Psychiatry Elsevier
113
Research, 33: 1 I3- 119
Pilocarpine, an Orally Active Muscarinic Cholinergic Agonist, Induces REM Sleep and Reduces Delta Sleep in Normal Volunteers Alan
Berkowitz,
Laura Sutton,
David
S. Janowsky,
and J. Christian
Gillin
Received October 23, 1989; revised version received March 2, 1990; accepted March 18, 1990. Abstract. The effect of oral pilocarpine,
a direct-acting muscarinic, cholinergic agonist, on polygraphic sleep parameters was studied in 13 healthy male volunteers. Subjects received placebo and oral pilocarpine (25 mg) in a doubleblind, counterbalanced, crossover design. Pilocarpine shortened the latency of rapid eye movement (REM) sleep and increased total REM time, REM%, and the duration of the first REM period. In addition, it reduced Stage 4 sleep and Delta sleep. Pulse rate was not significantly changed during the first hour of darkness after administration of pilocarpine. Subjective sleep experience and the subjects’ condition in the morning were not altered. These results suggest that pilocarpine has central effects (i.e., induction of REM sleep) that are similar to those of other centrally acting muscarinic cholinomimetic agents. Key Words. Rapid eye movement sleep, sleep stages, pilocarpine, receptors, human parasympathomimetic.
muscarinic
The exact neurophysiological and biochemical mechanisms that regulate sleep are poorly understood (McGinty and Drucker-Colin, 1982; Vertes, 1984; Hobson and Steriade, 1986). Nevertheless, results of in vivo animal studies indicate a possible role for the cholinergic nervous system in the induction and maintenance of rapid eye movement (REM) sleep (George et al., 1964; Domino et al., 1968; Baxter, 1969; Jouvet, 1975; Hobson et al., 1976; Vertes, 1984; Hobson and Steriade, 1986; Baghdoyan et al., 1989). In normal control subjects, Sitaram et al. (1976, 1978~) demonstrated that physostigmine, a cholinesterase inhibitor, and arecoline, a direct muscarinic agonist, hasten the onset of the following REM sleep period when infused during the preceding non-REM period. REM periods induced by physostigmine were of the same duration as and did not differ from those normally occurring with regard to subjective dream experience (Sitaram et al., 1977; Gillin et al., 1978). Furthermore, the effects of arecoline on REM sleep could be blocked by pretreatment with scopolamine, a peripherally and centrally acting direct muscarinic
Alan Berkowitz, M.D., was a UCSD Fellow in Clinical Psychopharmacology and Psychobiology when this research was conducted. Laura Sutton, R.N., is a Research Nurse. David S. Janowsky, M.D., is currently Chairman and Professor of Psychiatry, The University of North Carolina. J. Christian Gillin, M.D., is Professor of Psychiatry, UCSD, and Staff Psychiatrist, San Diego Veterans Administration Medical Center. (Reprint requests to Dr. J.C. Gillin, Dept. of Psychiatry (M-003), University of California, San Diego, La Jolla, CA 92093, USA.) 0165-1781/90/$03.50
@ 1990 Elsevier Scientific
Publishers
Ireland
Ltd.
114 cholinergic receptor antagonist (Sitaram et al., 19786, 1978~). In addition, RS-86, an experimental, orally active muscarinic agonist, induces REM sleep in man (Spiegel, 1984). Sagales et al. (1969) found that scopolamine when given alone at bedtime suppresses REM sleep. Antidepressants with anticholinergic activity inhibit REM sleep (Oswald, 1980). These observations suggest that blockade of central cholinergic neurotransmission inhibits REM sleep while cholinergic muscarinic facilitation promotes it. Physostigmine, arecoline, and RS-86 have had two major uses in human sleep studies: (1) These drugs have demonstrated cholinergic facilitation of REM sleep. (2) All three drugs have been used in the Cholinergic REM Induction Test (CRIT) in clinical studies (Sitaram et al., 1980, 1981, 1982; Berger et al., 1983, 1985, 1989; Riemann and Berger, 1989). In this test, the speed with which a cholinergic agonist induces REM sleep is measured, i.e., following administration of either i.v. arecoline during the second non-REM period or oral RS-86 before bedtime. Results with both drugs suggest that cholinomimetic muscarinic agonists induce REM sleep more quickly in depressed patients than in normal controls. Furthermore, Berger et al. (1983) reported that i.v. administration of physostigmine shortly after sleep onset awakened depressed patients significantly more often than normal controls, suggesting again that depressed patients are supersensitive to cholinergic agonist challenge. Unfortunately, physostigmine and arecoline have numerous experimental shortcomings that limit their usefulness; since both drugs have a very short duration of action, it is necessary to administer them by the i.v. route during non-REM sleep. Furthermore, although RS-86 can be administered orally, it is no longer available from the manufacturer. Therefore, we studied the effect of pilocarpine, a centrally acting muscarinic cholinergic agonist, and predicted that it would shorten REM latency and, possibly, increase total REM sleep because of its long duration of action. This is the first study of pilocarpine on human sleep. Pilocarpine, an old ophthalmologic preparation, is well absorbed at mucous membranes, such as the eye or gastrointestinal tract, and readily passes the bloodbrain barrier (Boyd and Fulford, 1961). Domino et al. (1968) reported both increased wakefulness and “fast wave sleep” (electroencephalographic desynchronization) in cats given systemic pilocarpine. These effects were abolished by pretreatment with systemic atropine while being only slightly decreased with methylatropine, which does not cross the blood-brain barrier. Several investigators have reported that systemic pilocarpine caused analgesia in mice, rats and rabbits via a central cholinergic mechanism since this effect was abolished by atropine but not by methyl-atropine. Suzuki et al. (1973) found that systemic pilocarpine significantly increased adrenal cortical secretions in rats, presumably through a central muscarinic effect. Pilocarpine has also been used to counteract anticholinergic effects such as dry mouth produced by tricyclic antidepressants. It was found in these studies and in others that it has a duration of action of 6-12 hours in man (Prutting, 1965).
