Sleep, 1:83-90, 1978
© Raven Press, New York
Induction and Resetting of REM Sleep Rhythm in Normal Man by Arecholine: Blockade by Scopolamine *N. Sitaram, *Angela M. Moore, and *tJ. Christian Gillin *Unit on Sleep Studies, Biological Psychiatry Branch, DCBR, IRP, National Institute of Mental Health, Bethesda, Maryland, and tLaboratory of Clinical Psychopharmacology, SMHR, IRP, National Institute of Mental Health, St. Elizabeths Hospital, WashinRton, D,C.
INTRODUCTION Cholinergic mechanisms may modulate rapid eye movement (REM) sleep in laboratory animals (Jouvet, 1975; Steriade and Hobson, 1976) and man (Sitaram et aI., 1976, 1977). We recently reported that infusion of physostigmine, an anticholinesterase agent, during non-REM sleep induced the onset of REM sleep without changing the duration of individual REM periods. Furthermore, the early induction of the first REM period advanced the appearance of successive REM -non-REM cycles and served to "reset" the normal ultraradian rhythm controlling REM sleep (Gillin et aI., in press). The data from the human studies with physostigmine support the hypothesis that cholinergic mechanisms modulate REM onset but are not conclusive. Physostigmine may induce REM sleep by nonspecific effects, since it affects brain serotonin and norepinephrine (Glisson et aI., 1974), and since it is not a direct cholinergic receptor agonist. Furthermore, because it presumably enhances both "muscarinic" and "nicotinic" actions of acetylcholine (ACh), physostigmine cannot be used to differentiate (if possible) the two sites of actions of ACh. In the present study, we sought to further clarify the role of ACh in the modulation of REM sleep by administering arecholine. In low doses, arecholine is reported to act as a specific muscarinic agonist (Baker et aI., 1971). For example, the electroencephalographic (EEG) arousal produced by arecholine is blocked selectively by an antimuscarinic agent (atropine) and not by antinicotinic agents (Riehl et aI., 1962; Pfeiffer et aI., 1967). Arecholine also increases brain ACh and cyclic guanosine 3' ,5' -monophosphate (cyclic GMP) in mice (Dinnendahl and Stock, 1975) and competitively displaces radioactive quinuclidinyl benzilate eHQNB) from binding to muscarinic receptors in rat brain with a potency intermediate to Address reprint requests to Dr. Sitaram, Building 10, Room 3N224, National Institutes of Health, Bethesda, Maryland 20014. Key words: Arecholine-Scopolamine-REM sleep-Ultra dian rhythms.
83
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N. SITARAM ET AL.
that of oxotremorine and ACh itself (Yamamura and Snyder, 1974). We studied the effects of intravenous infusions of arecholine on doses of 1.0 and 1.5 mg after subjects were pretreated with methscopolamine, a peripheral anticholinergic. In order to verify whether the effects (if any) of arecholine resulted from central "muscarinic" action, we attempted to block the effects with scopolamine, a well-known antimuscarinic agent (Safer and Allen, 1971). METHODS Ten paid normal volunteers (8 males, 2 females; mean age, 25.3 years, ranging from 21 to 31) participated in a controlled, double-blind experimental design. The administration of all drugs used in this study (arecholine, methscopolamine, and scopolamine) to normal volunteers was approved by the NIMH Clinical Research Review Committee and by the Director, The Clinical Center, National Institutes of Health. The Food and Drug Administration approved the study as an Investigational New Drug. Informed consent was obtained from each subject. After a night of adaptation, subjects were studied under four randomly ordered experimental conditions on four separate nonconsecutive nights. On each experimental night, subjects received an intramuscular injection of either methscopolamine or scopolamine prior to retiring (about 11:30 p.m.) followed by one intravenous infusion of either placebo or arecholine administered about 32 to 35 min after sleep onset. The drugs administered during the four experimental conditions were: (1) pretreatment with methscopolamine (0.5 mg i.m.) followed by placebo (isotonic saline i. v.); (2) pretreatment with methscopolamine (0.5 mg i.m.) followed by arecholine (1.0 mg i.v.); (3) pretreatment with methscopolamine (0.5 mg i.m.) followed by are choline (1.5 mg Lv.); (4) pretreatment with scopolamine (0.5 mg i.m.) followed by arecholine (1.5 mg i.v.). All intravenous infusions during the four conditions were given over a 3-min period and timed to end at about 35 min after sleep onset (identified by the first appearance of Stage 2). Subjects slept with an intravenous needle inserted into a forearm vein and attached to a 10-foot (3.3-meter) sterile tubing extending out of the room. A slow drip of isotonic saline was maintained to avoid coagulation. Pretreatment with methscopolamine, a peripheral anticholinergic that has previously been shown to have no effects on human sleep (Sagales et