700
Electroencephalography and Clinical Neurophysiology, 1977, 43 : 700--706
© Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECTS OF BENZODIAZEPINES ON PGO FIRINGS AND MULTIPLE UNIT ACTIVITY IN THE MIDBRAIN RETICULAR FORMATION IN CATS TOSHIRO TSUCHIYA and HIDEAKI FUKUSHIMA Research and Development Center, Pharmaceuticals Division, Sumitomo Chemical Co., Ltd., 4-2-1, Takatsukasa, Takarazuka, Hyogo 665 (Japan)
(Accepted for publication: March 22, 1977)
It is well-known that benzodiazepines affect the sleep-wakefulness cycle of the cat. On acute administration they increase arousal and decrease slow wave and paradoxical sleep (Lanoir and Killam 1968; Hern~ndez-PeSn et al. 1969; Schallek et al. 1972; Otsuka et al. 1973, 1975}. Many electroencephalographic studies indicate that these drugs inhibit the ascending reticular and hypothalamic activating systems, but have little or no effect on the diffuse thalamo-cortical projecting system (Monnier and Graber 1962; R~quin et al 1963; Arrigo et al. 1965; SchaUek and Kuhen 1965; Schallek et al. 1965; Otsuka et al. 1973, 1975). These inhibitory actions, which diminished the waking influence of the activating systems, do not explain the increased arousal with benzodiazepines. Studies on multiple unit activity may throw further light on the mode of action of benzodiazepines. Multiple unit activity in the midbrain reticular formation clearly changed at each state of consciousness in the cat (Winters et al. 1969). During paradoxical sleep, there were two types of increased activity; one was a tonic increase which continued as long as the cortical EEG maintained the desynchronized pattern and the other was a phasic one which was associated with the occurrence of rapid eye movement (Winters et al. 1967; Mori et al. 1972). PGO waves were the electrical signs preceding such paradoxical episodes. In most cases there was a close relationship between the occurrence of PGO waves and rapid eye movements during the
state of sleep (Michel et al. 1964). In the present experiments, the effects of benzodiazepines administered via the intraperitoneal route were studied on multiple unit activity in the midbrain reticular formation and on PGO firings during various stages of consciousness in cats. Changes in the sleepwakefulness cycle after direct injection of benzodiazepines into the midbrain reticular formation were also studied in order to assess the role of the reticular formation in the sleep-wakefulness cycle of the cat.
Methods The m e a s u r e m e n t o f m u l t i p l e unit activity Six adult cats (3.2--3.9 kg) of either sex with chronically implanted electrodes were used in this experiment. The procedures for the implantation of electrodes and measurements of EEGs were described in previous papers (Otsuka et al. 1973, 1975). Multiple unit activity was amplified by a Nihon-Kohden VC-8 oscilloscope Model AVB-2 preamplifier. The o u t p u t from the oscilloscope was passed through a band pass filter (1000--3000 c/sec: --6.8 dB, 800 and 4000 c/sec: --40 dB) and amplified by an oscilloscope Model AVH-2 preamplifier. The final o u t p u t from the oscilloscope was rectified and integrated at a time constant of 100 msec. The integrated sign of multiple unit activity was recorded together with EEGs and integrated EMG (time constant of 350 msec) by a medical-
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES
The procedures for the chronic implantation of electrodes were the same as those for the recording of multiple unit activity. Cannulas were also chronically implanted in the bilateral midbrain reticular formation. The cannula (diam.: 0.8 mm) served as a guide for insertion of the micro-syringe which was previously filled with the solution to be injected. Diazepam and fludiazepam were dissolved in 50% polyethylene glycol No. 400 solution (PEG) in saline. The volume was 25 pl/injection, and the dosage (injected into one side) of fludiazepam was 5, 10, 20 and 50 #g and that of diazepam 20 /~g. The procedures for the measurement of sleep-wakefulness cycle were described in previous papers (Otsuka et al. 1973, 1975). In all experiments, the statistical significance of differences between drug and control values was analyzed by Student's t-test (n =
corder (Nihon-Kohden). The value of multiple unit activity was arbitrarily designated as a multiple of noise level with a 10 k ~ resistor across the input. Diazepam and fludiazepam [Code number of fludiazepam is ID-540 (Asami et al. 1974).] synthesized in our laboratory were administered to the animals by the intraperitoneal route in suspension with 0.5% carboxymethyl cellulose (CMC). The dosage of diazepam and fludiazepam was 1 mg/kg and 0.5 mg/kg respectively which prolonged the onset of paradoxical sleep for 70--150 min (Otsuka et al. 1975).
Micro-injection into the midbrain reticular formation and the measurement of sleepwakefulness cycle The study was carried out with six chronic cat preparations with implanted electrodes.
i
MC-EEG
i
iI
,
701
,
I min
i
SWSwi~PGO
i
pre-PSi.