Methods Subjects. The results of this study are based upon data from 13 male subjects, ages 25-46 (mean = 34.8 years), who were without physical or psychiatric diagnoses after a complete
115 medical and psychiatric evaluation, including an and a formal psychiatric diagnostic interview, Schizophrenia (SADS; Spitzer et al., 1978). No days before the study. The subjects were paid for
electrocardiogram (EKG), laboratory tests, the Schedule for Affective Disorders and subject had taken any drugs for at least 30 participation.
Experimental Design and Drug Dosage. Subjects slept on the UCSD Mental Health Research Center in the San Diego Veterans Administration Medical Center. They had allnight polygraphic recordings of electroencephalogram (EEG), electro-oculogram (EOG), electromyogram, and EKG. During the first (adaptation) night, all subjects were screened for nocturnal myoclonus (with tibia1 EMG recordings) and finger oximetry to exclude those with sleep disorders, including sleep apnea or periodic leg movements during sleep. Subjects also had one or two baseline nights before entering the experimental phase of the study. No alcoholic beverages or drugs were permitted on the experimental day. Oral food intake was discouraged after 1600h on experimental days: an average of 6.5 hours elapsed between the last food and the administration of pilocarpine. Routine activities were otherwise encouraged. The subjects arrived at 2100h and were then given a baseline questionnaire about current health and psychiatric state. After a baseline EKG rhythm strip and vital signs on experimental nights, subjects received oral probanthine, 45 mg, with a small amount of fruit juice at about 2200h. Probanthine is a peripherally active anticholinergic agent that does not cross the blood-brain barrier; it was given to block peripheral cholinomimetic effects of pilocarpine. Subjects were allowed to read or watch TV for the following 50 min when vital signs were repeated. Subjects then drank cranberry juice containing either placebo (1.25 ml sterile water) or pilocarpine, 25 mg (1.25 ml of 2% pilocarpine HCl ophthalmic solution [20 mg/ml], CooperVision Pharmaceuticals, Inc.) 15 min before lights out in a randomized, counterbalanced, and double-blind fashion on different nights, separated by 48-72 hours. Subjects retired at approximately 2300h. Recordings were made on a Nihon/ Kohden Model EEG 5210 polysomnograph at a paper speed of 10 mm/set and scored blindly for sleep stages according to standardized methods (Rechtschaffen and Kales, 1968) as modified by Gillin et al. (1979). For statistical comparison between subjects and nights, a two-way analysis of variance (ANOVA) was performed.
