EFFECT OF ENFLURANE ON CEREBELLAR cGMP AND ON MOTOR ACTIVITY IN THE MOUSE

Br. J. Anaesth. (1983), 55, 79

EFFECT OF ENFLURANE ON CEREBELLAR cGMP AND ON MOTOR ACTIVITY IN THE MOUSE Y. VULLIEMOZ, M. VEROSKY, M . ALPERT AND L . TRINER SUMMARY

The results of studies of enflurane-induced muscle relaxation suggest that, in addition to an action at the neuromuscular junction, enflurane may act at other less distal sites along the motor pathways (Waud and Waud, 1979). Our recent study showed that halothane decreases motor activity by an action on the cerebellar control of motor pathways and that this effect is accompanied by dimished concentrations of cerebellar cyclic 3 ' , 5'-guanosine monophosphate (cGMP) (Triner et al., 1981). Since previous work showed certain similarities in the effects of halothane and enflurane on cyclic nucleotide metabolism in various tissues, including brain (Triner, Vulliemoz and Verosky, 1975; Woo et al., 1979), the effect of enflurane on cerebellar cGMP content and on the cerebellar control of motor activity was examined in mice. The results show that enflurane diminished mouse cerebellar cGMP content and prevented or delayed (depending on the dose) the drug-induced increase of the nucleotide in the cerebellum. These effects were accompanied by corresponding changes of motor activity, suggesting an effect of enflurane on the cerebellar control of motor pathways.

YVONNE VULLIEMOZ, PH.D.; MARIAGNES VEROSKY, B.A.; MYLES ALPERT, B_A.; LUBOS TRINER,* M.D.,PH.D.; Department of Anes-

thesiology, Columbia University, College of Physicians & Surgeons, New York, NY 10032, U.S.A. •Address for correspondence: Anesthesia Service, Presbyterian Hospital in the City of New York, New York, NY 10032, U.S.A. 0007-09122/83/010079-06 $01.00

MATERIALS AND METHODS l25

I-succinyl cyclic GMP-TME and cyclic GMP antiserum were obtained from Collaborative Research Inc.; 3 H-cGMP, from New England Nuclear Corp.; isonicotinic acid, hydrazide and picrotoxin, from the Sigma Chemical Co.; strychnine sulphate, from ICN Pharmaceuticals, Inc.; enflurane (Ethrane), from Ohio Medical Products. All other compounds were "reagent" grade. Male Swiss Webster mice weighing 20-25 g were allowed free access to Purina chow and water until 2 h before the experiment. The mice were housed in cages kept at 22 °C and maintained at a 12-h light-dark cycle. The enflurane concentration required to abolish the righting reflex in 50% of the animals was determined in mice placed in rotating wire mesh cages (lOrevmin" 1 ) inside a chamber. A continuous flow of the anaesthetic in air (total flow 5htremin"') through the chamber (20-litre volume) was maintained for 60min at concentrations sufficient to abolish the righting reflex in all the animals. The concentration was subsequently decreased gradually and maintained at each concentration for a minimum of 30min, or until the righting reflex response was reproduced three times. Results did not differ when the reverse sequence, from low to high concentration, was used. Enflurane concentration in the effluent gas from the chamber was continuously measured with a Beckman Medical Gas Analyzer, LB-2, calibrated with three standards (supplied by Matheson, a Division of Will Ross, © The MacmUlan Press Ltd 1983

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The effect of enflurane on the cercbellar content of the intracellular mediator cyclic 3 ' , 5'-guanosine monophosphate (cGMP) and on motor activity was studied in mice. Seizures, as an index of increased motor activity, associated with an increase in cerebellar cGMP content were induced with isoniazide or picrotoiin. Enflurane 0.28-1.68 vol% produced a dose-dependent, reversible decrease in cerebellar cGMP (by about 50% at 0.28 vol%) and delayed or prevented both the increase in cerebellar cGMP and the convulsions induced by isoniazide. Enflurane also protected against picrotoxin-induced convulsions, but not against strychnine-induced convulsions which presumably do not involve cerebellar mechanisms. These results indicate that enflurane affects the cerebellar mechanisms controlling motor activity and it is postulated that this action contributes to the decrease in muscle tone induced by enflurane.

