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Effects of the hypnotic drug etomidate in a model nervous system (buccal ganglia, helix pomatia)
Comp. Biochem. Physiol. Vol. 99C, No. 3, pp. 579-587, 1991 Printed in Great Britain
0306-4492/91 $3.00+ 0.00 © 1991 PergamonPresspie
EFFECTS OF THE HYPNOTIC D R U G ETOMIDATE IN A MODEL NERVOUS SYSTEM (BUCCAL GANGLIA,
HELIX POMATIA) U. ALTRUP,* A. LEHMENKOHLER'~and E.-J. SPECKMANN*t *Institut fiir Experimentelle Epilepsieforschung, Hfffferstr. 68, 4400 Mfinster, F.R.G.; and ~'Institut fiir Physiologie, Robert-Koch-Str. 27a, 4400 Miinster, F.R.G. (Telephone: (0251) 83-5530; Fax: (0251) 83-5551) (Received 3 December 1990)
Abstract--1. Effects of the hypnotic drug etomidate were studied with intracellular recordings of the identified neurons BI to B4 of the buecal ganglia of Helix pomatia. 2. At threshold doses of 10#mol/l, etomidate mainly affected interneuronal networks. 3. In concentrations above 200pmol/l, the drug induced typical epileptic activities (paroxysmal depolarization shifts, PDS). Neurons BI to B4 generated epileptic aetivites in differential concentration ranges. PDS were synchronized via electrical contacts. PDS could be blocked by the "calcium antagonist" verapamil but not by a block of chemical synaptic transmission. 4. In comparison with the epileptogenic drug pentylenetetrazol, effective doses of etomidat[ to induce PDS were about 100 times lower.
INTRODUCTION The hypnotic drug etomidate (ethyl-l-(alpha-methylbenzyl) imidazole-5-carboxylate) is often used in man for the induction of short-term narcosis. However, basic mechanisms of action of the drug are widely unknown. Elementary mechanisms underlying its effects are of special interest since it has been shown several times that etomidate-induced hypnosis can be accompanied by epileptic activities (Cohn et al., 1983; Krieger et al., 1985). Thus, epileptiform activities are often observed with long-term application of etomidate as a sedative (Grant and Hutchinson, 1983; Nickel and Schmickaly, 1985) and with treatment with etomidate for epilepsy surgery (Ebrahim et al., 1986). The present paper describes mechanisms of action of etomidate in the isolated buccal ganglia of Helix pomatia. The ganglia are used as a model nervous system since (i) they can be studied easily on a cellular level using microelectrodes, (ii) the ganglia contain identified cell individuals, neurons B l, B2, B3 and B4, and (iii) the known principles of function of nervous systems are also realized in these relatively "simple" ganglia. Thus the ganglia offer the opportunity of recording electrical activities from neurons which have known general and special properties as, for example, characteristics or synaptic inputs of known origin and meaning (Schulze et al., 1974; Altrup and Speckmann, 1982; Peters and Altrup, 1984; Altrup, 1987; Altrup et al., 1990). Furthermore, the ganglia have proved useful in experimental epileptology (Speckmann and Caspers, 1973; Altrup and Speckmann, 1988; Walden et at., 1988; Altrup et al., 1989; Madeja et al., 1990). *Please address correspondence to Dr U. Altrup.
The aim of the present investigation was to analyze the effects of etomidate especially as far as the hypnotic and epileptogenic effects are concerned. To this end the following were studied: (i) the spontaneous activity of neuronal networks ("feeding" depolarizations, synchronization of activities) as well as membrane properties of single neurons (resting membrane potential, membrane resistance, action potentials), and (ii) induction of epileptic activities (changes of the ionic microenvironment, comparison with known epileptogenic drugs). With these investigations the actions of different concentrations of the drug were especially taken into account. MATERIALSAND METHODS The buccal ganglia of Helix pomatia including its nerves and cerebrobuccal connectives were separated from the animal and mounted in an experimental chamber. The connective tissue covering the isolated ganglia was dissected, except for the rigid inner sheath which maintained ganglionic integrity. No proteolytic enzymes were used (Altrup et al., 1980). Several neurons were recorded simultaneously using conventional intracellular mieroelectrodes. Membrane potential was shifted by current injections via a second intracellular electrode. The experimental chamber was continuously perfused (ca. 3 mi/min, volume of the chamber: 2 ml) with a solution containing NaCI: 130; KCh 4.5; CaCI2: 9; Triw-Ch 5 mmol/1. Temperature was adjusted to 20°C and pH to 7.4. Etomidate was added to the control bath solution in concentrations of 1-1000/~mol/1. In some experiments C.aCI2 was reduced to 2 mmol/l and MgCI2 was added to give 9 mmol/l ("high Mg2+, low Ca2+ solution"). The organic "calciumantagonist" verapamil ( 10-100/~ mol/l) or the epileptogenic drug pentylenetetrazol (10-60 mmol/1) were added to the solutions without osmotic compensation. Extracellular potassium concentration was measured with ion sensitive microelectrodes based on the potassium ionophore
579
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I-cocktail B from Fluka (No. 60398). The results are based on a total number of ca. 200 intracellularly recorded cells treated with etomidate.