PS
GL-EEG EMG-INT
~
~L n ,,,I1F I
IO K.(}. DC-O
Fig. 1. Relationship between cortical and lateral geniculate EEGs, EMG and multiple unit activity in the midbrain reticular formation. MC-EEG and GL-EEG: electroencephalogram in the motor cortex and lateral geniculate body; EMG-INT: integrated value of electromyogram; RF-MUA: multiple unit activity in the midbrain reticular fomation; SWS with PGO: slow wave sleep with PGO firings; pre-PS: preparatory period to paradoxical sleep; PS: paradoxical sleep. The lowest line (DC = O) represents the recording system balanced to zero; the 10 k ~ line represents the system noise level with a 1 0 k ~ resistor across the input.
702
T. T S U C H I Y A , H. F U K U S H I M A
4). After the experiment, stereotaxic placements of the electrodes and cannulas were verified histologically.
Multiple unit activity in the midbrain reticular formation was in order from the highest value to the lowest as follows: paradoxical sleep -- alert > awake > slow wave sleep. During slow wave sleep, the activity with PGO firings was higher than without PGO firings. Both diazepam and fludiazepam significantly decreased multiple unit activity during the 3 sleep states b u t had no significant effects during the 2 waking states (Fig. 2). There was a positive correlation between the PGO firing rate and the appearance on the phasic c o m p o n e n t of multiple unit activity in the midbrain reticular formation during paradoxical sleep (r = 0.793). When PGO firing rate was high, the appearance of the phasic c o m p o n e n t increased. Fludiazepam maintained the correlation (r = 0.837) but shifted the relation to lower values 2--5 h after administration {Fig. 3); that is, it reduced the appearance of phasic components of multiple unit activity in the midbrain reticular formation during paradoxical sleep. Diazepam showed the same effect.
The criteria for various stages of consciousness The criteria for various levels of consciousness in the cat, modified from the description by Jouvet {1967), were based on neocortical and lateral geniculate EEGs, neck muscle tone (EMG), multiple unit activity in the midbrain reticular formation, and on gross behavior. They were divided into four categories; alert, awake, slow wave sleep and paradoxical sleep. Slow wave sleep was divided into the following stages: slow wave sleep without PGO firings, slow wave sleep with PGO firings, and pre-paradoxical sleep (the preparatory period to paradoxical sleep during slow wave sleep) (Fig. 1).
Results
Effects of benzodiazepines on PGO firings and multiple unit activity in the midbrain reticular formation The PGO firing rate was much higher during paradoxical sleep than during slow wave or pre-paradoxical sleep. Fludiazepam (0.5 mg/kg) produced a statistically significant increase in PGO firing rate during pre-paradoxical sleep, b u t had no significant effect on firing rates during slow wave or paradoxical sleep. Diazepam (1 mg/kg) had no significant effects on PGO firing rates (Table I).
Micro-injection of benzodiazepines into the midbrain reticular formation Fig. 4 shows the effect of fludiazepam at doses of 5, 10, 20 and 50/~g/injection, which was injected bilaterally into the midbrain reticular formation, on each ratio of arousal, slow wave sleep and paradoxical sleep. Fludiazepare increased arousal and d e c r e a s e d slow wave sleep, b u t did not affect paradoxical sleep. The action of fludiazepam was marked for 2 h after injection, but disappeared after
TABLE I E f f e c t o f f l u d i a z e p a m a n d d i a z e p a m o n P G O firing rate (per rain) during slow wave sleep (SWS), p r e - p a r a d o x i c a l sleep (pre-PS) and paradoxical sleep (PS). SWS
pre-PS
PS
Control 0.5% CMC
23.7 ± 2.9
27.5 _+ 1.4
57.9 ± 4.2
Fludiazepam 0.5 mg/kg
29.7 ± 5.2
35.3 _+ 2.9 *
52.0 + 4.0
Diazepam 1.0 mg/kg
22.4 ± 7.2
29.1 ± 6.3
52.4 ± 7.4
* P < 0.05, drug vs. c o n t r o l , n = 4.
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES i0
703
10
9
8
7
6
5
5
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g
3
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2 1 0
0 • • PARADOXICAL SLEEP
ALERT
AWAKE
SLOWWAVE SLOWWAVE
SLEEP WITH SLEEP WITHOUT PGO PGO
A
PARADOXICAL
At~ERT
AWAKE
SLEEP
SLOWWAVE SLOWWAVE SLEEP WITH PGO
SLEEP WITHOUT P60
B
Fig. 2. C o m p a r i s o n o f m u l t i p l e u n i t activity in t h e m i d b r a i n r e t i c u l a r f o r m a t i o n d u r i n g p a r a d o x i c a l sleep, alert, awake, slow wave sleep w i t h P G O firings a n d slow wave sleep w i t h o u t P G O firings. O p e n squares are t h e c o n t r o l states and hatched squares are the states after a single administration of fludiazepam, 0.5 m g / k g i.p. (A) and diazepam, 1 m g / k g i,p. (B). n = 4. * P < 0.05. R F - M U A value was arbitrarily designated as a multiple of noise level with a 10 k ~ resistor across the input.
3 h. Diazepam at 20 #g also increased arousal and decreased slow wave sleep.
