smallest a m o n g three phases (X~ in Table 1; see also X2~-3 in Table 2). During S the index was, in m a n y cases, greatest a m o n g three phases (Xl in Table 1 and 2). Tri-phasic group occupied 20.2% of the total response. A b o u t 70% of this group were lacking a significant r e s p o n s e in one phase (0>Xq. During P the index was, in most occa-
groups (Table 1): ( 1 ) m o n o - p h a s i c group: significant r e s p o n s e is present only in one phase and absent in o t h e r two phases; (2) bi-phasic group: the r e s p o n s e is present at least in two phases, but the indexes of two phases are not significantly different; (3) tri-phasic group: the indexes during three phases of sleep and wakefulness are significantly different with one another; (4) no change: significant r e s p o n s e is present throughout three phases, but no change in the index. Mono-phasic group occupied 24.5% of the total response. R e s p o n s e s occurring only during W or S w e r e e n c o u n t e r e d at a c o m p a r a b l e f r e q u e n c y , while it was m u c h less f r e q u e n t to h a v e the r e s p o n s e only during P. The numbers of excita-
TABLE 2 RELATION OF EXCITATORY AND INHIBITORY RESPONSES TO THE BEHAVIOR OF THE INDEX. FOR ABBREVIATIONS REFER TO TABLE 1 Mono-phasic
Bi-phasic
X
X,
Tfi-phasic
X'~+:, excit inhib
excit
inhib
excit
inhib
W
10
9
2
3
1
S
10
5
10
4
P
1
5
1
21
19
13
Tot~
X
No change X"
excit
inhib
excit
inhib
1
9
2
2
2
3
4
19
3
0
1
1
33
13
0
0
26
2
8
37
18
28
5
28
5
excit
inhib
5
9
744
SATOH, EGUCHI AND WATABE
sions, under the level of significance or smallest among three phases (X" in Table 1 and 2). During S the index was frequently greatest among three phases (X in tri-phasic group in Table 1 and 2). Inhibitory responses were relatively small in number (Table 2). Responses with unaltered index throughout three phases were found in 8.5% of the total response. The proportion of the inhibitory response was relatively high in this group (Table 2). From Table 1 it did not seem that the site of stimulation, the site of recording or certain combinations of them were preferentially related with a particular behavior of the index. Therefore it was examined whether the behavior of the indexes is correlated with other factors like the magnitude of the index during W, the BDR during W, the latency of the response, or the polarity of the response, i.e. excitatory or inhibitory. As shown in Table 2, the polarity of the response seemed to be somewhat correlated with sleep phases. Smallest indexes (X2~+X'~ were most often found during P (=74) as compared to W(=6) and S(=8). Among these responses during P with smallest index, 79.7% were excitatory. Monophasic responses during P were, as indicated already, mostly inhibitory. During S there was a tendency that excitatory responses had greatest index among three phases, as seen in Xl of bi-phasic responses and X of mono- and tri-phasic responses. DISCUSSION The latencies of excitatory and inhibitory responses were not greatly different among different combinations of various structures stimulated and recorded, and were similar to the values reported on cat brainstem reticular formation [6, 8,
111. The BDR of the MRF neurons during sleep-wakefulness cycle was consistent with other reports [9,12]. One half of the LC neurons had a very low BDR and, hence, they are presumed to be noradrenaline-containing neurons. There were also neurons with higher discharge rate which were described by Chu and Bloom [5]. One half of the neurons in the RM was slowly discharging, suggesting that they are serotonin-containing neurons. Their discharge rates during sleep-wakefulness cycle were generally in agreement with the report by Sheu et al. [15], excepting slightly lower rate during W in our record which was obtained during quite
wakefulness without gross movement. The behavior of the indexes of the presumably monoamine-containing neurons was not markedly different from that of other neurons in the LC and RM. During P it was very infrequent for the index to be greatest among three phases (X in mono- and tri-phasic responses plus X, in bi-phasic responses in Table 2 = 6+0+2=8), while it was frequent during W(19 +11+5=35) and S(15+22+ 14=51). Smallest index among three phases was most often found during P(X'~+:~in bi-phasic and X" in tri-phasic responses in Table 2=46+28=74), while it was much less often during W(2+4=6) and S(7+ 1=8). It seemed, therefore, that reduction in the effectiveness of information transmission between different centers is one of the outstanding features of P. As the BDR is greatly increased during P in most neurons, the release of the activity of individual neurons from the control by other centers might be the basis of characteristic events occurring phasically or tonically during P; for example, PGO-spikes, REMs, desynchronized EEG, fiat EMG and so on. During S the index tended to be greatest among three phases. However, this tendency was far less remarkable as compared to the change during P. Generally speaking, S was more like W in terms of the behavior of the index. There were communication channels between different centers which were activated only during a particular phase and inactivated in other phases. However, there were far more numerous channels of which activity was modulated differentially in two or three phases. These communication channels with phase-specific and multi-phasic properties have been found between all centers studied. The proportion of the channels which were not influenced by sleepwakefulness phases was relatively small. There seemed to be no particular combination of stimulated and recorded sites which was correlated very well with a particular behavior of the indexes. Only the polarity of the response seemed to have some correlation with phase-dependent change in the index. The conclusion which may be reached by the present experiment is that different phases of sleep and wakefulness are realized by a complicated interplay of many sleepwakefulness centers which would be communicating with one another not only through channels which are activated in a phase-specific manner, but also through a larger number of channels of which activity is modulated differentially in different phases of sleep and wakefulness.
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