RESULTS With application of etomidate to the bath solution several effects were observed in the neurons depending on the concentration of the drug (Figs l and 3). (i) The normal activity of intraganglionic networks and membrane properties of the studied neurons changed with a threshold concentration of the drug of about l0/tmol/l. (ii) Epileptic discharges occurred with drug concentrations of above 200 gmol/1. These differential effects are described subsequently (cf. also Altrup et al., 1990). Changes o f normal network activities and membrane properties
Interneuronal networks in the buccal ganglia of different snails are well known (cf. Benjamin et al., 1985). In Helix pomatia there are at least two spontaneously active networks. They evoke typical synaptic responses in the neurons BI through B4 (Altrup et al., 1979; 1990; Altrup and Speckmann, 1982; 1984; Peters and Altrup, 1984; Altrup, 1987). Consequently, the activity of the networks can be monitored through the membrane potential of the identified neurons mentioned. The first network depolarizes neurons Bl through B4 nearly simultaneously (of. Altrup et al., 1990). In the isolated ganglia the depolarizations occurred at a rate of about 1/min (Fig. l, first line). These spontaneous inputs to neurons B1 through B4 were de-
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pressed by etomidate. At threshold concentrations (ca. 10 #mol/1) this depression reversed within some minutes of washing. It outlasted the application for more than 1 hr when higher concentrations (above 100 #mol/l) were applied. The second intraganglionic network evokes a typical long-lasting hyperpolarization in neuron B1, depolarization in neuron B2, depolarization mostly followed by a hyperpolarization in neuron B3 and a suppression of action potentials in neuron B4 (Altrup and Speckmann, 1982; 1984; Altrup, 1987; Altrup et al., 1990). These simultaneously appearing fluctuations in neurons Bl through B4 were found only occasionally under control conditions. During etomidate application they occurred regularly (Fig. 2). With increasing etomidate concentration, frequency of occurrence of fluctuations increased. In the higher concentration range the fluctuations in neurons BI through B3 became intermingled with epileptic discharges. Thus, the described depolarizations in neurons B2 and B3 could trigger epileptic discharges and the hyperpolarizations in neuron BI could prevent them. As a whole, with increasing concentration of etomidate, the first effects consisted of a conversion of network activities in that the main excitatory synaptic inputs were depressed and the normally silent ones were activated. In neurons B1 through B4 which are efferent with respect to the aforementioned intraganglionic networks, changes in membrane properties became apparent at etomidate concentrations of higher than 10#mol/l. Generally, in a dose-dependent manner, membrane potential depolarized transiently for up to 5 mV at first. Then it turned into a permanent and
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Fig. 2. Effects of etomidate (100/imol/l) on neuronal networks in the buccal ganglia of Helix pomatia. Simultaneous recordings of the membrane potential of neurons B1 to B4. Numbers and arrows in the inkwriter recordings in A point to fluctuations of membrane potential shown in an extended time scale in B. Bar and dotted lines mark duration of etomidate application in A. small depolarization or hypcrpolarization of up to 5 mV (Fig. 1). These membrane potential changes did not depend upon the initial membrane potential nor on the type of neuron. They also appeared in
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Fig. 3. Fluctuations of membrane potential of neurons BI to B4 in the buccal ganglia of Helix pomatia under control conditions and during etomidate application. Concentration of the substance are indicated. The 4 recordings in each line are simultaneously recorded. Action potentials are truncated by the recording system.