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BRITISH JOURNAL OF ANAESTHESIA TABLE I. Effect of enflurane on the cGMP content of mouse cerebellar cortex, tpmol cGMP/mg protein (mean + SEM). Numbers in parentheses indicate number of mice. 'Significant change from control (P <0.05) Enflurane (vol%) Control

0.28

0.56

0.84

1.12

1.68

3.82t ±0.32 (34)

2.20* ±0.31 (13)

1.02* ±0.11 (13)

0.56* ±0.09 (6)

0.57* ±0.08 (7)

0.59* ±0.02 (15)

0.28 vol%, concentrations smaller than the concentration at which 50% of the mice lose the "righting reflex" (ED50RR = 1.12 ± 0.03 (SEM) vol%). The time-course of the effect of enflurane on cerebellar cGMP is shown in figure 1. When the animals were exposed to 1.68 vol% enflurane, about 75% of the effect was obtained within 5 min of exposure and in about 20 min the effect was fully developed. The cGMP content remained constant during continued exposure to enflurane for at least 60 min. After enflurane had been discontinued the cGMP content returned towards pre-anaesthetic control values in about 20 min. Biggio, Costa and Guidotti (1977) and Costa and colleagues (1975) showed that isoniazide-induced convulsions are associated with an increase in cerebellar cGMP content and that compounds which antagonize the increase in cGMP induced by isoniazide also protect against convulsions. The effect of enflurane on isoniazide-induced increase in cerebellar cGMP is shown in figure 2. Before the onset of convulsion, isoniazide 200mgkg~1 s.c. caused an increase in cerebellar cGMP content of

r

Enflurane

1.68 vol I

< off

The significance of the results at a 95% confidence limit was evaluated by Student's t test. RESULTS

Enflurane produced a dose-dependent decrease in the cGMP content of mouse cerebellar cortex (table I). The maximum decrease in cGMP was elicited by 0.84 vol% enflurane and a 50% effect, by about

30

60

90

Time (min)

FIG. 1. Time-course of cerebellar cGMP content in mice during and after enflurane exposure. Values are mean ± SEM from seven to nine mice. *Significant change from time "0" (.P-C0.05).

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Inc.) and maintained constant (Goldman et al., 1980). The temperature of the chamber was adjusted to maintain at 37 °C the rectal temperature of a restrained mouse placed in the chamber with a rectal temperature probe. The frequency of convulsion in response to varying doses (s.c.) of convulsive drugs (isoniazide, picrotoxin, and strychnine) was determined in the absence and presence of enf lurane and was used as an index of motor activity. The method described by Ipsen and Feigl (1970) was used to calculate the ED50 convulsive dose. The cGMP content of the cerebellar cortex was determined in mice exposed to varying concentrations of enflurane at constant temperature for a defined period of time. The anaesthetic concentration in the effluent gas from the chamber containing the mice was maintained constant and continuously monitored (see above). The mice were then sacrificed by microwave radiation focused to the head for 2.5 s, a procedure which stops all enzyme activity, as confirmed by measurement of phosphodiesterase activity in the tissue. The cerebellum was immediately dissected out, macroscopically evident white matter removed and the tissue stored at -60°C until processed. The specimens were homogenized in 5% trichloroacetic acid 1.5 ml. After centrifugation, trichloroacetic acid in the supernatant was extracted with water-saturated ether and the cGMP content was measured in aliquots of the supernatant by radioimmunoassay; the sensitivity of the assay was increased by succinylation of the nucleotide (Steiner, Parker and Kipnis, 1972; Frandsen and Krishna, 1976). The good agreement in the cGMP content from varying volumes of supernatant and in recovering known amounts of cGMP added to the assay indicated that purification and separation of cGMP in the sample before the immunoassay was not necessary. Reagent blanks and appropriate standards were carried through the whole procedure. Determinations were made in triplicate, and the results expressed in pmol cGMP/mg protein. The pellet was used for protein determination by the method of Lowry and others (1951).

ENFLURANE AND CEREBELLAR cGMP AND MOTOR CONTROL C-Control I-Isoniazide E-Enflurane

i

1 6.