Results Side Effects of Oral Pilocarpine. Pilocarpine was generally well tolerated by the normal control subjects in this study. One subject experienced some nausea and as a result was dropped from the study. Another subject noted a mild increase in salivation. No other side effects attributable to pilocarpine were noted. Effects of Oral Pilocarpine. As seen in Table 1, compared with placebo, pilocarpine had the following statistically significant effects: (1) It increased REM% (Fz7.92; df = 1, 12;p=O.O2) and REM Sleep Time (F= 7.82; df= 1, 12;p=O.O2) and decreased REM latency (F = 7.93; df = 1, 12; p = 0.02). The length of the first REM period (Rl duration) also increased (F = 5.96; df = 1, 12; p = 0.02). (2) Pilocarpine tended to decrease Stage 3 sleep (F= 3.71; df = 1, 12;~ = 0.09) and significantly decreased Stage 3% (F= 5.01; df = 1, 12; p = 0.05). (3) It significantly decreased both Stage 4 sleep (F= 5.09; df = 1, 12;~ = 0.05) and Stage 4% (F= 7.58; df = 1, 12; p = 0.02). (4) It significantly reduced both Delta (Stage 3 and 4) sleep (F=6.05;df=l, 12;p=0.02)andDelta%(F=7.82;df=l, 12;p=O.O2). In contrast to the effects it had on sleep, pilocarpine did not significantly alter heart rate, which was measured continuously by an EKG. Heart rate increased from
116
Table 1. Effect of pilocarpine on human sleep ANOVA
Placebo
Pilocarpine, 25 mg
F
P
Sleep onset time
23:19 + 00:27
23:24
f
00:27
NS
REM onset time
00:34 f 00:33
00:20
f
00:58
NS
Sleep latency
17+
16
17+
NS
14
Total sleep time
354 + 41
368 + 33
1.02
NS
Sleep efficiency %
88.1 + 7.5
89.9 + 4.9
0.68
NS
WAS0
28 k 28
23+18
0.84
NS
Stage 1
17*9
15f8
0.59
NS
Stage 1%
5.0 + 2.5
Stage 2
197+47
Stage 2%
53.5 + 10.5
0.87
NS
211 f29
0.73
NS
57.7 L’C8.7
0.41
NS
20fl8
3.71
0.09
5.1 + 4.2
5.01
0.05
5.09
0.05
4.1 + 2.3
Stage 3
28 + 20
Stage 3%
8.3 III 6.7
Stage 4
25 k 21
Stage 4%
7.4 + 6.6
3.7 * 6.4
7.58
0.02
Delta sleep
53 + 39
34 * 42
6.05
0.03
7.82
0.02
7.82
0.02
7.92
0.02
56 + 42
7.93
0.02
Delta % REM sleep REM %
15-t26
15.7 k 12.7
8.9 *
86 zt 24
108+30
24.0 zt 5.3
10.1
29.3 + 2.1
REM latency
74 + 31
REM density
2.0 + 0.5
1.8 f
0.6
1.56
NS
Rl density
1.4 f 0.6
1.5 * 0.7
0.24
NS
5.96
0.031
0.07
NS
Rl duration Rl cycle length
17+5 llOk32
34 + 25 114+3
Note. Data are presented as mean f SD. Values are in min except for clock times, REM density (scored on a 0 to 8 scaleimin), and percentages. ANOVA = analysis of variance. REM = rapid eye movement. WAS0 = wake time after sleep onset. Rl = REM period 1. df = 1,12.
a mean of 72 beats/ min before pilocarpine to 76 beats/ min just after lights out, to 75 beats/min 30 min after lights out, and 75 beats/min 60 min after lights out.
Discussion In this investigation, pilocarpine significantly shortened REM latency and increased REM sleep, REM% time, and the duration of the first REM period. These results are consistent with earlier animal studies showing that intracerebral application of carbachol, oxotremorine, and other muscarinic agonists shortens the latency to REM sleep and induces prolonged REM sleep periods. They are also consistent with human studies demonstrating that administration of physostigmine, arecoline, and RS-86 induces REM sleep. Our results conflict, however, with our earlier study with physostigmine, infused i.v. over I hour, which shortened REM latency but did not alter the duration of the REM period in normal controls (Gillin et al., 1978). Nevertheless, our current results are consistent with the observation that orally administered RS-86 significantly prolonged the duration of the first REM period (Spiegel, 1984; Berger et al., 1989; Riemann and Berger, 1989). We found a significant decrease in Delta sleep dfter pilocarpine treatment
117
compared with placebo. Sleep latency was unchanged, suggesting that pilocarpine at this dose did not increase arousal. Our observation of Delta sleep with pilocarpine is consistent with those of Spiegel (1984), Berger et al. (1989), and Riemann and Berger (1989), who administered RS-86. Borbely (1982) hypothesized that REM latency is directly correlated with Slow Wave Sleep (SWS). In our study pilocarpine produced both decreased Delta sleep and short REM latency, which could be consistent with this view. Nevertheless, Van den Hoofdakker and Beersma (1985) were unable to find a significant correlation between SWS and REM latency. This led Berger et al. (1989) to conclude that the REM-stimulating effect of RS-86 results directly from cholinergic facilitation of REM sleep rather than inhibition of Delta sleep. These theoretical issues cannot be resolved without further research. Our group has recently shown that direct administration of the M, agonists cisdioxolan and oxotremorine into the medial pontine reticular formation of the cat induced REM sleep; the M, agonist MacN-383, however, did not induce REM sleep (Velazquez-Moctezuma et al., 1989). Pilocarpine and arecoline are probably mixed M, / M, agonists, as is RS-86, which may nevertheless have a predilection for the M, receptor as compared with the M, receptor (Palacios et al., 1986). Pilocarpine, like RS-86, is generally well tolerated. In this study, we administered it following a peripherally acting anticholinergic agent, probanthine, to block peripheral cholinomimetic side effects. It may be a valuable tool for further human pharmacologic and clinical investigations in that it induces REM sleep, is orally active, and is long acting and readily available. It will be interesting to test whether it differentially enhances REM sleep measures in depressed patients compared with normal controls. This research was supported in part by the UCSD Mental Health Clinical Research Center (MH-30914), the UCSD Fellowship in Clinical Pharmacology and Psychobiology (MH-18399), MH-38738, and the Medical Research Service of the Department of Veteran Affairs. We are indebted for assistance to Rick Starch, Caroline Ruiz, Shah Golshan, Ph.D., Anna Demodena, Jenifer Verba, Regina Ciambrone, Steve Funk, Ph.D., Lorraine Goyette, and Kathy Resovsky, R.N.