Discussion
The reduction of paradoxical sleep Moruzzi (1963) suggested that paradoxical sleep could be subdivided into tonic and phasic events. The most precise measure for phasic activity in the cat has been the large slow potential recorded from the pontine reticular formation, the lateral geniculate bodies and the occipital cortex, that is PGO waves (Jeannerod et al. 1965). Ferguson et al. (1968) have suggested that the phasic activity as measured by PGO firings may be the crucial factor in paradoxical sleep and the tonic activity may be irrelevant. This PGO phenom-
enon, although it predominantly occurred during paradoxical sleep, also occurred sporadically during slow wave sleep. PGO waves were consistently seen during slow wave sleep some 30--60 sec prior to the tonic activity of paradoxical sleep. Thus, considerable temporal overlap exists between slow wave sleep process and the primary phasic phenomenon of paradoxical sleep (Henriksen et al. 1974). Furthermore, the interrelation between PGO firings and the increase of multiple unit activity during slow wave and paradoxical sleep may be close. The occurrence of paradoxical sleep can be forecast from the appearance of multiple unit activity in the midbrain reticular formation. There are some reports with respect to the function of the midbraln reticular formation during paradoxical sleep (Evarts 1964; Candia
704
T. TSUCHIYA, H. FUKUSHIMA CONTROL (SOLVENT PEG) FLUDIAZEPAM 5 ~UG FLUDIAZEPAM 10 ~UG FLUDIAZEPAM 20 ~G FLUDIAZEPAM 50 .UG
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y=0.115X-2.798 (r=(~.793)
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+=0+074X-3. 007 ~+=0.83+,
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--
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,
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m°--+-O 50
o
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,
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,
,
60
70
80
90
i00
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PG0 FIRING RATE (PERM]N) ~
Fig. 3. Interrelation between PGO firing rate and the appearance of phasic components of multiple unit activity in the midbrain reticular formation during paradoxical sleep. Each point indicates the observed value of four cats between 2 and 5 h after administration of 0.5%CMC (O) and fludiazepam, 0.5 mg/kg i.p. (o).
20
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2o
et al. 1962; Faure et al. 1962; Yamamoto and Kido 1962). It is suggested that the midbrain reticular formation may not play a primary role o n paradoxical sleep b u t has a facilitatory influence on the appearance of paradoxical sleep. We have shown in this paper that benzodiazepines decrease multiple unit activity in the midbrain reticular formation. We have found that these compounds also reduced increased multipe unit activity in the preoptic area and m o t o r cortex (unpublished data). We suggest that one of the principal actions of benzodiazepines is a general inhibition of increksed neuronal activity which is triggered from the pons. The increase o f arousal Considering the prominent role which the midbrain reticular formation plays in EEG arousal, it is to some extent surprising that arousal in sleep-wakefulness cycle was increased by benzodiazepines, even though EEG arousal responses by reticular and hypothalamic stimulation and multiple unit activ-
0-i
HR
1-2 HR
2-3 HR
3+4 HR
Fig. 4. Effect of fludiazepam and the solvent PEG on sleep-wakefulness cycle after bilateral injection into the midbrain reticular formation. The ratio of arousal, slow wave sleep and paradoxical sleep is indicated every 1 h. n = 4. * P ~ 0.05.
ity in the midbrain reticular formation at various stages of sleep were decreased by them. The increase of arousal by benzodiazepines could be considered to be induced by some action on the midbrain reticular formation because bilateral injection of benzodiazepines into the reticular formation increased arousal. The reduction of EEG arousal response, elicited by stimulation of the midbrain reticular formation, by benzodiazepines might be induced by inhibition of other areas of neuronal connection which might affect the excitability of the midbrain reticular formation. Several studies of brain lesion, transection and stimulation (Kawamura et al. 1961;
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES Feldman and Waller 1962; Schlag and Chaillet 1963; Kawamura and Domino 1968) have demonstrated that tonic EEG arousal in the cortex was enhanced b y a powerful inflow of impulses from the medial hypothalamus to the midbrain formation. F u k u d a et al. (1974) suggested that 1,4-benzodiazepines suppressed the tonic neocortical EEG arousal because of the reduction of this powerful inflow of impulses from the hypothalamus to the midbrain reticular formation. The suppression of multiple unit activity in the midbrain reticular formation after administration of benzodiazepines would be considered to be due to the same reduction. T h e fact that the suppression of multiple unit activity was observed only during sleep allows us to speculate that the physiological and pharmacological properties of reticular neuronal activity differ at various levels of consciousness. The benzodiazepines seem to facilitate the reticular formation under certain circumstances and depress it under other circumstances. The possible dependence of these actions on the state of consciousness of the animal deserves further study.
Summary
Effects of benzodiapines administered b y the intraperitoneal route on PGO firings and multiple unit activity in the midbrain reticular formation in chronic cat preparations were investigated at various levels of consciousness. Changes in the sleep-wakefulness cycle induced b y direct injection of benzodiazepines into the reticular formation were also investigated. Benzodiazepines markedly decreased multiple unit activity in the midbrain reticular formation during each stage of sleep, b u t had little effect during behavioral and EEG arousal. Benzodiazepines did not affect PGO firing.rate, b u t attenuated all increase of multiple unit activity following PGO firings. The bilateral injection of benzodiazepines into the midbrain reticular formation induced an increase of arousal and a decrease
705
of slow wave sleep, but did not change the a m o u n t of paradoxical sleep. It is concluded that benzodiazepines show a mixture of depressant and facilitatory effects, which seem to vary with the state of consciousness of the animal.