aI., 1967; confirmed by Mendelson et aI., in preparation), was used to block peripheral cholinergic effects of arecholine. Scopolamine, in contrast, is both a peripheral and central anticholinergic agent acting on muscarinic receptor sites (Safer and Allen, 1971). Continuous all-night recordings of the EEG, electrooculogram (EOG), and electromyogram (EMG) were obtained, and all records were scored according to standard criteria (Rechtschaffen and Kales, 1968) by a single investigator who did not know the drug status of subjects. Eight out of the 10 subjects participated in all four experimental conditions and their data were analyzed using a one-way analysis of variance (ANOV A) with repeated measures. Newman-Keul's comparisons were made only after a significant ANOV A (p < 0.05). The two additional subjects who had participated in only the placebo and arecholine 1.5 mg conditions could not be. included in the ANOV A. A separate analysis (paired t-tests) of the placebo and arecholine 1.5 mg
Sleep, Vol. I. No. I, September 1978
ARECHOLINE AND REM SLEEP
85
conditions alone with all 10 subjects served only to increase the level of statistical significance on all the measures, which were already significant on the ANOV A followed by Newman-Keul's test with N = 8 subjects. In the interest ofa consistent statistical design, only data from the 8 subjects who underwent all the conditions will be presented hereafter. RESULTS As shown in Table 1 and Fig. 2, our data indicated that infusions of are choline (both 1.0 mg and 1.5 mg) significantly reduced REM latency and the time from infusion to onset of first REM period as compared to placebo. REM sleep was triggered, within an average of 15.9 min and 20.1 min after infusions of 1.0 mg and 1.5 mg of are choline , respectively. This is shown in Fig. I, in which a subject was in Stage 4 at the time of the are choline infusion. By the end of the infusion, one of the components of REM sleep (EMG suppression) was already evident, and within 4 min he was in clear REM sleep (low-voltage, mixed-frequency EEG, EMG suppression). Unequivocal rapid eye movements began 2 min later. The number of REM periods increased significantly with both are choline 1.0 mg and 1.5 mg compared to placebo (Table I). The duration of individual REM periods were, however, not different between placebo and arecholine (Fig. 2). The total REM time and REM percent increased with both are choline 1.5 mg and 1.0 mg but the increase reached statistical significance only with the 1.5-mg dose. Other sleep parameters, such as total recording time, total sleep time, total nonREM time, and time spent in Stages 1 and 2 and delta (Stages 3 and 4) sleep, were unchanged (Table I). The earlier onset of the first REM period after arecholine infusion served to "reset" the onset of subsequent REM periods. As shown in Fig. 2 and Table 1, once the onset of the "ultradian clock" had been advanced by arecholine, there were no subsequent effects on its intrinsic rhythmicity; neither duration of the REM - REM cycles nor the second, third, and fourth non-REM periods were changed. There was no correlation between the stage of sleep during which arecholine infusions were given and the time taken from infusion to REM onset. Pretreatment with scopolamine 0.5 mg reversed all of the above effects of arecholine 1.5 mg. After six of the eight infusions of arecholine, we noted a decrease of EMG amplitude (compared with preinfusion amplitude) occurring within 15 min of the infusion. The preinfusion EMG amplitudes in the other two infusions were too low for any further decrease to become apparent after the infusion. In addition, although scopolamine plus arecholine significantly increased REM latency, it did not alter the duration of the first and second REM periods (Fig. 2). The number of REM periods, total REM time, and REM percent decreased significantly (Table I). It is of interest that Sagales et al. (1975) also showed that scopolamine (in a similar dose) increased the REM latency from 126 ± 8.4 min (placebo) to 301.2 ± 9 min (scopolamine) without altering the duration of the first REM period after placebo (15 ± 5.5 min) as compared with scopolamine (10.3 ± 4.1 min). Our data with scopolamine plus arecholine indicated that the decrease of REM sleep was accompanied by a significant enhancement of delta sleep (Stages 3 and
Sleep, Vol. I, No. I, September 1978
TABLE 1. The effect of arecholine on various sleep parameters
'"
;;-
Treatment condition (Mean
~
~
.~ .-
'" ":I"
" -
"'
© Raven Press, New York
Induction and Resetting of REM Sleep Rhythm in Normal Man by Arecholine: Blockade by Scopolamine *N. Sitaram, *Angela M. Moore, and *tJ. Christian Gillin *Unit on Sleep Studies, Biological Psychiatry Branch, DCBR, IRP, National Institute of Mental Health, Bethesda, Maryland, and tLaboratory of Clinical Psychopharmacology, SMHR, IRP, National Institute of Mental Health, St. Elizabeths Hospital, WashinRton, D,C.
