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Effects of muscimol and flurazepam on the sleep EEG in the rat
Life Sciences, Vol. 47, pp. PL-99-PL-101 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Co.m~nication
EFFECTS
OF M U S C I M O L
Wallace
AND FLURAZEPAM IN T H E R A T
B. M e n d e l s o n *
ON T H E SLEEP
EEG
and J o s e p h V. M a r t i n
Dept. of Psychiatry, State U n i v e r s i t y of N e w Y o r k at S t o n y Brook, H e a l t h S c i e n c e s Center, T-10, S t o n y Brook, NY 11794-8101 USA (Submitted July 31, 1990; accepted August 15, 1990; received in final form August 30, 1990)
Abstract. In order to assess the possible role of GABAreceptor function in the hypnotic property of benzodiazepines, we have examined the sleep EEG in rats given the GABAagonist musch~.ol, alone and in combination with flurazepam. Muscimol0.05 and 0.1 mg/kg IP failed to alter sleep latency or total sleep time, and did not interact with the sleep-enhancing properties of flurazepam 20 mg/kg IP. These observations, in conjunction with a previous study of bicuculllne, suggest that the hypnotic property of benzodiazeoines may not be mediated by alteration of OABAeraic qctivfly. In~oduction The traditional description of the benzodiazepine receptor complex is of a macromolecular structure with three interacting moieties: recognition sites for benzodiazepines and gamma-aminobutyric acid (GABA), and a chloride ionophore (1). The mechanism by which these components interact to produce the pharmacologic effects of BZs remains, however, to be fully elucidated. Although a great deal of data indicate that GABA-mediated enhancement of chloride channel function is involved in pharmacologic actions such as the muscle relaxation and anticonflict effects, the mechanism of some other properties remains unclear. In certain circumstances, GABA agonists may induce different pharmacological responses than benzodiazepines. The GABAA receptor agonist muscimol and the GABA analog and receptor agonist THIP, for example, enhance petit real-like seizures which occur spontaneously (2) or which are induced by a cholesterol synthesis inhibitor (3), while benzodiazepines have the opposite effect. Similarly, both THIP and the GABA agonist progabide have been reported to not alter the anti-pentylenetetrazole seizure property of chloridazepoxide and triazolam (4). In an in vitro study, GABA and muscimol were observed to increase transient chloride currents in frog sensory neurons, while diazepam did not (5). Just as GABA agonists may not reproduce some bensodiazepine properties, drugs which bind at or near the chloride ionophore site, as well as GABA blockers, may fail to prevent some benzodiazepine effects. Thus picrotoxin has been reported to not block benzodiazepine-decreased motor activity in mice (6), and the GABA receptor blocker bicuculline may not alter sleep in rats, at subconvulsant doses (Y). The common element of these disparate studies seems to be that in some circumstances, some specific benzodiazepine effects are not mimicked or blocked by GABA agonists or antagonists respectively, or by a drug altering chloride channel function. In order to examine possible GABA mediation of the hypnotic properties of BZs, we are reporting *Correspondence
and r e p r i n t
requests.
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press ple
PL-IO0
Muscimol and Flurazepam on Sleep EEG
Vol. 47, No. 19, 1990
here on the effects of muscimol on sleep in the rat. Muscimol has properties similar to GABA insofar as it induces a transient CI" current in voltage-clamp preparations (5), and alters ~f~nity for ligands to the BZ recognition site (8). In this study, we have administered it alone and in combination with the benzodiazepine hypnotic flurazepam. Methods Seventeen male Sprague-Dawley 200-225 gm rats were implanted with stainless screw electrodes for recording of bifrontal and fronto-occipital electroencephalogram and nuchal electromyogram. After a one week recovery period in chambers with lights on from 8:00 AM until 8:00 PM, they were acclimatized to a recording chamber overnight. The following morning at 10:00 AM they were administered muscimol 0.05 or 0.1 mg/kg IP, alone or in combination with flurazepam 20 mg/kg IP, and two hour polygraphic recordings were performed. Treatments were administered in random order, in studies separated by one week each. Recordings were interpreted by one "blind" investigator, who made a determination for each 30 second epoch as to whether the rat was awake or in nonREM or REM sleep according to standard criteria (9). Data were analyzed by a repeated measures analysis of variance (ANOVA) which determined the effects on standard sleep parameters of each drug, and their possible interaction. Parameters measured included sleep latency (the time from injection of drug until sleep onset), REM latency (the time from sleep onset until the beginning of REM sleep), intermittent waking time (cumulative amount of waking time after the initial sleep onset), and total amounts of all sleep, REM sleep and nonREM sleep. Results
As seen in the Table, flurazepam had the expected hypnotic effects of shortening sleep latency and increasing total sleep. REM sleep time was decreased, and REM latency was enhanced. In contrast, muscimol failed to alter either of these parameters. There was no significant interaction between muscimol and flurazepam on the ANOVA. The higher dose of muscimol produced occasional bursts of high amplitude episodic EEG activity. Muscimol is known to cross the blood brain barrier to a significant degree (10); this EEG activity suggests that despite the lack of alteration of sleep architecture, muscimol was indeed having CNS effects. Discussion In summary, flurazepam had a significant hypnotic effect as measured by the sleep EEG, while muscimol had none. Moreover, co-administration of muscimol failed to alter the hypnotic actions of flurazepam. These data suggest that the hypnotic properties of flurazepam may not be mediated by altering GABAergic function. This hypothesis is supported further by the observation that subconvulsant doses of the GABA antagonist bicuculline administered intraperitoneally do not alter EEG measures of sleep in the rat (7). It remains possible, of course, that potent GABA antagonists may indirectly affect sleep by inducing EEG spiking or motor seizure activity, which may have a disruptive effect on sleep. Similarly, it should be noted that the description of "hypnotic activit¢' as used here refers to EEG measures of sleep and waking; it is not implied that this finding generalizes to the ataxia or decreased motor activity during waking which can be induced by benzodiazepines. Within these limitations, however, these data continue to raise the possibility that, although many actions of
Vol.