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Fig. 4. Effects of "high Mg, low Ca" solution on synaptically evoked spontaneous depolarizations under control conditions (control) and during etomidate induced epileptic activity (etomidate, 700 #mol/l) in neuron B3 of the buccal ganglia of Helix pomatia. date for up to 50% of its initial value. It re-decreased with washing, showing a slight undershoot. With higher etomidate concentration (above 100 #mol/l), width of action potentials increased with the amplitude being hardly changed and after-hyperpolarizations decreased. Appearance o f epileptic activity With concentration of etomidate of above 200 #mol/l epileptic discharges occurred. They were intermingled with the effects of etomidate described for lower concentrations in the above chapter. Concerning the induction of epileptic activities, neurons BI to 134 were differentially sensitive to etomidate (Fig. 3). Neuron B3 generated typical paroxysmal depolarization shifts (PDS; Goldenson and Purpura, 1963, Matsumoto and Ajmone Marsan, 1964, Chalazonitis and Takeuchi, 1968) at concentrations of above 200 #mol/l and neurons B1 and B2 at concentrations of above 400 #mol/l. In neuron B4 epileptiform activities were recorded in the higher concentration range of ca. 1000 #mol/1. They consisted of small depolarizations as compared with those of the other neurons. In all neurons, the
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epileptogenic effects of etomidate declined over ca. 30 min after washing with control solution. The epileptic activities induced by etomidate were studied in several respects: (a) elementary mechanisms underlying PDS, (b) synchronization of epileptic activities in the ganglia and (c) comparison of the effects of etomidate with those of the well-known epileptogenic substance pentylenetetrazol. (a) Elementary mechanisms underlying etomidate induced PDS. As already mentioned above, PDS in neurons B1 through B3 were sometimes found to be triggered by synaptic inputs from the intraganglionic networks (cf. Fig. 3, B3, 300 #tool/l). These typical synaptic inputs have been shown elsewhere to be chemically mediated since they can be blocked by the application of a "high Mg, low Ca solution" (Fig. 4, upper line; Altrup and Speckmann, 1984). Therefore, in the present study it was tested whether or not a "high Mg, low Ca solution" was able to block PDS. As shown in Fig. 4 (lower line) the blockade of chemical synaptic transmission failed to suppress the appearance of PDS. In these cases frequency of PDS often increased especially with higher concentrations of etomidate. This effect may
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Effects of etomidate on buccal ganglia be interpreted as to be due to a suppression of synaptic inhibition or to a predominance of non-inactivating depolarizing currents with respect to calcium dependent repolarizing currents (Walden et al., 1988, Lficke et al., 1990). The dependence of the occurrence and shape of PDS on the actual resting membrane potential was studied in a further series of experiments. When membrane potential of a neuron was shifted to higher levels by current injection into the soma via a second intracellular electrode, frequency of occurrence of PDS decreased (Fig. 5, 1-3). During the first hyperpolarizing steps the amplitude of PDS was increased. Apparently, the increase in amplitude mainly resulted from the fact that the initial membrane potential increased due to the current injection whereas the plateau of the PDS remained at the same ceiling level of membrane potential (cf. Witte et al., 1985). With further hyperpolarizing current injection (Fig. 5, 4-6) the PDS failed abruptly. Steady-state depolarization of the neurons exerted the inverse effects. In further experiments it was tested whether the organic "calcium-antagonist" verapamil can block etomidate-induced PDS as has been shown for PDS induced by other epileptogenic drugs in vertebrate and in invertebrate neurons (cf. Speckman et al., 1986). A typical recording is shown in Fig. 6. The bath solution contained 500/zmol/1 etomidate resulting in PDS in neuron B3 (upper recordings in Fig. 6A) whereas the spontaneous control activity of neuron B4 was only slightly changed. The addition of
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verapamil to the bath solution decreased the PDS duration until only action potentials remained whereas no effects were encountered in the B4 neuron which showed no PDS. The verapamil-induced depression of PDS could be washed out within several hours. (b) Synchronization o f epileptic activities. Simultaneous recordings from several neurons showed that the induced PDS appeared synchronized in typical neuronal aggregates. Thus, in the neurons B3, which have been shown to be electrically coupled via interneurons (Madeja et al., 1989), the PDS occurred locked in time. As has already been shown for pentylenetratrazol-induced PDS (Madeja et al., 1989), the coupling in time of etomidate-induced PDS was based on a small "coupling depolarization" induced in the "follower-neuron" by the PDS in a "leader-neuron" (Fig. 7A). Within the electrically coupled network the most depolarized neuron was the "leader-neuron". PDS were also synchronized in both neurons B2 (Fig. 7B) known to be directly coupled electrically (Altrup, 1987). Simultaneous recordings from several motoneurons revealed, furthermore, that motoneurons also generated PDS coupled in time. However, recordings from a motoneuron, a neuron B2, and a neuron B3 showed that there was no synchronization of PDS among these networks beside the above-mentioned occasional triggering of PDS by the intraganglionic networks. Different neuronal networks within the buccal ganglia were found to generate PDS synchronized only within the proper network. Therefore, some