R~sum~ Effets des benzodiazdpines sur les ddcharges PGO et l'activitd multi-unitaire de la formation rdticulaire mdsocdphalique du chat Les auteurs ont ~tudi~ l'action des benzodiazepines administr~es par voie intrap~riton~ale sur les d~charges PGO et l'activit~ multiunitaire de la formation r~ticulaire mdsoc~phalique du chat en preparation chronique, divers niveaux de vigilance, ainsi que les modifications du cycle veille-sommeil induites par injection directe de benzodiaz~pines dans la formation r~ticulaire. Les benzodiaz~pines diminuent de faqon marquee l'activitd multiunitaire de la formation r~ticulaire m~soc~phalique ~ chaque stade de sommeil, mais n ' o n t que peu d'effet au cours de l'~veil EEG et comportemental. Les benzodiazdpines n'affectent pas le taux de d~charges PGO, mais att~nuent toute augmentation de l'activit~ multi-unitaire consecutive aux d~charges PGO. L'injection bilat~rale de benzodiaz~pines dans la formation r~ticulaire m~soc~phalique induit une augmentation de l'~veil et une diminution du sommeil ~ ondes lentes, mais ne modifie pas la quantit~ de sommeil paradoxal. Les auteurs concluent que les benzodiaz~pines pr~sentent un ensemble d'effets ddpresseurs et facflitateurs qui paraissent varier avec l'~tat de vigilance de l'animal.
References Arrigo, A., Jann, G. and Tonali, P. Some aspects of the action of Valium and of Librium on the electrical activity of the rabbit brain. Arch. int. Pharmacodyn., 1965, 154: 364--373.
706 Asami, Y., Otsuka, M., Hirohashi, T., Inaba, S. and Yamamoto, H. The synthesis and pharmacology of a novel benzodiazepine derivative, 1-methyl-5(o-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4-benzodiazepin-2-one (ID-540). Arzneim.-Forsch., 1974, 24: 1563--1568. Candia, O., Favale, E., Giussani, A. and Rossi, G.F. Blood pressure during stimulation of the brain stem reticular formation. Arch. ital. Biol., 1962, 100: 216--233. Evarts, E.V. Temporal patterns of discharge of pyramidal tract neurons during sleep and waking in the monkey. J. Neurophysiol., 1964, 27: 152--172. Faure, J., Bensch, C. et Vincent, D. RSle d'un syst6me m6senc6phalo-limbique dans la "phase paradoxale" du sommeil chez le lapin. C. R. Soc. Biol. (Paris), 1962, 156: 70--73. Feldman, S.M. and Waller, H.J. Dissociation of electrocortical activation and behavioral arousal. Nature (Lond.), 1962, 196: 1320--1322. Ferguson, J., Henriksen, S., McGarr, K., Belensky, G., Mitchell, G., Gonda, W., Cohen, H. and Dement, W. Phasic event deprivation in the cat. Psychophysiology, 1968, 5: 238--239. Fukuda, N., Saji, Y. and Nagawa, Y. Neuropharmacological studies of effect of new central depressant, 8-chloro-6-phenyl-4H-s-triazole [4,3-a] [1,4] benzodiazepine (D-40TA) on EEG and central sympathetic activating mechanism in cats. Jap. J. Pharmacol., 1974, 24: 75--88. Henriksen, S., Dement, W. and Brachas, J. The role of serotonin in the regulation of a phasic event of rapid eye movement sleep: The ponto-geniculo-occipital waves. In E. Costa, G.L. Gessa and M. Sandler (Eds.), Advances in Biochemical Psychopharmacology, Raven Press, New York, 1974, 11: 169--179. Hern~ndez-Pe6n, R., Goldberg, L. and Rojas-Ramirez, J.A. Physiology and psychosomatic medicine: Neurophysiological models of emotional behavior and of action of psychotrophic d~ugs. In A. Pletseher and A. Marino (Eds.), Psychotropic Drugs in Internal Medicine, Exc. Med. Found., Amsterdam, 1969: 16--46. Jeannerod, M., Mouret, J. et Jouvet, M. Effects secondaires de la d~aff~rentiation visuelle sur l'activit6 phasique pont-g~niculo-occipital du sommeU paradoxical. J. Physiol. (Paris), 1965, 57: 255--256. Jouvet, M., Neurophysiology of the states of sleep. Physiol. Rev., 1967, 47: 117--177. Kawamura, H., and Domino, E.F. Hippocampal slow ("arousal") wave activation in the rostral midbrain transected cat. Electroenceph. clin. Neurophysiol., 1968, 25: 471--480. Kawamura, H., Nakamura, Y. and Tokizane, T. Effect of acute brain stem lesions on the electrical activities of the limbic system and neocortex. Jap. J. Physiol., 1961, 11: 564--575. Lanoir, J. and Killam, E.K. Alteration in the sleepwakefulness patterns by benzodiazepines in the cat. Electroenceph. clin. Neurophysiol., 1968, 25: 530--542.