Acknowledgments.
References Baghdoyan, H.A.; Lydic, R.; Callaway, C.W.; and Hobson, J.A. The carbachol-induced enhancement of desynchronized sleep signs is dose dependent and antagonized by centrally administered atropine. Neuropsychopharmacology, 2:67-80, 1989. Baxter, B.L. Induction of both emotional behavior and a novel form of REM sleep by chemical stimulation applied to cat mesencephalon. Experimental Neurology, 23:220-229, 1969.
Berger, M.; Hochli, D.; Zulley, J.; Laver, C.; and von Zerssen, D. Cholinomimetic drug RS-86, REM sleep, and depression. Luncet, 1:1385-1386, 1985. Berger, M.; Lund, R.; Bronisch, T.; and von Zerssen, D. REM latency in neurotic and endogenous depression and the cholinergic REM induction test. Psychiatry Research, 10: 113123, 1983. Berger, M.; Riemann, D.; Hochli, D.; and Spiegel, R. The cholinergic rapid eye movement sleep induction test with RS-86. Archives of General Psychiatry, 46:421-428, 1989. BorbBly, A.A. A two process model of sleep regulation. Human Neurobiology, 1: 195-204, 1982.
118 Boyd, E.M., and Fulford, R.A. Pilocarpine-induced convulsions and delayed psychotic-like reaction. Canadian Journal of Biochemistry and Physiology, 39:1287-1294, 1961. Domino, E.F.; Yamamoto, K.; and Drew, A.T. Role of cholinergic mechanisms in states of wakefulness and sleep. Progress in Brain Research, 28: 113-133, 1968. George, R.; Haslett, W.L.; and Jenden, D.J. A cholinergic mechanism in the brainstem reticular formation: Induction of paradoxical sleep. International Journal of Neuropharmacology, 3:541-552, 1964. Gillin, J.C.; Duncan, W.; Pettigrew, K.D.; Frankel, B.L.; and Snyder, F. Successful separation of depressed, normal and insomniac patients by EEG sleep data. Archives of General Psychiatry, 36:85-90, 1979. Gillin, J.C.; Sitaram, N.; Mendelson, W.B.; and Wyatt, R.J. Physostigmine alters onset but not duration of REM sleep in man. Psychopharmacology, 58: 111-l 14, 1978. Hobson, J.A.; McCarley, R.W.; and McKenna, T.M. Cellular evidence bearing on the pontine brainstem hypothesis of desynchronized sleep control. Progress in Neurobiology, 6:279-376, 1976. Hobson, J.A., and Steriade, M. Neuronal basis of behavioral state control. In: Mountcastle, V.B.; Bloom, F.E.; and Gieger, S.R., eds. Handbook of Physiology. Bethesda, MD: American Physiologic Society, 4:701-823, 1986. Jouvet, M. Cholinergic mechanisms and sleep. In: Waser, P.G., ed. Cholinergic Mechanisms. New York: Raven Press, 1975. McGinty, D.J., and Drucker-Colin, R.R. Sleep mechanisms: Biology and control of REM sleep. International Review of Neurobiology, 23:391-436, 1982. Oswald, I. Sleep studies in clinical pharmacology. British Journal of Clinical Pharmacology, 10:317-326, 1980. Palacios, J.M.; Bolliger, G.; Closse, A.; Enz, A.; Gmelin, G.; and Malanowski, J. The pharmacologic assessment of RS-86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion hydrobromide): A potent, specific muscarinic acetylcholine receptor agonist. European Journal of Pharmacology, 125:45-62, 1986. Prutting, J. Pilocarpine nitrate and psychostimulants. Journal of the American Medical Association, 191:603, 1965. Rechtschaffen, A., and Kales, A., eds. A Manual of Standardized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects. Washington, DC: Superintendent of Documents, U.S. Government Printing Office, 1968. Riemann, D., and Berger, M. EEG sleep in depression and in remission and the REM sleep response to the cholinergic agonist RS-86. Neuropsychopharmacology, 21145-152, 1989. Sagales, T.; Erill, S.; and Domino, E.F. Differential effects of scopolamine and chlorpromazine on REM and NREM sleep in normal male subjects. Clinical Pharmacology and Therapeutics, 10:522-529, 1969. Sitaram, N.; Mendelson, W.B.; Wyatt, R.J.; and Gillin, J.C. The time-dependent induction of REM sleep and arousal by physostigmine infusion during normal human sleep. Brain Research, 122:562-567, 1977. Sitaram, N.; Moore, A.M.; and Gillin, J.C. The effect of physostigmine on normal human sleep and dreaming. Archives of General Psychiatry, 35:1239-1243, 1978a.
