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Diazepam and midazolam increase light slow-wave sleep (SWS1) and decrease wakefulness in rats
Brain Research, 303 (1984) 194-196 Elsevier
194 BRE 20236
Diazepam and midazolam increase light slow-wave sleep (SWSl) and decrease wakefulness in rats M. RADULOVACKI, G. SRECKOVIC, R. ZAK and G. ZAHREBELSKI
Department of Pharmacology, Collegeof Medicine, Universityof Illinois at Chicago, Chicago, IL 60680 (U.S.A.) (Accepted February 21st, 1984)
Key words: diazepam - - midazolam - - slow-wave sleep (SWS1) - - rat
Rats implanted with electrodes for polygraphic recordings were injected with diazepam (5 mg/kg, i.p.) or midazolam (10 mg/kg, i.p.) and recorded for 6 h during the 8 h of darkness of a 16 h light/8 h dark cycle. The results show that administration of diazepam reduced SWS1 latency by 92%, and increased SWS1 and total sleep by 255% and 59%, respectively, in comparison to control. Administration of midazolam increased SWS1 by 158% and total sleep by 57% when compared to control. These findings correlate well with the effects of benzodiazepines on sleep stage 2 in humans and indicate that benzodiazepine hypnotics increase only the behaviorally lighter stage of SWS in rats as well as in human subjects.
Non-rapid eye movement (non-REM) or slowwave sleep (SWS) in rats is divided on light SWS1 and deep SWS2 according to the amount of high-amplitude waves, with SWS2 having 50% or more of this activity 3. In humans, n o n - R E M sleep is divided into 4 stages, where stages 3 and 4 are characterized by high amplitude waves of 1-4 c/s 6. Barbiturates and benzodiazepines increase stage 2 and total sleep in humans but they do not increase behaviorally deeper delta sleep 7. Hypnotic drugs have been shown to increase SWS in ratsl.2,8, 9 but since SWS in those studies was not divided into behaviorally lighter SWS1 and behaviorally deeper SWS2, the precise effect of these hypnotics on sleep stages remained unknown. Our aim here was to determine the effects of two benzodiazepine drugs, diazepam and midazolam, on SWS1 and SWS2 in rats in an insomnia model previously described 1. The results of the study show that both of these benzodiazepines significantly increased only SWS1 and total sleeping time. Adult male S p r a g u e - D a w l e y rats (300 g) were implanted with cortical and dorsal neck muscle electrodes for E E G and E M G recordings. After surgery, each animal was maintained in a separate cage for 10
days in a room under 16 h light and 8 h dark cycle, with the dark period from 10.00 h to 18.00 h. This experimental design was chosen because Halperin et al.~ have shown that a 16 h light/8 h dark lighting schedule can be employed as a model insomnia for rats which are then intensely awake during the 8 h dark period. In this model, animals were awake for 100 rain during the first 2 h of polygraphic recording, whereas rats on 12 h light/12 h dark cycle spend half of that time in wakefulness during the first 3 h of E E G recording4, 5. On the day of experiment, animals were divided into the following groups: control (saline, i.p.), diazepam (5 mg/kg, i.p.) and midazolam (10 mg/kg, i.p.). After receiving saline and drugs animals were continuously recorded for 6 h during the 8 h dark period. The E E G records were scored in 1-min epochs as wakefulness, SWS1 (spindles and less than 50% delta slow waves), SWS2 (50% or more delta slow waves) and R E M sleep 3. SWS1 and SWS2 latencies were defined as the time (min) from the injection of saline or drugs to the appearance of the first 2 min SWS1 or SWS2 episode, respectively. R E M sleep latency was described as the time (min) from the injection of saline or drugs to the appear-
Correspondence: M. Radulovacki, Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcon, St., Chicago, IL 60680, U.S.A. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
195 TABLE I
ance of the first 1-min R E M sleep episode. Total
The effects of diazeparn (5 rng/kg, i.p.) and midazolam (10 mg/kg, i.p.) on wakefulness, SWS1, SWS2, REM sleep and TS in rats
sleep (TS) included SWS1, SWS2 and R E M sleep. D u n n e f s test was performed on SWS1, SWS2 and R E M sleep latencies. The same test was also performed on 2 h time periods within each behavioral
The results are means ± S.D. (min). There were 6 animals in each group. All statistical comparisons between means were made using the Dunnett test.