T. TSUCHIYA, H. FUKUSHIMA Michel, F., Rechestchaffen, A. et Vimont, P. Activit~ dlectrique des muscles occulairees extrims~ques au cours du cycle veille-sommeil. C. R. Soc. Biol. (Paris), 1964, 158: 106--109. Monnier, M. et Graber, S. Classification ~lectrophysiologique des substances psycholeptiques. Arch. int. Pharmacodyn., 1962, 140: 206--216. Mori, K., Mitani, H., Fujita, M. and Winters, W.D. Multiple unit activity of dorsal cochlear nucleus and midbrain reticular formation during paradoxical phase of sleep. IV. A supplementary note. Electroenceph, clin. Neurophysiol., 1972, 33: 104-106. Moruzzi, G. Active processes in the brain stem during sleep. In the Harvey Lectures, Academic Press, New York, 1963, Set. 58: 233- 397. Otsuka, M., Tsuchiya, T. and Kitagawa, S. Electroencephalographic and behavioral studies on the central action of nimetazepam (S-1530) in cats. Arzneimittel-Forsch., 1973, 23: 645--652. Otsuka, M., Tsuchiya, T. and Kitagawa, S. Electrophysiological and behavioral effects of 1-methyl-5(o-fluorophenyl)-7-chloro-1,3-dihydro-2H-1,4-benzodiazepin-2-one (ID-540) in cats and rabbits. Arzneimittel-Forseh., 1975, 25: 755--760. Rdquin, S., Lanoir, J., Plas, R. and Naquet, R. Etude comparative des effets neurophysiologique du Librium et du Valium. C. R. Soc. Biol. (Paris), 1963, 157: 2015--2019. Schallek, W. and Kuhen, A. Effects of benzodiazepines on spontaneous EEG and arousal responses of cats. In K. Akert, C. Bally and J.P. Schad~ (Eds.) Sleep Mechanisms, Progr. Brain Res. Vol. 18, Elsevier, Amsterdam, 1965: 231--238. Schallek, W., Kuhen, A. and Kovocs, J. Effects of chlordiazepoxide hydrochloride on discrimination responses and sleep cycles in cats. Neuropharmacology, 1972, 11: 69--79. Schallek, W., Thomas, J., Kuhen, A. and Zabransky, F. Effects of Mogadon on responses to stimulation of sciatic nerve, amygdala and hypothalamus of cat. Int. J. Neuropharmacol., 1965, 4: 317--326. Schlag, J. and Chaillet, F. Thalamic mechanisms involved in cortical desynchronization and recruiting responses. Electroenceph. clin. Neurophysiol., 1963, 15: 39--62. Winters, W.D., Mori, K., Spooner, C.E. and Kado, R.T. Correlation of reticular and cochlear multiple unit activity with auditory evoked responses during wakefulness and sleep. I. Electroenceph. clin. Neurophysiol., 1967, 23 : 539--545. Winters, W.D., Mori, K. Wallach, M.B., Marcus, R.J. and Spooner, C.E. Reticular multiple unit activity during a progression of states induced b y CNS excitants. III. E l e c t r o e n c e p h . clin. Neurophysiol., 1969, 27: 514--522. Yamamoto, K. and Kido, R. [Neurophysiological studies on the nature of sleep -- Neural-mechanism related to "Activated Sleep" --] (in Japanese). Clin. Psychiat. 1962, 4: 821--830.
Electroencephalography and Clinical Neurophysiology, 1977, 43 : 700--706
© Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECTS OF BENZODIAZEPINES ON PGO FIRINGS AND MULTIPLE UNIT ACTIVITY IN THE MIDBRAIN RETICULAR FORMATION IN CATS TOSHIRO TSUCHIYA and HIDEAKI FUKUSHIMA Research and Development Center, Pharmaceuticals Division, Sumitomo Chemical Co., Ltd., 4-2-1, Takatsukasa, Takarazuka, Hyogo 665 (Japan)
(Accepted for publication: March 22, 1977)
It is well-known that benzodiazepines affect the sleep-wakefulness cycle of the cat. On acute administration they increase arousal and decrease slow wave and paradoxical sleep (Lanoir and Killam 1968; Hern~ndez-PeSn et al. 1969; Schallek et al. 1972; Otsuka et al. 1973, 1975}. Many electroencephalographic studies indicate that these drugs inhibit the ascending reticular and hypothalamic activating systems, but have little or no effect on the diffuse thalamo-cortical projecting system (Monnier and Graber 1962; R~quin et al 1963; Arrigo et al. 1965; SchaUek and Kuhen 1965; Schallek et al. 1965; Otsuka et al. 1973, 1975). These inhibitory actions, which diminished the waking influence of the activating systems, do not explain the increased arousal with benzodiazepines. Studies on multiple unit activity may throw further light on the mode of action of benzodiazepines. Multiple unit activity in the midbrain reticular formation clearly changed at each state of consciousness in the cat (Winters et al. 1969). During paradoxical sleep, there were two types of increased activity; one was a tonic increase which continued as long as the cortical EEG maintained the desynchronized pattern and the other was a phasic one which was associated with the occurrence of rapid eye movement (Winters et al. 1967; Mori et al. 1972). PGO waves were the electrical signs preceding such paradoxical episodes. In most cases there was a close relationship between the occurrence of PGO waves and rapid eye movements during the
state of sleep (Michel et al. 1964). In the present experiments, the effects of benzodiazepines administered via the intraperitoneal route were studied on multiple unit activity in the midbrain reticular formation and on PGO firings during various stages of consciousness in cats. Changes in the sleepwakefulness cycle after direct injection of benzodiazepines into the midbrain reticular formation were also studied in order to assess the role of the reticular formation in the sleep-wakefulness cycle of the cat.