119
Sitaram, N.; Moore, A.M.; and Gillin, J.C. Experimental acceleration and slowing of REM sleep ultradian rhythm by cholinergic agonist and antagonist. Nature, 274:490-492, 1978b. Sitaram, N.; Moore, A.M.; and Gillin, J.C. Induction and resetting of REM sleep rhythm in normal man by arecoline: Blockade by scopolamine. Sleep, 1:83-90, 1978~. Sitaram, N.; Moore, A.M.; Vanskiver, C.; Blendy, J.; Nurnberger, J.I.; Gershon, ES.; and Gillin, J.C. Hypersensitive cholinergic functioning in primary affective illness. In: Pepeu, G., and Ladinsky, H., eds. Cholinergic Mechanisms. New York: Raven Press, 1981. pp. 947-962. Sitaram, N.; Nurnberger, J.I.; Gershon, E.S.; and Gillin, J.C., Faster cholinergic REM sleep induction in euthymic patients with primary affective illness. Science, 208:200-202, 1980. Sitaram, N.; Nurnberger, J.I.; Gershon, E.S.; and Gillin, J.C. Cholinergic regulation of mood and REM sleep: Potential model and marker of vulnerability to affective disorder. American Journal of Psychiatry, 134571-576, 1982. Sitaram, N.; Wyatt, R.J.; Dawson, S.; and Gillin, J.C. REM sleep induction by physostigmine infusion during sleep. Science, 191: 1281-1283, 1976. Spiegel, R. Effects of RS-86, an orally active cholinergic agonist, on sleep in man, Psychiatry Research, 1 l:l-13, 1984. Spitzer, R.L.; Endicott, J.; and Robins, E. Research Diagnostic Criteria: Rationale and reliability. Archives of General Psychiatry, 35773-782, 1978. Suzuki, T.D.; Abe, K.; and Hirose, T. Adrenal cortical secretion in response to pilocarpine in dogs with hypothalamic lesions. Neuroendocrinology, 17:75-82, 1973. Van den Hoofdakker, R.W., and Beersma, D.G.M. On the exploration of short REM latencies in depression. Psychiatry Research, 16:155-163, 1985. Velazquez-Moctezuma, J.; Gillin, J.C.; and Shiromani, P. Effects of specific Ml, M2 muscarinic receptor agonists on REM sleep generation. Brain Research, 503: 128-13 1, 1989. Vertes, R.P. Brainstem control of the events of REM sleep. Progress in Neurobiology, 22:241-288, 1984.
113
Research, 33: 1 I3- 119
Pilocarpine, an Orally Active Muscarinic Cholinergic Agonist, Induces REM Sleep and Reduces Delta Sleep in Normal Volunteers Alan
Berkowitz,
Laura Sutton,
David
S. Janowsky,
and J. Christian
Gillin
Received October 23, 1989; revised version received March 2, 1990; accepted March 18, 1990. Abstract. The effect of oral pilocarpine,
a direct-acting muscarinic, cholinergic agonist, on polygraphic sleep parameters was studied in 13 healthy male volunteers. Subjects received placebo and oral pilocarpine (25 mg) in a doubleblind, counterbalanced, crossover design. Pilocarpine shortened the latency of rapid eye movement (REM) sleep and increased total REM time, REM%, and the duration of the first REM period. In addition, it reduced Stage 4 sleep and Delta sleep. Pulse rate was not significantly changed during the first hour of darkness after administration of pilocarpine. Subjective sleep experience and the subjects’ condition in the morning were not altered. These results suggest that pilocarpine has central effects (i.e., induction of REM sleep) that are similar to those of other centrally acting muscarinic cholinomimetic agents. Key Words. Rapid eye movement sleep, sleep stages, pilocarpine, receptors, human parasympathomimetic.