I

212

m

I

E

I+E

FIG. 2. Effect of enflurane on isoniazide-induced increase in cerebellar cGMP content. Isoniazide 200mgkg~' s.c. and 1.68 vol% enflurane were administered 45 min before sacrifice of the mice. Values are mean ± SEM from five to 13 mice. 'Significant change from control. Numbers in column indicate number of mice.

167% (.P<0.05) greater than the control value of 1.96 + 0.12 pmol cGMP/mg protein. When the mice were given the same dose of isoniazide and simultaneously exposed to 1.68 vol% enflurane the increase in cGMP was not only prevented but the cerebellar cGMP content remained below the control value at a concentration not different from that found in mice exposed to enflurane only (that is,

10

20

85% less than control (P<0.001)). While all mice given isoniazide alone convulsed 45-50 min after injection, none of the mice given isoniazide and exposed to enflurane convulsed within 2 h following injection of isoniazide 200mgkg~' s.c. When the animals were given a larger dose of isoniazide (500mgkg"' s.c.) and exposed to enflurane at a smaller concentration (0.28 vol%), the cerebellar cGMP content did increase to a concentration which was not different from that of mice treated only with isoniazide 500mgkg~' s . c ; the increase in cGMP, however, was delayed by 12 min, as shown by the shift to the right along the time axis of the cGMP response at the ED50 of the effect in the mice exposed to enflurane, compared with mice given isoniazide alone (fig. 3). All the animals given isoniazide 500 mg kg"' s. c. exposed or not exposed to enflurane convulsed; however, the convulsion latency was longer in the mice exposed to 0.28 vol% enflurane (35 ± 2 (SEM) min) than in the mice receiving only isoniazide (23 ± 1 min). Enflurane caused a similar delay in the increase in cGMP and in the onset of convulsions induced by isoniazide—12 and 14 min, respectively, as estimated from the time response curves on figure 3 at the 50% of the effects. The protective effect of enflurane against convulsions induced by isoniazide is further illustrated by the increase caused by enflurane in the isoniazide

40

Time (min) FIG. 3. Effect of enflurane on the time-course of the increase in cerebellar cGMP content and of the frequency of convulsions induced by isoniazide. Enflurane 0.28 vol% was administered 20 min before injection of isoniazide 500mgkg~' s.c. cGMP content in the absence (O O) and in the presence ( • • ) of enflurane; values represent mean ± SEM from six to 16 mice. Frequency of convulsion in the absence (cross-hatching) and in the presence (dots) of enflurane.

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C

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BRITISH JOURNAL OF ANAESTHESIA

82

2 200 .2

100 0

0.5 Enflurane (vol2)

1

FIG. 4. Effect of enflurane on the EDso convulsive dose of isoniazide. Values are mean ± SEM from 14 to 16 mice for each point.

seizure threshold (fig. 4). The ED,, convulsive dose of isoniazide increased linearly with the increase in enflurane concentration. At a concentration of 0.84vol% enflurane the EDJO convulsive dose of isoniazide was about doubled. Figure 5 shows that there is an inverse proportional relationship between the enflurane-induced increase in the ED50 convulsive dose of isoniazide and the decrease in cerebellar cGMP content caused by enflurane; the cGMP dose-response curve to enflurane is the mirror image of the ED50 convulsive dose-response curve to isoniazide. 300

200 CD

."5

100 0.5 Enflurane (vol 1)

1

FlG. 5. Comparison of the effect of enflurane on cerebellar cGMP content and on the EDJO convulsive dose of isoniazide. Values are from table I and figure 4.