Methods The m e a s u r e m e n t o f m u l t i p l e unit activity Six adult cats (3.2--3.9 kg) of either sex with chronically implanted electrodes were used in this experiment. The procedures for the implantation of electrodes and measurements of EEGs were described in previous papers (Otsuka et al. 1973, 1975). Multiple unit activity was amplified by a Nihon-Kohden VC-8 oscilloscope Model AVB-2 preamplifier. The o u t p u t from the oscilloscope was passed through a band pass filter (1000--3000 c/sec: --6.8 dB, 800 and 4000 c/sec: --40 dB) and amplified by an oscilloscope Model AVH-2 preamplifier. The final o u t p u t from the oscilloscope was rectified and integrated at a time constant of 100 msec. The integrated sign of multiple unit activity was recorded together with EEGs and integrated EMG (time constant of 350 msec) by a medical-
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES
The procedures for the chronic implantation of electrodes were the same as those for the recording of multiple unit activity. Cannulas were also chronically implanted in the bilateral midbrain reticular formation. The cannula (diam.: 0.8 mm) served as a guide for insertion of the micro-syringe which was previously filled with the solution to be injected. Diazepam and fludiazepam were dissolved in 50% polyethylene glycol No. 400 solution (PEG) in saline. The volume was 25 pl/injection, and the dosage (injected into one side) of fludiazepam was 5, 10, 20 and 50 #g and that of diazepam 20 /~g. The procedures for the measurement of sleep-wakefulness cycle were described in previous papers (Otsuka et al. 1973, 1975). In all experiments, the statistical significance of differences between drug and control values was analyzed by Student's t-test (n =
corder (Nihon-Kohden). The value of multiple unit activity was arbitrarily designated as a multiple of noise level with a 10 k ~ resistor across the input. Diazepam and fludiazepam [Code number of fludiazepam is ID-540 (Asami et al. 1974).] synthesized in our laboratory were administered to the animals by the intraperitoneal route in suspension with 0.5% carboxymethyl cellulose (CMC). The dosage of diazepam and fludiazepam was 1 mg/kg and 0.5 mg/kg respectively which prolonged the onset of paradoxical sleep for 70--150 min (Otsuka et al. 1975).
Micro-injection into the midbrain reticular formation and the measurement of sleepwakefulness cycle The study was carried out with six chronic cat preparations with implanted electrodes.
i
MC-EEG
i
iI
,
701
,
I min
i
SWSwi~PGO
i
pre-PSi.
PS
GL-EEG EMG-INT
~
~L n ,,,I1F I
IO K.(}. DC-O
Fig. 1. Relationship between cortical and lateral geniculate EEGs, EMG and multiple unit activity in the midbrain reticular formation. MC-EEG and GL-EEG: electroencephalogram in the motor cortex and lateral geniculate body; EMG-INT: integrated value of electromyogram; RF-MUA: multiple unit activity in the midbrain reticular fomation; SWS with PGO: slow wave sleep with PGO firings; pre-PS: preparatory period to paradoxical sleep; PS: paradoxical sleep. The lowest line (DC = O) represents the recording system balanced to zero; the 10 k ~ line represents the system noise level with a 1 0 k ~ resistor across the input.
702
T. T S U C H I Y A , H. F U K U S H I M A
4). After the experiment, stereotaxic placements of the electrodes and cannulas were verified histologically.
Multiple unit activity in the midbrain reticular formation was in order from the highest value to the lowest as follows: paradoxical sleep -- alert > awake > slow wave sleep. During slow wave sleep, the activity with PGO firings was higher than without PGO firings. Both diazepam and fludiazepam significantly decreased multiple unit activity during the 3 sleep states b u t had no significant effects during the 2 waking states (Fig. 2). There was a positive correlation between the PGO firing rate and the appearance on the phasic c o m p o n e n t of multiple unit activity in the midbrain reticular formation during paradoxical sleep (r = 0.793). When PGO firing rate was high, the appearance of the phasic c o m p o n e n t increased. Fludiazepam maintained the correlation (r = 0.837) but shifted the relation to lower values 2--5 h after administration {Fig. 3); that is, it reduced the appearance of phasic components of multiple unit activity in the midbrain reticular formation during paradoxical sleep. Diazepam showed the same effect.
The criteria for various stages of consciousness The criteria for various levels of consciousness in the cat, modified from the description by Jouvet {1967), were based on neocortical and lateral geniculate EEGs, neck muscle tone (EMG), multiple unit activity in the midbrain reticular formation, and on gross behavior. They were divided into four categories; alert, awake, slow wave sleep and paradoxical sleep. Slow wave sleep was divided into the following stages: slow wave sleep without PGO firings, slow wave sleep with PGO firings, and pre-paradoxical sleep (the preparatory period to paradoxical sleep during slow wave sleep) (Fig. 1).