muscarinic
The exact neurophysiological and biochemical mechanisms that regulate sleep are poorly understood (McGinty and Drucker-Colin, 1982; Vertes, 1984; Hobson and Steriade, 1986). Nevertheless, results of in vivo animal studies indicate a possible role for the cholinergic nervous system in the induction and maintenance of rapid eye movement (REM) sleep (George et al., 1964; Domino et al., 1968; Baxter, 1969; Jouvet, 1975; Hobson et al., 1976; Vertes, 1984; Hobson and Steriade, 1986; Baghdoyan et al., 1989). In normal control subjects, Sitaram et al. (1976, 1978~) demonstrated that physostigmine, a cholinesterase inhibitor, and arecoline, a direct muscarinic agonist, hasten the onset of the following REM sleep period when infused during the preceding non-REM period. REM periods induced by physostigmine were of the same duration as and did not differ from those normally occurring with regard to subjective dream experience (Sitaram et al., 1977; Gillin et al., 1978). Furthermore, the effects of arecoline on REM sleep could be blocked by pretreatment with scopolamine, a peripherally and centrally acting direct muscarinic
Alan Berkowitz, M.D., was a UCSD Fellow in Clinical Psychopharmacology and Psychobiology when this research was conducted. Laura Sutton, R.N., is a Research Nurse. David S. Janowsky, M.D., is currently Chairman and Professor of Psychiatry, The University of North Carolina. J. Christian Gillin, M.D., is Professor of Psychiatry, UCSD, and Staff Psychiatrist, San Diego Veterans Administration Medical Center. (Reprint requests to Dr. J.C. Gillin, Dept. of Psychiatry (M-003), University of California, San Diego, La Jolla, CA 92093, USA.) 0165-1781/90/$03.50
@ 1990 Elsevier Scientific
Publishers
Ireland
Ltd.
114 cholinergic receptor antagonist (Sitaram et al., 19786, 1978~). In addition, RS-86, an experimental, orally active muscarinic agonist, induces REM sleep in man (Spiegel, 1984). Sagales et al. (1969) found that scopolamine when given alone at bedtime suppresses REM sleep. Antidepressants with anticholinergic activity inhibit REM sleep (Oswald, 1980). These observations suggest that blockade of central cholinergic neurotransmission inhibits REM sleep while cholinergic muscarinic facilitation promotes it. Physostigmine, arecoline, and RS-86 have had two major uses in human sleep studies: (1) These drugs have demonstrated cholinergic facilitation of REM sleep. (2) All three drugs have been used in the Cholinergic REM Induction Test (CRIT) in clinical studies (Sitaram et al., 1980, 1981, 1982; Berger et al., 1983, 1985, 1989; Riemann and Berger, 1989). In this test, the speed with which a cholinergic agonist induces REM sleep is measured, i.e., following administration of either i.v. arecoline during the second non-REM period or oral RS-86 before bedtime. Results with both drugs suggest that cholinomimetic muscarinic agonists induce REM sleep more quickly in depressed patients than in normal controls. Furthermore, Berger et al. (1983) reported that i.v. administration of physostigmine shortly after sleep onset awakened depressed patients significantly more often than normal controls, suggesting again that depressed patients are supersensitive to cholinergic agonist challenge. Unfortunately, physostigmine and arecoline have numerous experimental shortcomings that limit their usefulness; since both drugs have a very short duration of action, it is necessary to administer them by the i.v. route during non-REM sleep. Furthermore, although RS-86 can be administered orally, it is no longer available from the manufacturer. Therefore, we studied the effect of pilocarpine, a centrally acting muscarinic cholinergic agonist, and predicted that it would shorten REM latency and, possibly, increase total REM sleep because of its long duration of action. This is the first study of pilocarpine on human sleep. Pilocarpine, an old ophthalmologic preparation, is well absorbed at mucous membranes, such as the eye or gastrointestinal tract, and readily passes the bloodbrain barrier (Boyd and Fulford, 1961). Domino et al. (1968) reported both increased wakefulness and “fast wave sleep” (electroencephalographic desynchronization) in cats given systemic pilocarpine. These effects were abolished by pretreatment with systemic atropine while being only slightly decreased with methylatropine, which does not cross the blood-brain barrier. Several investigators have reported that systemic pilocarpine caused analgesia in mice, rats and rabbits via a central cholinergic mechanism since this effect was abolished by atropine but not by methyl-atropine. Suzuki et al. (1973) found that systemic pilocarpine significantly increased adrenal cortical secretions in rats, presumably through a central muscarinic effect. Pilocarpine has also been used to counteract anticholinergic effects such as dry mouth produced by tricyclic antidepressants. It was found in these studies and in others that it has a duration of action of 6-12 hours in man (Prutting, 1965).