Behavioral state W
SWS1
SWS2
REM
TS
Hours
Control
Diazepam
Midazolam
0-2 2-4 4-6 0-6
88 + 71 + 90 ± 250 ±
24 16 16 35
64 ± 60 ± 66 ± 185 ±
22 20 26 59*
58 ± 58 ± 71 ± 187 +
18" 15 20 29**
0-2 2-4 4-6 0-6
11 ± 12 + 11 ± 34 ±
5 8 5 15
54 ± 36 ± 31 + 121 +
19"** 19" 27 59**
40 ± 27 ± 21 ± 88 ±
21"* 13" 10" 34**
0-2 2-4 4-6 0-6
20 ± 34 ± 17 ± 71 +
19 11 12 24
7+ 20 ± 20 + 48 +
10 12 13 24
22 ± 32 ± 23 ± 76 ±
22 17 15 34
0-2 2-4 4-6 0-6
2±3 3±2 2±2 6±6
0±0 3±5 4±3 7±7
0-6
110 + 35
175 + 58*
0±0 4±3 5±3 9±3 173 + 29**
State. Results in Table I show that administration of diazepam and midazolam reduced wakefulness in comparison to control by 26% (P < 0.05) and 25.2% (P < 0.01), respectively, during the 0 - 6 h time period. Midazolam also decreased wakefulness during the 0 - 2 h time period by 34% (P < 0.05). Diazepam increased SWS1 during 0 - 2 , 2 - 4 and 0 - 6 h time periods. The increase during the 0 - 6 h time period was 255.8% (P < 0.01) when compared to control. Midazolam increased SWS1 during all time periods. The increase during the 0 - 6 h time period was 158.8% (P < 0.01) in comparison to control. Both drugs increased TS during the 0 - 6 h time period by 59% (P < 0.05) and 57.2% (P < 0.01), respectively. Neither diazepam nor midazolam had any effect on SWS2 or R E M sleep. Table II shows that only diazepam reduced SWS1 latency by 92.5% in comparison to control (P < 0.01). There were no effects on SWS2 or R E M sleep laten-
*P < 0.05, **P < 0.01, ***P < 0.001 significantly different from control.
cy by diazepam or midazolam.
TABLE II
rats, as well as h u m a n s , benzodiazepine hypnotics increase only the behaviorally lighter stage of SWS. As in humans, diazepam's hypnotic action was rapid and
The effects of diazepam (5 rng/kg, i.p.) and midazolam (10 mg/kg, i.p. ) in sleep latencies in rats The results are means + S.D. (min). There were 6 animals in each group. All statistical comparisons between means were made using the Dunnet test.
Sleep latency
Control
Diazepam
Midazolam
SWS1 SWS2 REM sleep
66 + 45 62 + 49 146 + 70
5 + 4* 72 + 65 213 + 102
27 + 22 59 + 28 184 + 72
*P < 0.01, significantly different from control.
1 Halperin, J. M., Miller, D. and Iorio, L. C., Sleep-inducing effects of three hypnotics in a new model of insomnia in rats, Pharmacol. Biochem. Behav., 14 (1981) 811-814. 2 Klygu, T. A., Kadlecova, O. and Vikhliaev, I., The effect of long-term nitrazepam administration on sleep cycles in rats, Bull. exp. Biol. Med., 81 (1976) 188-190.
Results obtained correlate well with k n o w n benzodiazepine or barbiturate-induced increases in nonR E M sleep stage 2 in h u m a n s 7. They indicate that in
was related only to SWS1 which was evidenced by the greatly reduced latency to SWS1. Being less lipophilic than diazepam, midazolam failed to reduce SWS1 latency in the dose used. It appears therefore that pharmacologically induced increase in total sleeping time in rats results from an increase in SWS1. Supported by O N R Contract N000 14-79-C-0420.
3 Larsen, M. and Ursin, R., Sleep is enhanced 5 h following intracerebroventricular injection of delta sleep inducing peptide in rats, Sleep Res., 10 (1981) 67. 4 Radulovacki, M., Virus, R. M., Djuricic-Nedelson, M. and Green, R. D., Hypnotic effects of deoxycoformycin in rats, Brain Research, 271 (1983) 392-395.
196 5 Radulovacki, M., Virus, R. M., Djuricic-Nedelson, M. and Green, R. D., Adenosine analogs and sleep in rats, J. Pharmacol, exp. Ther., 228 (1984) 268-274. 6 Rechtschaffen, A. and Kales, A. D., A Manual of Standard-
ized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects, Brain Information Service/Brain Research Institute, Los Angeles, 1968. 7 Anonymous, Sleeping pills, Insomnia and Medical Practice,
Institute of Medicine, National Academy of Sciences, Washington, DC, 1979. 8 Staudacherova, D., Mares, P. and Trojan, S., Ontogenetic development of pentobarbital-induced EEG pattern and sleeping time in rats, Develop. Psychobiol., 12 (1979) 11-25. 9 Voronina, T. A. and Nerobkova, L. N., Effect of phenazepare on sleep cycles in rats, Farmakol. Toksikol., 44 (1981) 18-20.