Results
Effects of benzodiazepines on PGO firings and multiple unit activity in the midbrain reticular formation The PGO firing rate was much higher during paradoxical sleep than during slow wave or pre-paradoxical sleep. Fludiazepam (0.5 mg/kg) produced a statistically significant increase in PGO firing rate during pre-paradoxical sleep, b u t had no significant effect on firing rates during slow wave or paradoxical sleep. Diazepam (1 mg/kg) had no significant effects on PGO firing rates (Table I).
Micro-injection of benzodiazepines into the midbrain reticular formation Fig. 4 shows the effect of fludiazepam at doses of 5, 10, 20 and 50/~g/injection, which was injected bilaterally into the midbrain reticular formation, on each ratio of arousal, slow wave sleep and paradoxical sleep. Fludiazepare increased arousal and d e c r e a s e d slow wave sleep, b u t did not affect paradoxical sleep. The action of fludiazepam was marked for 2 h after injection, but disappeared after
TABLE I E f f e c t o f f l u d i a z e p a m a n d d i a z e p a m o n P G O firing rate (per rain) during slow wave sleep (SWS), p r e - p a r a d o x i c a l sleep (pre-PS) and paradoxical sleep (PS). SWS
pre-PS
PS
Control 0.5% CMC
23.7 ± 2.9
27.5 _+ 1.4
57.9 ± 4.2
Fludiazepam 0.5 mg/kg
29.7 ± 5.2
35.3 _+ 2.9 *
52.0 + 4.0
Diazepam 1.0 mg/kg
22.4 ± 7.2
29.1 ± 6.3
52.4 ± 7.4
* P < 0.05, drug vs. c o n t r o l , n = 4.
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES i0
703
10
9
8
7
6
5
5
g
g
3
3
2 1 0
0 • • PARADOXICAL SLEEP
ALERT
AWAKE
SLOWWAVE SLOWWAVE
SLEEP WITH SLEEP WITHOUT PGO PGO
A
PARADOXICAL
At~ERT
AWAKE
SLEEP
SLOWWAVE SLOWWAVE SLEEP WITH PGO
SLEEP WITHOUT P60
B
Fig. 2. C o m p a r i s o n o f m u l t i p l e u n i t activity in t h e m i d b r a i n r e t i c u l a r f o r m a t i o n d u r i n g p a r a d o x i c a l sleep, alert, awake, slow wave sleep w i t h P G O firings a n d slow wave sleep w i t h o u t P G O firings. O p e n squares are t h e c o n t r o l states and hatched squares are the states after a single administration of fludiazepam, 0.5 m g / k g i.p. (A) and diazepam, 1 m g / k g i,p. (B). n = 4. * P < 0.05. R F - M U A value was arbitrarily designated as a multiple of noise level with a 10 k ~ resistor across the input.
3 h. Diazepam at 20 #g also increased arousal and decreased slow wave sleep.
Discussion
The reduction of paradoxical sleep Moruzzi (1963) suggested that paradoxical sleep could be subdivided into tonic and phasic events. The most precise measure for phasic activity in the cat has been the large slow potential recorded from the pontine reticular formation, the lateral geniculate bodies and the occipital cortex, that is PGO waves (Jeannerod et al. 1965). Ferguson et al. (1968) have suggested that the phasic activity as measured by PGO firings may be the crucial factor in paradoxical sleep and the tonic activity may be irrelevant. This PGO phenom-
enon, although it predominantly occurred during paradoxical sleep, also occurred sporadically during slow wave sleep. PGO waves were consistently seen during slow wave sleep some 30--60 sec prior to the tonic activity of paradoxical sleep. Thus, considerable temporal overlap exists between slow wave sleep process and the primary phasic phenomenon of paradoxical sleep (Henriksen et al. 1974). Furthermore, the interrelation between PGO firings and the increase of multiple unit activity during slow wave and paradoxical sleep may be close. The occurrence of paradoxical sleep can be forecast from the appearance of multiple unit activity in the midbrain reticular formation. There are some reports with respect to the function of the midbraln reticular formation during paradoxical sleep (Evarts 1964; Candia
704
T. TSUCHIYA, H. FUKUSHIMA CONTROL (SOLVENT PEG) FLUDIAZEPAM 5 ~UG FLUDIAZEPAM 10 ~UG FLUDIAZEPAM 20 ~G FLUDIAZEPAM 50 .UG
8O
y=0.115X-2.798 (r=(~.793)
60
40
20
,
•
+ ,~
P
•
•
O
o
+
80
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•
1
o °o°/-
+=0+074X-3. 007 ~+=0.83+,
•
6O
0
..%
--
,
,
,
20
30
~0
m°--+-O 50
o
o ,
,
,
,
,
60
70
80
90
i00
4O
PG0 FIRING RATE (PERM]N) ~
Fig. 3. Interrelation between PGO firing rate and the appearance of phasic components of multiple unit activity in the midbrain reticular formation during paradoxical sleep. Each point indicates the observed value of four cats between 2 and 5 h after administration of 0.5%CMC (O) and fludiazepam, 0.5 mg/kg i.p. (o).