Methods Subjects. The results of this study are based upon data from 13 male subjects, ages 25-46 (mean = 34.8 years), who were without physical or psychiatric diagnoses after a complete
115 medical and psychiatric evaluation, including an and a formal psychiatric diagnostic interview, Schizophrenia (SADS; Spitzer et al., 1978). No days before the study. The subjects were paid for
electrocardiogram (EKG), laboratory tests, the Schedule for Affective Disorders and subject had taken any drugs for at least 30 participation.
Experimental Design and Drug Dosage. Subjects slept on the UCSD Mental Health Research Center in the San Diego Veterans Administration Medical Center. They had allnight polygraphic recordings of electroencephalogram (EEG), electro-oculogram (EOG), electromyogram, and EKG. During the first (adaptation) night, all subjects were screened for nocturnal myoclonus (with tibia1 EMG recordings) and finger oximetry to exclude those with sleep disorders, including sleep apnea or periodic leg movements during sleep. Subjects also had one or two baseline nights before entering the experimental phase of the study. No alcoholic beverages or drugs were permitted on the experimental day. Oral food intake was discouraged after 1600h on experimental days: an average of 6.5 hours elapsed between the last food and the administration of pilocarpine. Routine activities were otherwise encouraged. The subjects arrived at 2100h and were then given a baseline questionnaire about current health and psychiatric state. After a baseline EKG rhythm strip and vital signs on experimental nights, subjects received oral probanthine, 45 mg, with a small amount of fruit juice at about 2200h. Probanthine is a peripherally active anticholinergic agent that does not cross the blood-brain barrier; it was given to block peripheral cholinomimetic effects of pilocarpine. Subjects were allowed to read or watch TV for the following 50 min when vital signs were repeated. Subjects then drank cranberry juice containing either placebo (1.25 ml sterile water) or pilocarpine, 25 mg (1.25 ml of 2% pilocarpine HCl ophthalmic solution [20 mg/ml], CooperVision Pharmaceuticals, Inc.) 15 min before lights out in a randomized, counterbalanced, and double-blind fashion on different nights, separated by 48-72 hours. Subjects retired at approximately 2300h. Recordings were made on a Nihon/ Kohden Model EEG 5210 polysomnograph at a paper speed of 10 mm/set and scored blindly for sleep stages according to standardized methods (Rechtschaffen and Kales, 1968) as modified by Gillin et al. (1979). For statistical comparison between subjects and nights, a two-way analysis of variance (ANOVA) was performed.
Results Side Effects of Oral Pilocarpine. Pilocarpine was generally well tolerated by the normal control subjects in this study. One subject experienced some nausea and as a result was dropped from the study. Another subject noted a mild increase in salivation. No other side effects attributable to pilocarpine were noted. Effects of Oral Pilocarpine. As seen in Table 1, compared with placebo, pilocarpine had the following statistically significant effects: (1) It increased REM% (Fz7.92; df = 1, 12;p=O.O2) and REM Sleep Time (F= 7.82; df= 1, 12;p=O.O2) and decreased REM latency (F = 7.93; df = 1, 12; p = 0.02). The length of the first REM period (Rl duration) also increased (F = 5.96; df = 1, 12; p = 0.02). (2) Pilocarpine tended to decrease Stage 3 sleep (F= 3.71; df = 1, 12;~ = 0.09) and significantly decreased Stage 3% (F= 5.01; df = 1, 12; p = 0.05). (3) It significantly decreased both Stage 4 sleep (F= 5.09; df = 1, 12;~ = 0.05) and Stage 4% (F= 7.58; df = 1, 12; p = 0.02). (4) It significantly reduced both Delta (Stage 3 and 4) sleep (F=6.05;df=l, 12;p=0.02)andDelta%(F=7.82;df=l, 12;p=O.O2). In contrast to the effects it had on sleep, pilocarpine did not significantly alter heart rate, which was measured continuously by an EKG. Heart rate increased from
116
Table 1. Effect of pilocarpine on human sleep ANOVA
Placebo
Pilocarpine, 25 mg
F
P
Sleep onset time
23:19 + 00:27
23:24
f
00:27
NS
REM onset time
00:34 f 00:33
00:20
f
00:58
NS
Sleep latency
17+
16
17+
NS
14
Total sleep time
354 + 41
368 + 33
1.02
NS
Sleep efficiency %
88.1 + 7.5
89.9 + 4.9
0.68
NS
WAS0
28 k 28
23+18
0.84
NS
Stage 1
17*9
15f8
0.59
NS
Stage 1%
5.0 + 2.5
Stage 2
197+47
Stage 2%
53.5 + 10.5
0.87
NS