47,
No.
19,
1990
and
Muscimol
Flurazepam
on
Sleep
EEG
PL-101
~%BLE I Effects of Muscimol and Fluraze~em on Sleep in the Rat (N = 17) MuscimclV+V
V+F
Sleep L a t e r ~
24.3 2.6
12.2.* 2.4
Non R~4
60.4 2.7
75.4** 2.6
~ ~ Waking Time
30.2 2.2
30.5 2.3
R~4
4.9 •85
2.6 .75
M~mcimol Effect (~)
Flurazep~ F I ~ Effect Interactiun
(~)
(p<)
0.05M+F
0.1M+F
0.0~M+V
0.1M+V
9.9** 2.2
12.3-* 1.8
21.3 2.7
20.5 2.5
.45
.0009
•60
74.4* 2.5
77.4** 2.3
60.5 3.1
60.5 2.6
.77
.0001
.86
32.6 2.9
27.5 1.7
32.0 3.3
35.0 2.4
.75
.25
.14
.81
.05
.58
2.4 .76
3.0 .67
4.4 1.0
3.6 .97
R~4 I ~ t e ~
47.7 9.4
78.5 9.8
91.9"* 7.4
80.6* 9.4
66.8 9.9
64.9 11.3
.19
•007
.71
Total Sleep
64.7 2.9
76.9* 2.4
77.4* 2.6
80.4** 2.3
64.9 3.2
63.8 2.4
.81
.0001
•68
*P<0.05 compared to vehicle-vehicle ccnditiun **P<0.01 cumpared to vehlcle-vehicle condition V=vehicle 0.0~, 0.1M = muscimol 0.0Smg/~j, 0.i m W ] g F = f l ~ 2O m g / ~
benzodiazepines are mediated by enhanced GABAergic activity, the hypnotic properties may be due to non-GABA mechanisms. Acknowledqements This study was partially supported by National Science Foundation grant DNS 8919229. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0.
P. SKOLNICK, W.B. MENDELSON, and S.M. PAUL, Psvchouharmacoloav of Sle~n D. Wheatley (ed), 117-134, Raven Press, New York, (1981). C. MARESCAUX, G. MICHELETTI, M. VERGNES, L.RUMBACH, and J.M. WARTER, Eur. J. Pharmacol. 113 19-24 (1985). K.A SMITH and G.C. BIERKAMPER, Eur. J. Pharmacol. 176 45-55 (1990). AY. CHWEH, E3L SWINYARD, H.H., WOLF, and H.J. Kupferberg, Life Sciences 36:737-744 (1985). T. MARUYAMA, J.C. BEHRENDS, and N. AKAIKE, Neurosci. Res. _6 97-108 (1988). P. SOUBRIE, P. SIMON and J. R. BOISSIER, Neuropharmacology 15:773-776 (1976). J.M. MONTI, H. ALTIER and L. D'ANGELO, Pharmaooloav of the states of alertness, P. Passouant and I. Oswald (eds), Pergamon Press, Oxford, 68-73 (1979). B.K. KOE, E. KONDRATOS, L.L. RUSSO, Eur. J. Pharmacol. 14~ 373-384 (1987). W.B. MENDELSON, It GUTHRIE, G. FREDERICK, and R.J. WYATT, Pharmaool. Biochem. Behav. 2 553-556 (1974). J.R. COOPER, F.E. BLOOM, R.H. ROTH, The Biochemical Basis 9[ Neurouharmacoloav, 141-143, Oxford University Press, New York (1986).