DISCUSSION Cyclic GMP is considered to be the intraceilular mediator of the excitatory stimuli reaching the cerebellar Purkinje cells (Mao, Guidotti and Costa, 1974a; Biggio and Guidotti, 1976). The cerebellar cortex cGMP content represents primarily the cyclic nucleotide of the Purkinje cells (Mao, Guidotti and Landis, 1975), which provide the major cerebellar output controlling the motor pathways (Eccles, 1977). The activity of Purkinje cells, reflected by the changes in cGMP, is determined by the balance between excitatory and inhibitory input. Consequently, increasing Purkinje cell activity is accompanied by increasing cerebellar cortex cGMP and by increasing motor activity, which may progress to seizure activity. Conversely, reduced excitatory or enhanced cerebellar GAB A-ergic inhibitory input to Purkinje cells is accompanied by a decrease in cerebellar cortex cGMP content and by a decrease in motor activity (Costa, Guidotti and Mao, 1975; Biggio, Costa, and Guidotti, 1977). In this respect, using the cerebellum as a model in the present study provided the possibility of correlating cellular events and behavioural changes induced by enflurane. Enflurane at low subanaesthetic concentrations, which markedly decreased cerebellar cGMP content, protected the mice against convulsions induced by isoniazide, as shown by the dosedependent increase in the seizure threshold, and antagonized the increase in cerebellar cGMP induced by isoniazide. This compound increases cerebellar cGMP and induces convulsions because it reduces the content of the inhibitory transmitter GAB A in the cerebellum (Biggio, Costa and Guidotti, 1977). Our data demonstrate a quantitative and temporal correlation between the anticonvulsant and the biochemical effect of enflurane; enflurane prevented or delayed to a comparable extent both the cGMP and the motor response to isoniazide.

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Enflurane also protected the mice against convulsions induced by picrotoxin, which, like isoniazide, increases cGMP content in the cerebellum (Mao, Guidotti, and Costa, 1974a). The picrotoxin ED*, convulsive dose increased by 215% in the presence of 1.12 vol% enflurane, from 1.9 ±0.2 (SEM) to 6.0±1.0mgkg- 1 s.c. (P<0.005). In contrast, enflurane did not affect the ED50 convulsive dose of strychnine (0.77 ±0.04 (SEM) and 0.66 ±0.03 mgkg" 1 s.c, in the absence and presence of 1.12 vol% enflurane respectively).

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ENFLURANE AND CEREBELLAR cGMP AND MOTOR CONTROL

REFERENCES

Angel, A. (1980). Effect of anesthetics on nervous pathways; in General Anesthesia, Vol. 1, 4th edn (eds T. C. Gray, J. E. Nunn, and J. E. Utting), p. 117. Boston: Butterworth Inc. Biggio, G., Costa, E., and Guidotti, A (1977). Pharmacologically induced changes in the 3', 5'-cyclicguanosinemonophosphate content of rat cerebellar cortex: Difference between apomorphine, haloperidol and harmaline. / . Pharmacol. Exp. Ther., 200,207. Guidotti, A. (1976). Climbing fiber activation and 3', 5'-cyclic guanosine monophosphate content in cortex and deep nuclei of cerebellum. Brain Res., 107, 365. Costa, E., Guidotti, A., and Mao, C. C. (1975). Evidence for involvement of GABA in the action of benzodiazepines: Studies on rat cerebellum; in Mechanism of Action of Benzo-

diazepines (eds E. Costa and P. Greengard), p. 113. New York: Raven Press. Suria, A. (1975). New concepts on the mechanism of action of benzodiazepines, Life Sci., 17,167. Curtis, D. R., Duggan, A. W., and Johnson, G. A. R. (1971). The specificity of strychnine as a glycine antagonist in the mammalian spinal cord. Exp. Brain Res., 12, 547. Ecdes, J. C. (1977). The Understanding of the Brain, 2nd edn, p. 104. New York: McGraw-Hill. Frandsen, E. K., and Krishna, G. (1976). A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. Life Sci., 18, 529. Galindo, A. (1969). Effects of procaine, pentobarbital and halothane on synaptic transmission in the CNS. / . Pharmacol.