INTRODUCTION Cholinergic mechanisms may modulate rapid eye movement (REM) sleep in laboratory animals (Jouvet, 1975; Steriade and Hobson, 1976) and man (Sitaram et aI., 1976, 1977). We recently reported that infusion of physostigmine, an anticholinesterase agent, during non-REM sleep induced the onset of REM sleep without changing the duration of individual REM periods. Furthermore, the early induction of the first REM period advanced the appearance of successive REM -non-REM cycles and served to "reset" the normal ultraradian rhythm controlling REM sleep (Gillin et aI., in press). The data from the human studies with physostigmine support the hypothesis that cholinergic mechanisms modulate REM onset but are not conclusive. Physostigmine may induce REM sleep by nonspecific effects, since it affects brain serotonin and norepinephrine (Glisson et aI., 1974), and since it is not a direct cholinergic receptor agonist. Furthermore, because it presumably enhances both "muscarinic" and "nicotinic" actions of acetylcholine (ACh), physostigmine cannot be used to differentiate (if possible) the two sites of actions of ACh. In the present study, we sought to further clarify the role of ACh in the modulation of REM sleep by administering arecholine. In low doses, arecholine is reported to act as a specific muscarinic agonist (Baker et aI., 1971). For example, the electroencephalographic (EEG) arousal produced by arecholine is blocked selectively by an antimuscarinic agent (atropine) and not by antinicotinic agents (Riehl et aI., 1962; Pfeiffer et aI., 1967). Arecholine also increases brain ACh and cyclic guanosine 3' ,5' -monophosphate (cyclic GMP) in mice (Dinnendahl and Stock, 1975) and competitively displaces radioactive quinuclidinyl benzilate eHQNB) from binding to muscarinic receptors in rat brain with a potency intermediate to Address reprint requests to Dr. Sitaram, Building 10, Room 3N224, National Institutes of Health, Bethesda, Maryland 20014. Key words: Arecholine-Scopolamine-REM sleep-Ultra dian rhythms.
83
84
N. SITARAM ET AL.
that of oxotremorine and ACh itself (Yamamura and Snyder, 1974). We studied the effects of intravenous infusions of arecholine on doses of 1.0 and 1.5 mg after subjects were pretreated with methscopolamine, a peripheral anticholinergic. In order to verify whether the effects (if any) of arecholine resulted from central "muscarinic" action, we attempted to block the effects with scopolamine, a well-known antimuscarinic agent (Safer and Allen, 1971). METHODS Ten paid normal volunteers (8 males, 2 females; mean age, 25.3 years, ranging from 21 to 31) participated in a controlled, double-blind experimental design. The administration of all drugs used in this study (arecholine, methscopolamine, and scopolamine) to normal volunteers was approved by the NIMH Clinical Research Review Committee and by the Director, The Clinical Center, National Institutes of Health. The Food and Drug Administration approved the study as an Investigational New Drug. Informed consent was obtained from each subject. After a night of adaptation, subjects were studied under four randomly ordered experimental conditions on four separate nonconsecutive nights. On each experimental night, subjects received an intramuscular injection of either methscopolamine or scopolamine prior to retiring (about 11:30 p.m.) followed by one intravenous infusion of either placebo or arecholine administered about 32 to 35 min after sleep onset. The drugs administered during the four experimental conditions were: (1) pretreatment with methscopolamine (0.5 mg i.m.) followed by placebo (isotonic saline i. v.); (2) pretreatment with methscopolamine (0.5 mg i.m.) followed by arecholine (1.0 mg i.v.); (3) pretreatment with methscopolamine (0.5 mg i.m.) followed by are choline (1.5 mg Lv.); (4) pretreatment with scopolamine (0.5 mg i.m.) followed by arecholine (1.5 mg i.v.). All intravenous infusions during the four conditions were given over a 3-min period and timed to end at about 35 min after sleep onset (identified by the first appearance of Stage 2). Subjects slept with an intravenous needle inserted into a forearm vein and attached to a 10-foot (3.3-meter) sterile tubing extending out of the room. A slow drip of isotonic saline was maintained to avoid coagulation. Pretreatment with methscopolamine, a peripheral anticholinergic that has previously been shown to have no effects on human sleep (Sagales et aI., 1967; confirmed by Mendelson et