194 BRE 20236
Diazepam and midazolam increase light slow-wave sleep (SWSl) and decrease wakefulness in rats M. RADULOVACKI, G. SRECKOVIC, R. ZAK and G. ZAHREBELSKI
Department of Pharmacology, Collegeof Medicine, Universityof Illinois at Chicago, Chicago, IL 60680 (U.S.A.) (Accepted February 21st, 1984)
Key words: diazepam - - midazolam - - slow-wave sleep (SWS1) - - rat
Rats implanted with electrodes for polygraphic recordings were injected with diazepam (5 mg/kg, i.p.) or midazolam (10 mg/kg, i.p.) and recorded for 6 h during the 8 h of darkness of a 16 h light/8 h dark cycle. The results show that administration of diazepam reduced SWS1 latency by 92%, and increased SWS1 and total sleep by 255% and 59%, respectively, in comparison to control. Administration of midazolam increased SWS1 by 158% and total sleep by 57% when compared to control. These findings correlate well with the effects of benzodiazepines on sleep stage 2 in humans and indicate that benzodiazepine hypnotics increase only the behaviorally lighter stage of SWS in rats as well as in human subjects.
Non-rapid eye movement (non-REM) or slowwave sleep (SWS) in rats is divided on light SWS1 and deep SWS2 according to the amount of high-amplitude waves, with SWS2 having 50% or more of this activity 3. In humans, n o n - R E M sleep is divided into 4 stages, where stages 3 and 4 are characterized by high amplitude waves of 1-4 c/s 6. Barbiturates and benzodiazepines increase stage 2 and total sleep in humans but they do not increase behaviorally deeper delta sleep 7. Hypnotic drugs have been shown to increase SWS in ratsl.2,8, 9 but since SWS in those studies was not divided into behaviorally lighter SWS1 and behaviorally deeper SWS2, the precise effect of these hypnotics on sleep stages remained unknown. Our aim here was to determine the effects of two benzodiazepine drugs, diazepam and midazolam, on SWS1 and SWS2 in rats in an insomnia model previously described 1. The results of the study show that both of these benzodiazepines significantly increased only SWS1 and total sleeping time. Adult male S p r a g u e - D a w l e y rats (300 g) were implanted with cortical and dorsal neck muscle electrodes for E E G and E M G recordings. After surgery, each animal was maintained in a separate cage for 10
days in a room under 16 h light and 8 h dark cycle, with the dark period from 10.00 h to 18.00 h. This experimental design was chosen because Halperin et al.~ have shown that a 16 h light/8 h dark lighting schedule can be employed as a model insomnia for rats which are then intensely awake during the 8 h dark period. In this model, animals were awake for 100 rain during the first 2 h of polygraphic recording, whereas rats on 12 h light/12 h dark cycle spend half of that time in wakefulness during the first 3 h of E E G recording4, 5. On the day of experiment, animals were divided into the following groups: control (saline, i.p.), diazepam (5 mg/kg, i.p.) and midazolam (10 mg/kg, i.p.). After receiving saline and drugs animals were continuously recorded for 6 h during the 8 h dark period. The E E G records were scored in 1-min epochs as wakefulness, SWS1 (spindles and less than 50% delta slow waves), SWS2 (50% or more delta slow waves) and R E M sleep 3. SWS1 and SWS2 latencies were defined as the time (min) from the injection of saline or drugs to the appearance of the first 2 min SWS1 or SWS2 episode, respectively. R E M sleep latency was described as the time (min) from the injection of saline or drugs to the appear-
Correspondence: M. Radulovacki, Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcon, St., Chicago, IL 60680, U.S.A. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
195 TABLE I
ance of the first 1-min R E M sleep episode. Total
The effects of diazeparn (5 rng/kg, i.p.) and midazolam (10 mg/kg, i.p.) on wakefulness, SWS1, SWS2, REM sleep and TS in rats
sleep (TS) included SWS1, SWS2 and R E M sleep. D u n n e f s test was performed on SWS1, SWS2 and R E M sleep latencies. The same test was also performed on 2 h time periods within each behavioral
The results are means ± S.D. (min). There were 6 animals in each group. All statistical comparisons between means were made using the Dunnett test.