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Co.m~nication
EFFECTS
OF M U S C I M O L
Wallace
AND FLURAZEPAM IN T H E R A T
B. M e n d e l s o n *
ON T H E SLEEP
EEG
and J o s e p h V. M a r t i n
Dept. of Psychiatry, State U n i v e r s i t y of N e w Y o r k at S t o n y Brook, H e a l t h S c i e n c e s Center, T-10, S t o n y Brook, NY 11794-8101 USA (Submitted July 31, 1990; accepted August 15, 1990; received in final form August 30, 1990)
Abstract. In order to assess the possible role of GABAreceptor function in the hypnotic property of benzodiazepines, we have examined the sleep EEG in rats given the GABAagonist musch~.ol, alone and in combination with flurazepam. Muscimol0.05 and 0.1 mg/kg IP failed to alter sleep latency or total sleep time, and did not interact with the sleep-enhancing properties of flurazepam 20 mg/kg IP. These observations, in conjunction with a previous study of bicuculllne, suggest that the hypnotic property of benzodiazeoines may not be mediated by alteration of OABAeraic qctivfly. In~oduction The traditional description of the benzodiazepine receptor complex is of a macromolecular structure with three interacting moieties: recognition sites for benzodiazepines and gamma-aminobutyric acid (GABA), and a chloride ionophore (1). The mechanism by which these components interact to produce the pharmacologic effects of BZs remains, however, to be fully elucidated. Although a great deal of data indicate that GABA-mediated enhancement of chloride channel function is involved in pharmacologic actions such as the muscle relaxation and anticonflict effects, the mechanism of some other properties remains unclear. In certain circumstances, GABA agonists may induce different pharmacological responses than benzodiazepines. The GABAA receptor agonist muscimol and the GABA analog and receptor agonist THIP, for example, enhance petit real-like seizures which occur spontaneously (2) or which are induced by a cholesterol synthesis inhibitor (3), while benzodiazepines have the opposite effect. Similarly, both THIP and the GABA agonist progabide have been reported to not alter the anti-pentylenetetrazole seizure property of chloridazepoxide and triazolam (4). In an in vitro study, GABA and muscimol were observed to increase transient chloride currents in frog sensory neurons, while diazepam did not (5). Just as GABA agonists may not reproduce some bensodiazepine properties, drugs which bind at or near the chloride ionophore site, as well as GABA blockers, may fail to prevent some benzodiazepine effects. Thus picrotoxin has been reported to not block benzodiazepine-decreased motor activity in mice (6), and the GABA receptor blocker bicuculline may not alter sleep in rats, at subconvulsant doses (Y). The common element of these disparate studies seems to be that in some circumstances, some specific benzodiazepine effects are not mimicked or blocked by GABA agonists or antagonists respectively, or by a drug altering chloride channel function. In order to examine possible GABA mediation of the hypnotic properties of BZs, we are reporting *Correspondence
and r e p r i n t
requests.
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press ple
PL-IO0
Muscimol and Flurazepam on Sleep EEG
Vol. 47, No. 19, 1990
here on the effects of muscimol on sleep in the rat. Muscimol has properties similar to GABA insofar as it induces a transient CI" current in voltage-clamp preparations (5), and alters ~f~nity for ligands to the BZ recognition site (8). In this study, we have administered it alone and in combination with the benzodiazepine hypnotic flurazepam. Methods Seventeen male Sprague-Dawley 200-225 gm rats were implanted with stainless screw electrodes for recording of bifrontal and fronto-occipital electroencephalogram and nuchal electromyogram. After a one week recovery period in chambers with lights on from 8:00 AM until 8:00 PM, they were acclimatized to a recording chamber overnight. The following morning at 10:00 AM they were administered muscimol 0.05 or 0.1 mg/kg IP, alone or in combination with flurazepam 20 mg/kg IP, and two hour polygraphic recordings were performed. Treatments were administered in random order, in studies separated by one week each. Recordings were interpreted by one "blind" investigator, who made a determination for each 30 second epoch as to whether the rat was awake or in nonREM or REM sleep according to standard criteria (9). Data were analyzed by a repeated measures analysis of variance (ANOVA) which determined the effects on standard sleep parameters of each drug, and their possible interaction. Parameters measured included sleep latency (the time from injection of drug until sleep onset), REM latency (the time from sleep onset until the beginning of REM sleep), intermittent waking time (cumulative amount of waking time after the initial sleep onset), and total amounts of all sleep, REM sleep and nonREM sleep. Results
As seen in the Table, flurazepam had the expected hypnotic effects of shortening sleep latency and increasing total sleep. REM sleep time was decreased, and REM latency was enhanced. In contrast, muscimol failed to alter either of these parameters. There was no significant interaction between muscimol and flurazepam on the ANOVA. The higher dose of muscimol produced occasional bursts of high amplitude episodic EEG activity. Muscimol is known to cross the blood brain barrier to a significant degree (10); this EEG activity suggests that despite the lack of alteration of sleep architecture, muscimol was indeed having CNS effects. Discussion In summary, flurazepam had a significant hypnotic effect as measured by the sleep EEG, while muscimol had none. Moreover, co-administration of muscimol failed to alter the hypnotic actions of flurazepam. These data suggest that the hypnotic properties of flurazepam may not be mediated by altering GABAergic function. This hypothesis is supported further by the observation that subconvulsant doses of the GABA antagonist bicuculline administered intraperitoneally do not alter EEG measures of sleep in the rat (7). It remains possible, of course, that potent GABA antagonists may indirectly affect sleep by inducing EEG spiking or motor seizure activity, which may have a disruptive effect on sleep. Similarly, it should be noted that the description of "hypnotic activit¢' as used here refers to EEG measures of sleep and waking; it is not implied that this finding generalizes to the ataxia or decreased motor activity during waking which can be induced by benzodiazepines. Within these limitations, however, these data continue to raise the possibility that, although many actions of
Vol.