aI., in preparation), was used to block peripheral cholinergic effects of arecholine. Scopolamine, in contrast, is both a peripheral and central anticholinergic agent acting on muscarinic receptor sites (Safer and Allen, 1971). Continuous all-night recordings of the EEG, electrooculogram (EOG), and electromyogram (EMG) were obtained, and all records were scored according to standard criteria (Rechtschaffen and Kales, 1968) by a single investigator who did not know the drug status of subjects. Eight out of the 10 subjects participated in all four experimental conditions and their data were analyzed using a one-way analysis of variance (ANOV A) with repeated measures. Newman-Keul's comparisons were made only after a significant ANOV A (p < 0.05). The two additional subjects who had participated in only the placebo and arecholine 1.5 mg conditions could not be. included in the ANOV A. A separate analysis (paired t-tests) of the placebo and arecholine 1.5 mg
Sleep, Vol. I. No. I, September 1978
ARECHOLINE AND REM SLEEP
85
conditions alone with all 10 subjects served only to increase the level of statistical significance on all the measures, which were already significant on the ANOV A followed by Newman-Keul's test with N = 8 subjects. In the interest ofa consistent statistical design, only data from the 8 subjects who underwent all the conditions will be presented hereafter. RESULTS As shown in Table 1 and Fig. 2, our data indicated that infusions of are choline (both 1.0 mg and 1.5 mg) significantly reduced REM latency and the time from infusion to onset of first REM period as compared to placebo. REM sleep was triggered, within an average of 15.9 min and 20.1 min after infusions of 1.0 mg and 1.5 mg of are choline , respectively. This is shown in Fig. I, in which a subject was in Stage 4 at the time of the are choline infusion. By the end of the infusion, one of the components of REM sleep (EMG suppression) was already evident, and within 4 min he was in clear REM sleep (low-voltage, mixed-frequency EEG, EMG suppression). Unequivocal rapid eye movements began 2 min later. The number of REM periods increased significantly with both are choline 1.0 mg and 1.5 mg compared to placebo (Table I). The duration of individual REM periods were, however, not different between placebo and arecholine (Fig. 2). The total REM time and REM percent increased with both are choline 1.5 mg and 1.0 mg but the increase reached statistical significance only with the 1.5-mg dose. Other sleep parameters, such as total recording time, total sleep time, total nonREM time, and time spent in Stages 1 and 2 and delta (Stages 3 and 4) sleep, were unchanged (Table I). The earlier onset of the first REM period after arecholine infusion served to "reset" the onset of subsequent REM periods. As shown in Fig. 2 and Table 1, once the onset of the "ultradian clock" had been advanced by arecholine, there were no subsequent effects on its intrinsic rhythmicity; neither duration of the REM - REM cycles nor the second, third, and fourth non-REM periods were changed. There was no correlation between the stage of sleep during which arecholine infusions were given and the time taken from infusion to REM onset. Pretreatment with scopolamine 0.5 mg reversed all of the above effects of arecholine 1.5 mg. After six of the eight infusions of arecholine, we noted a decrease of EMG amplitude (compared with preinfusion amplitude) occurring within 15 min of the infusion. The preinfusion EMG amplitudes in the other two infusions were too low for any further decrease to become apparent after the infusion. In addition, although scopolamine plus arecholine significantly increased REM latency, it did not alter the duration of the first and second REM periods (Fig. 2). The number of REM periods, total REM time, and REM percent decreased significantly (Table I). It is of interest that Sagales et al. (1975) also showed that scopolamine (in a similar dose) increased the REM latency from 126 ± 8.4 min (placebo) to 301.2 ± 9 min (scopolamine) without altering the duration of the first REM period after placebo (15 ± 5.5 min) as compared with scopolamine (10.3 ± 4.1 min). Our data with scopolamine plus arecholine indicated that the decrease of REM sleep was accompanied by a significant enhancement of delta sleep (Stages 3 and
Sleep, Vol. I, No. I, September 1978
TABLE 1. The effect of arecholine on various sleep parameters
'"
;;-
Treatment condition (Mean
~
~
.~ .-
'" ":I"
" -
"'