Exp. Ther.,U9,185. Goldman, E., Sherrill, D., de Campo, T., and Aldrete, J. A. (1980). Calibration curves of enflurane using a Beckman LB2 Gas Analyzer with halothane head. Anesthesiology, 53, 79. Ipsen, J.,andFeigl, P. (1970). Bancroft's Introduction to Biostatistics, 2nd edn, p. 164. New York: Harper Row. de Jong, R. H., Hershey, W. N., and Wagman, I. H. (1967). Measurement of a spinal reflex response (H-Reflex) during general anesthesia in man. Anesthesiology, 28, 382. Lebowitz, M. H., Blitt, C. D., and Walts, L. F. (1970). Depression of twitch response to stimulation of the ulnar nerve during Ethrane anesthesia in man. Anesthesiology, 33, 52. Lowry, D. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent, / . Biol. Chem., 193, 265. Mao, C. C , Guidotti, A., and Costa, E. (1974a). The regulation of cyclic guanosine monophosphate in rat cerebellum: Possible involvement of putative amino acid neurotransmitters. Brain Res., 79, 510. (1974b). Interactions between gammaaminobutyric acid and guanosine cyclic 3', 5'-monophosphate in rat cerebellum, Mol. Pharmacol., 10, 736. Landis, S. (1975). Cyclic GMP: Reduction of cerebellar concentration in nervous mice. Brain Res., 90, 335. Ngai, S. H., Hanks, E. C , and Fahrie, S. E. (1965). Effects of anesthetics on neuromuscular transmission and somatic reflexes. Anesthesiology, 26,162. Obata, K., Takeda, K., and Shinozaki, H. (1970). Further studies on the pharmacological properties of the cerebellarinduced inhibition of Deiter's neurons. Exp. Brain Res., 11, 327. Somjen, G. G., and Gill, M. (1963). The mechanism of the blockade of synaptic transmission in the mammalian spinal cord by diethyl ether. / . Pharmacol. Exp. Ther., 140,19. Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972). Radioimmunoassay for cyclic nucleotides. I: Preparation of antibodies and iodinated cyclic nucleotides. / . Biol. Chem., 247,1106. Triner, L., Vulliemoz, Y., and Verosky, M. (1975). Effects of halothane, enflurane and isoflurane on bronchial tone and cAMP. Fed. Proc., 34, 798. Alpert, M. (1981). Halothane effect on cGMP and control of motor activity in mouse cerebellum. Anesthesiology, 54,193. Waud, B. E., and Waud, D. R. (1975). Comparison of the effects of general anesthetics on the end-plate of skeletal muscle. Anesthesiology, 43, 540. (1979). Effect of volatile anesthetics on directly and indirectly stimulated skeletal muscle. Anesthesiology, 50, 103.

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These results suggest that there is a close relationship between the decrease in cerebellar cGMP induced by enflurane and the decrease in motor activity. It is well documented that picrotoxin and isoniazide inhibit the action and the synthesis of the inhibitory transmitter GABA in the cerebellum, while strychnine blocks the glycine receptors primarily in the spinal cord (Obata, Takeda and Shinozaki, 1970; Curtis, Duggan and Johnson, 1971; Young and Snyder, 1973; Mao, Guidottiand Costa, 1974b; Biggio, Costa and Guidotti, 1977). The difference between the effect of enflurane on convulsions induced by isoniazide or picrotoxin, on one hand, and those induced by strychnine, on the other hand, indicates that the anticonvulsant effect of enflurane is exerted primarily on the cerebellar processes controlling muscle tone and motor activity. Numerous studies have shown that muscular relaxation induced by inhalation anaesthetics such as halothane, ether and methoxyflurane, is attributable to depression of motor neurons and spinal reflexes brought about by a direct effect on spinal mechanisms, indirectly by affecting supraspinal structures modulating spinal mechanisms, or both (Somjen and Gill, 1963; Ngai, Hanks and Fahrie, 1965; de Jong, Hershey and Wagman, 1967; Galindo, 1969; Angel, 1980). Since the concentrations of enflurane required to decrease cerebellar cGMP content are smaller than those needed to diminish myoneural transmission and muscle twitch in vivo and in vitro (Lebowitz, Blitt and Walts, 1970; Waud and Waud, 1975, 1979), it would seem that the effect of enflurane on cerebellar neuronal activity may play a role in the decrease in muscle tone and motor activity produced by enflurane.

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84 Woo, S. Y., Vcrosky, M., Vulliemoz, Y., and Triner, L. (1979). Dopamine-sensitive adenylate cyclase activity in rat caudate nucleus during exposure to halothane and enflurane. Antsthesiology, 51, 27.