20
o 6o
qo
2o
et al. 1962; Faure et al. 1962; Yamamoto and Kido 1962). It is suggested that the midbrain reticular formation may not play a primary role o n paradoxical sleep b u t has a facilitatory influence on the appearance of paradoxical sleep. We have shown in this paper that benzodiazepines decrease multiple unit activity in the midbrain reticular formation. We have found that these compounds also reduced increased multipe unit activity in the preoptic area and m o t o r cortex (unpublished data). We suggest that one of the principal actions of benzodiazepines is a general inhibition of increksed neuronal activity which is triggered from the pons. The increase o f arousal Considering the prominent role which the midbrain reticular formation plays in EEG arousal, it is to some extent surprising that arousal in sleep-wakefulness cycle was increased by benzodiazepines, even though EEG arousal responses by reticular and hypothalamic stimulation and multiple unit activ-
0-i
HR
1-2 HR
2-3 HR
3+4 HR
Fig. 4. Effect of fludiazepam and the solvent PEG on sleep-wakefulness cycle after bilateral injection into the midbrain reticular formation. The ratio of arousal, slow wave sleep and paradoxical sleep is indicated every 1 h. n = 4. * P ~ 0.05.
ity in the midbrain reticular formation at various stages of sleep were decreased by them. The increase of arousal by benzodiazepines could be considered to be induced by some action on the midbrain reticular formation because bilateral injection of benzodiazepines into the reticular formation increased arousal. The reduction of EEG arousal response, elicited by stimulation of the midbrain reticular formation, by benzodiazepines might be induced by inhibition of other areas of neuronal connection which might affect the excitability of the midbrain reticular formation. Several studies of brain lesion, transection and stimulation (Kawamura et al. 1961;
PGO AND UNIT ACTIVITY FOLLOWING BENZODIAZEPIDES Feldman and Waller 1962; Schlag and Chaillet 1963; Kawamura and Domino 1968) have demonstrated that tonic EEG arousal in the cortex was enhanced b y a powerful inflow of impulses from the medial hypothalamus to the midbrain formation. F u k u d a et al. (1974) suggested that 1,4-benzodiazepines suppressed the tonic neocortical EEG arousal because of the reduction of this powerful inflow of impulses from the hypothalamus to the midbrain reticular formation. The suppression of multiple unit activity in the midbrain reticular formation after administration of benzodiazepines would be considered to be due to the same reduction. T h e fact that the suppression of multiple unit activity was observed only during sleep allows us to speculate that the physiological and pharmacological properties of reticular neuronal activity differ at various levels of consciousness. The benzodiazepines seem to facilitate the reticular formation under certain circumstances and depress it under other circumstances. The possible dependence of these actions on the state of consciousness of the animal deserves further study.
Summary
Effects of benzodiapines administered b y the intraperitoneal route on PGO firings and multiple unit activity in the midbrain reticular formation in chronic cat preparations were investigated at various levels of consciousness. Changes in the sleep-wakefulness cycle induced b y direct injection of benzodiazepines into the reticular formation were also investigated. Benzodiazepines markedly decreased multiple unit activity in the midbrain reticular formation during each stage of sleep, b u t had little effect during behavioral and EEG arousal. Benzodiazepines did not affect PGO firing.rate, b u t attenuated all increase of multiple unit activity following PGO firings. The bilateral injection of benzodiazepines into the midbrain reticular formation induced an increase of arousal and a decrease
705
of slow wave sleep, but did not change the a m o u n t of paradoxical sleep. It is concluded that benzodiazepines show a mixture of depressant and facilitatory effects, which seem to vary with the state of consciousness of the animal.
R~sum~ Effets des benzodiazdpines sur les ddcharges PGO et l'activitd multi-unitaire de la formation rdticulaire mdsocdphalique du chat Les auteurs ont ~tudi~ l'action des benzodiazepines administr~es par voie intrap~riton~ale sur les d~charges PGO et l'activit~ multiunitaire de la formation r~ticulaire mdsoc~phalique du chat en preparation chronique, divers niveaux de vigilance, ainsi que les modifications du cycle veille-sommeil induites par injection directe de benzodiaz~pines dans la formation r~ticulaire. Les benzodiaz~pines diminuent de faqon marquee l'activitd multiunitaire de la formation r~ticulaire m~soc~phalique ~ chaque stade de sommeil, mais n ' o n t que peu d'effet au cours de l'~veil EEG et comportemental. Les benzodiazdpines n'affectent pas le taux de d~charges PGO, mais att~nuent toute augmentation de l'activit~ multi-unitaire consecutive aux d~charges PGO. L'injection bilat~rale de benzodiaz~pines dans la formation r~ticulaire m~soc~phalique induit une augmentation de l'~veil et une diminution du sommeil ~ ondes lentes, mais ne modifie pas la quantit~ de sommeil paradoxal. Les auteurs concluent que les benzodiaz~pines pr~sentent un ensemble d'effets ddpresseurs et facflitateurs qui paraissent varier avec l'~tat de vigilance de l'animal.
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