211 f29
0.73
NS
57.7 L’C8.7
0.41
NS
20fl8
3.71
0.09
5.1 + 4.2
5.01
0.05
5.09
0.05
4.1 + 2.3
Stage 3
28 + 20
Stage 3%
8.3 III 6.7
Stage 4
25 k 21
Stage 4%
7.4 + 6.6
3.7 * 6.4
7.58
0.02
Delta sleep
53 + 39
34 * 42
6.05
0.03
7.82
0.02
7.82
0.02
7.92
0.02
56 + 42
7.93
0.02
Delta % REM sleep REM %
15-t26
15.7 k 12.7
8.9 *
86 zt 24
108+30
24.0 zt 5.3
10.1
29.3 + 2.1
REM latency
74 + 31
REM density
2.0 + 0.5
1.8 f
0.6
1.56
NS
Rl density
1.4 f 0.6
1.5 * 0.7
0.24
NS
5.96
0.031
0.07
NS
Rl duration Rl cycle length
17+5 llOk32
34 + 25 114+3
Note. Data are presented as mean f SD. Values are in min except for clock times, REM density (scored on a 0 to 8 scaleimin), and percentages. ANOVA = analysis of variance. REM = rapid eye movement. WAS0 = wake time after sleep onset. Rl = REM period 1. df = 1,12.
a mean of 72 beats/ min before pilocarpine to 76 beats/ min just after lights out, to 75 beats/min 30 min after lights out, and 75 beats/min 60 min after lights out.
Discussion In this investigation, pilocarpine significantly shortened REM latency and increased REM sleep, REM% time, and the duration of the first REM period. These results are consistent with earlier animal studies showing that intracerebral application of carbachol, oxotremorine, and other muscarinic agonists shortens the latency to REM sleep and induces prolonged REM sleep periods. They are also consistent with human studies demonstrating that administration of physostigmine, arecoline, and RS-86 induces REM sleep. Our results conflict, however, with our earlier study with physostigmine, infused i.v. over I hour, which shortened REM latency but did not alter the duration of the REM period in normal controls (Gillin et al., 1978). Nevertheless, our current results are consistent with the observation that orally administered RS-86 significantly prolonged the duration of the first REM period (Spiegel, 1984; Berger et al., 1989; Riemann and Berger, 1989). We found a significant decrease in Delta sleep dfter pilocarpine treatment
117
compared with placebo. Sleep latency was unchanged, suggesting that pilocarpine at this dose did not increase arousal. Our observation of Delta sleep with pilocarpine is consistent with those of Spiegel (1984), Berger et al. (1989), and Riemann and Berger (1989), who administered RS-86. Borbely (1982) hypothesized that REM latency is directly correlated with Slow Wave Sleep (SWS). In our study pilocarpine produced both decreased Delta sleep and short REM latency, which could be consistent with this view. Nevertheless, Van den Hoofdakker and Beersma (1985) were unable to find a significant correlation between SWS and REM latency. This led Berger et al. (1989) to conclude that the REM-stimulating effect of RS-86 results directly from cholinergic facilitation of REM sleep rather than inhibition of Delta sleep. These theoretical issues cannot be resolved without further research. Our group has recently shown that direct administration of the M, agonists cisdioxolan and oxotremorine into the medial pontine reticular formation of the cat induced REM sleep; the M, agonist MacN-383, however, did not induce REM sleep (Velazquez-Moctezuma et al., 1989). Pilocarpine and arecoline are probably mixed M, / M, agonists, as is RS-86, which may nevertheless have a predilection for the M, receptor as compared with the M, receptor (Palacios et al., 1986). Pilocarpine, like RS-86, is generally well tolerated. In this study, we administered it following a peripherally acting anticholinergic agent, probanthine, to block peripheral cholinomimetic side effects. It may be a valuable tool for further human pharmacologic and clinical investigations in that it induces REM sleep, is orally active, and is long acting and readily available. It will be interesting to test whether it differentially enhances REM sleep measures in depressed patients compared with normal controls. This research was supported in part by the UCSD Mental Health Clinical Research Center (MH-30914), the UCSD Fellowship in Clinical Pharmacology and Psychobiology (MH-18399), MH-38738, and the Medical Research Service of the Department of Veteran Affairs. We are indebted for assistance to Rick Starch, Caroline Ruiz, Shah Golshan, Ph.D., Anna Demodena, Jenifer Verba, Regina Ciambrone, Steve Funk, Ph.D., Lorraine Goyette, and Kathy Resovsky, R.N.
Acknowledgments.
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