Young, A. B., and Snyder, S. H. (1973). Strychnine binding associated with glycine receptors of the central nervous system. Pwc. Nat. Acad. Sex. U.S.A., 70, 2832.

RESUME

Nous avons etudie l'effet de renflurane sur le contenu intracellulaire en mediateur 3', 5-guanosine monophosphate cyclique (cGMP) et sur l'activite mortice chez la souris. Des crises convulsives, utilisees comme stigmates d'augmentttion d'activite motrice, assodees a une augmentation du contenu c£re"belleux en cGMP ont 6t6 induites par l'isoniazide ou la picrotcurine. L'enflurane (0,28 a 1,68 vol%) a provoque une diminution reversible, dose-dependente du cGMP cerebelleux (d'environ 50% a 0,28 vol%) et a retard^ ou prtvenu a la fois l'augmentation du cGMP cerfbelleux et les convulsions induites par l'isoniazide. L'enflurane protegeait aussi contre les convulsions induites par la picrotoxine, mais pas contre les convulsions induites par la strychnine qui n'impUquent probablement pas de mecanismes cerebelleux. Ce* resultats indiquent que l'enflurane affecte les mecanismes cerebelleux qui contrdlent l'activite motrice et il est postule que cette action contribue a la diminution de tonus musculaire induite par l'enflurane.

DIE WIRKUNG VON ENFLURANE AUF DIE CEREBELLARE cGMP UND AUF DIE MOTORISCHE AKTTVITAT BEI DER MAUS ZUSAMMEKFASSUNG

Die Wirkung von Enflurane auf den Gehalt des Kleinhirns an intrazellularem mediatorischem, zyklischem 3,5-Guanosin-

EFECTO DEL ENFLURANO SOBRE EL cGMP CEREBELOSO Y LA ACTTVIDAD MOTRIZ EN LOS RATONES SUMARIO

En ratones, se estudio el efecto del enflurano sobre el contenido cerebeloso de monofosfato de guanosina-3',5' (cGMP) ciclico mediador intracelular y la actividad motriz. Se indujeron con isoniacida o picrotoxina crisis como indice del aumento de la actividad motriz, asociadas con un aumento del contenido cGMP cerebeloso. El enflurano, endosisde al 0,28 a 1,68 vol%, produjo una disminucion reversible dependiente de la dosis del cGMP cerebeloso (en un 50% aproximadamente al 0,28 vol%) y atraso o previno tanto el aumento en el cGMP cerebeloso como las convulsiones inducidas por la isoniacida. El enflurano tambien facilit6 una proteccion contra les convulsiones inducidas por la picrotoxina, pero no asi contra las convulsiones inducidas por la estricnina que, se supone, no involucran a los mecanismos cerebelosos. Estos resultados indican que el enflurano afecta los mecanismos cerebelosos que controlan la actividad motriz y se postula que esa accion contribuye al descenso del tono muscular inducido por el enflurano.

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EFFET DE L'ENFLURANE SUR LE cGMP CEREBELLEUX ET SUR L'ACTIVITE MOTRICE CHEZ LA SOURIS

monophosphat (cGMP) und auf die motorische Aktivitat wurde an Mausen untersucht. Anfalle, als Anzeichen von verstarkter motorischer Aktivitat in Verbindung mit erhdhtem Gehalt des Kleinhims an cGMP wurden mit Isoniazid oder Picrotoxin induziert. Enflurane 0,28—1,68 vol% rief einen dosisabhangigen, reversiblen Abfall der Zerebellaren cGMP (etwa 50% bei 0,28 vol%) hervor, und verzogerte bzw. verhinderte sowohl den Anstieg der zerebellaren cGMP als auch die durch Isoniazid hervorvorgerufenen Krampfe. Enflurane schutzte auch vor durch Picrotoxin induzierten Krfimpfen, aber nicht vor durch Strychnin induzierten Kr&mpfen, an denen unserer Annahmf nach keine zerebellaren Mechanismen beteiligt sind. Diese Ergebnisse zeigen, dafi Enflurane die zerebellaren Mechanismen beinfluSt, die die motorische Aktivitat steuern und es wird postuliert, dafi diese Wirkung beitragt zu dem durch Enflurane induzierten Abfall des Muskeltonus.