47,
No.
19,
1990
and
Muscimol
Flurazepam
on
Sleep
EEG
PL-101
~%BLE I Effects of Muscimol and Fluraze~em on Sleep in the Rat (N = 17) MuscimclV+V
V+F
Sleep L a t e r ~
24.3 2.6
12.2.* 2.4
Non R~4
60.4 2.7
75.4** 2.6
~ ~ Waking Time
30.2 2.2
30.5 2.3
R~4
4.9 •85
2.6 .75
M~mcimol Effect (~)
Flurazep~ F I ~ Effect Interactiun
(~)
(p<)
0.05M+F
0.1M+F
0.0~M+V
0.1M+V
9.9** 2.2
12.3-* 1.8
21.3 2.7
20.5 2.5
.45
.0009
•60
74.4* 2.5
77.4** 2.3
60.5 3.1
60.5 2.6
.77
.0001
.86
32.6 2.9
27.5 1.7
32.0 3.3
35.0 2.4
.75
.25
.14
.81
.05
.58
2.4 .76
3.0 .67
4.4 1.0
3.6 .97
R~4 I ~ t e ~
47.7 9.4
78.5 9.8
91.9"* 7.4
80.6* 9.4
66.8 9.9
64.9 11.3
.19
•007
.71
Total Sleep
64.7 2.9
76.9* 2.4
77.4* 2.6
80.4** 2.3
64.9 3.2
63.8 2.4
.81
.0001
•68
*P<0.05 compared to vehicle-vehicle ccnditiun **P<0.01 cumpared to vehlcle-vehicle condition V=vehicle 0.0~, 0.1M = muscimol 0.0Smg/~j, 0.i m W ] g F = f l ~ 2O m g / ~
benzodiazepines are mediated by enhanced GABAergic activity, the hypnotic properties may be due to non-GABA mechanisms. Acknowledqements This study was partially supported by National Science Foundation grant DNS 8919229. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0.
P. SKOLNICK, W.B. MENDELSON, and S.M. PAUL, Psvchouharmacoloav of Sle~n D. Wheatley (ed), 117-134, Raven Press, New York, (1981). C. MARESCAUX, G. MICHELETTI, M. VERGNES, L.RUMBACH, and J.M. WARTER, Eur. J. Pharmacol. 113 19-24 (1985). K.A SMITH and G.C. BIERKAMPER, Eur. J. Pharmacol. 176 45-55 (1990). AY. CHWEH, E3L SWINYARD, H.H., WOLF, and H.J. Kupferberg, Life Sciences 36:737-744 (1985). T. MARUYAMA, J.C. BEHRENDS, and N. AKAIKE, Neurosci. Res. _6 97-108 (1988). P. SOUBRIE, P. SIMON and J. R. BOISSIER, Neuropharmacology 15:773-776 (1976). J.M. MONTI, H. ALTIER and L. D'ANGELO, Pharmaooloav of the states of alertness, P. Passouant and I. Oswald (eds), Pergamon Press, Oxford, 68-73 (1979). B.K. KOE, E. KONDRATOS, L.L. RUSSO, Eur. J. Pharmacol. 14~ 373-384 (1987). W.B. MENDELSON, It GUTHRIE, G. FREDERICK, and R.J. WYATT, Pharmaool. Biochem. Behav. 2 553-556 (1974). J.R. COOPER, F.E. BLOOM, R.H. ROTH, The Biochemical Basis 9[ Neurouharmacoloav, 141-143, Oxford University Press, New York (1986).