Behavioral state W
SWS1
SWS2
REM
TS
Hours
Control
Diazepam
Midazolam
0-2 2-4 4-6 0-6
88 + 71 + 90 ± 250 ±
24 16 16 35
64 ± 60 ± 66 ± 185 ±
22 20 26 59*
58 ± 58 ± 71 ± 187 +
18" 15 20 29**
0-2 2-4 4-6 0-6
11 ± 12 + 11 ± 34 ±
5 8 5 15
54 ± 36 ± 31 + 121 +
19"** 19" 27 59**
40 ± 27 ± 21 ± 88 ±
21"* 13" 10" 34**
0-2 2-4 4-6 0-6
20 ± 34 ± 17 ± 71 +
19 11 12 24
7+ 20 ± 20 + 48 +
10 12 13 24
22 ± 32 ± 23 ± 76 ±
22 17 15 34
0-2 2-4 4-6 0-6
2±3 3±2 2±2 6±6
0±0 3±5 4±3 7±7
0-6
110 + 35
175 + 58*
0±0 4±3 5±3 9±3 173 + 29**
State. Results in Table I show that administration of diazepam and midazolam reduced wakefulness in comparison to control by 26% (P < 0.05) and 25.2% (P < 0.01), respectively, during the 0 - 6 h time period. Midazolam also decreased wakefulness during the 0 - 2 h time period by 34% (P < 0.05). Diazepam increased SWS1 during 0 - 2 , 2 - 4 and 0 - 6 h time periods. The increase during the 0 - 6 h time period was 255.8% (P < 0.01) when compared to control. Midazolam increased SWS1 during all time periods. The increase during the 0 - 6 h time period was 158.8% (P < 0.01) in comparison to control. Both drugs increased TS during the 0 - 6 h time period by 59% (P < 0.05) and 57.2% (P < 0.01), respectively. Neither diazepam nor midazolam had any effect on SWS2 or R E M sleep. Table II shows that only diazepam reduced SWS1 latency by 92.5% in comparison to control (P < 0.01). There were no effects on SWS2 or R E M sleep laten-
*P < 0.05, **P < 0.01, ***P < 0.001 significantly different from control.
cy by diazepam or midazolam.
TABLE II
rats, as well as h u m a n s , benzodiazepine hypnotics increase only the behaviorally lighter stage of SWS. As in humans, diazepam's hypnotic action was rapid and
The effects of diazepam (5 rng/kg, i.p.) and midazolam (10 mg/kg, i.p. ) in sleep latencies in rats The results are means + S.D. (min). There were 6 animals in each group. All statistical comparisons between means were made using the Dunnet test.
Sleep latency
Control
Diazepam
Midazolam
SWS1 SWS2 REM sleep
66 + 45 62 + 49 146 + 70
5 + 4* 72 + 65 213 + 102
27 + 22 59 + 28 184 + 72
*P < 0.01, significantly different from control.
1 Halperin, J. M., Miller, D. and Iorio, L. C., Sleep-inducing effects of three hypnotics in a new model of insomnia in rats, Pharmacol. Biochem. Behav., 14 (1981) 811-814. 2 Klygu, T. A., Kadlecova, O. and Vikhliaev, I., The effect of long-term nitrazepam administration on sleep cycles in rats, Bull. exp. Biol. Med., 81 (1976) 188-190.
Results obtained correlate well with k n o w n benzodiazepine or barbiturate-induced increases in nonR E M sleep stage 2 in h u m a n s 7. They indicate that in
was related only to SWS1 which was evidenced by the greatly reduced latency to SWS1. Being less lipophilic than diazepam, midazolam failed to reduce SWS1 latency in the dose used. It appears therefore that pharmacologically induced increase in total sleeping time in rats results from an increase in SWS1. Supported by O N R Contract N000 14-79-C-0420.
3 Larsen, M. and Ursin, R., Sleep is enhanced 5 h following intracerebroventricular injection of delta sleep inducing peptide in rats, Sleep Res., 10 (1981) 67. 4 Radulovacki, M., Virus, R. M., Djuricic-Nedelson, M. and Green, R. D., Hypnotic effects of deoxycoformycin in rats, Brain Research, 271 (1983) 392-395.
196 5 Radulovacki, M., Virus, R. M., Djuricic-Nedelson, M. and Green, R. D., Adenosine analogs and sleep in rats, J. Pharmacol, exp. Ther., 228 (1984) 268-274. 6 Rechtschaffen, A. and Kales, A. D., A Manual of Standard-
ized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects, Brain Information Service/Brain Research Institute, Los Angeles, 1968. 7 Anonymous, Sleeping pills, Insomnia and Medical Practice,
Institute of Medicine, National Academy of Sciences, Washington, DC, 1979. 8 Staudacherova, D., Mares, P. and Trojan, S., Ontogenetic development of pentobarbital-induced EEG pattern and sleeping time in rats, Develop. Psychobiol., 12 (1979) 11-25. 9 Voronina, T. A. and Nerobkova, L. N., Effect of phenazepare on sleep cycles in rats, Farmakol. Toksikol., 44 (1981) 18-20.