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5s Fig. 7. Simultaneously recorded epileptic discharges in several neurons of the buccal ganglion of Helix pomatia during application of etomidate (600 #mol/1). A: neurons B3 and neuron B2 from the left (1) and right (r) ganglion. B: neurons B2. Arrows and dotted lines indicate coupling in time of discharges. experiments were done to clarify the question whether or not the intraganglionic K+-concentration contributes to synchronization (cf. Yaari et al., 1986). For this purpose, intraganglionic K+-concen tration was measured via ion selective microelectrodes during etomidate induced epileptic activities. Measurements showed that, following PDS in the motoneurons, there was an increase in intraganglionic K+-concentration which, however, was mostly unable to synchronize the non-motoric neurons BI, B2 or B3 (Fig. 8A). In these cells the endogenous rhythms of PDS generation seemed to override the K+-induced depolarizations. To estimate the contribution of PDS in a single neuron to the increase of extracellular K+-concentration, K + was measured at the surface of the soma of the non-motoric neuron B3 (Fig. 8B, upper recording). The K+-changes were found to be below 0.1 mmol/l. This suggests that many motoneurons contributed to the extracellular K+-changes in the inner part of the ganglion (Fig. 8B, lower recording). As a whole, the K+-measurements led one to assume (i) that there was highly synchronized epileptic activity in the motoneuronal pools and (ii) that the synchronized PDS in the non-motoric B2 and B3 networks are not synchronized to the "mass-activity" in the ganglion via extracellular K+-changes.
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(c) Comparision o f actions o f etomidate and pentylenetetrazol. The experiments have shown that etomidate is obviously a valuable tool in experimental epileptology. Thus, it is of interest to compare the effects of etomidate with those of the well-known and commonly used epileptogenic drug pentylenetetrazol. Some differences in the effects of both drugs have been observed. (i) The effective doses of etomidate with respect to the induction of epileptic activities, were about 100 times lower than those for pentylenetetrazol. As can be derived from Fig. 3, concentrations of etomidate of between 0.2 and 0.5 mmol/l already induced PDS in a neuron B3 whereas with pentylenetetrazol ca. 40 mmol/1 of the drug were needed (Fig. 9). (ii) With the use of etomidate, only slight changes of membrane potential occurred whereas pentylenetetrazol application is generally accompanied by an enduring depolarization (Speckmann and Caspers, 1973). (iii) With the use of pentylenetetrazol, the activity changes of the intraganglionic networks appear to be less pronounced. (iv) The duration of PDS evoked by etomidate was shorter than the duration of PDS evoked by pentylenetetrazol (typically in the range of sec vs. min; Fig. 9). A mixture of the half-effective doses of both drugs induced PDS of unchanged amplitudes but of about medial duration.
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10s Fig. 8. Extracellular potassium concentration (K+)0 during etomidate-induced epileptic discharges of neurons of the buccal ganglia of Helix pomatia. Etomidate: 600 #mol/l. A: changes of 0K+)0 in the extracellular space of the ganglion simultaneously recorded to one unidentified motoneuron (MN) and to neuron B3. B: changes of (K+)0 on the surface of the soma of neuron B3 (upper recording) and in the ganglionic extracellular space (lower recording) simultaneously recorded to epileptic discharges in neuron B3 (middle recording).
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2rain (3oo ~ol/i) Fig. 9. Induction of epileptic discharges by pentylenetetrazol and etomidate in neuron B3 of the buceal ganglia of Helix pomatia. Effects of pentylenetetrazol and etomidate in effective doses (40 mmol/l, 600 #reel/l, respectively;left recordings), half-effectivedoses (20 retool/l, 300 #reel/l, respectively;middle recordings), and in a mixture of both half-effectivedoses (20 mmol/l plus 300/~mol/l; right recording). Inkwriter recordings. DISCUSSION
Bolus injections of the hypnotic drug etomidate are often used for the induction of narcosis in man. In these cases, etomidate concentrations are ca. 1-2#mol/kg body weight which is below the presently found threshold concentration of 10 #mol/I in neurons of the buccal ganglia of Helix pomatia. It should be mentioned, however, that with increasing etomidate concentration, the first observed effects concerned changes in the activities of networks of internenrons. Thus, etomidate may exert either effects on endogeneous properties of these neurons (of. Benjamin et al., 1985) or slight changes of, for example, membrane potential. These effects might accumulate to distinct changes in network activities and may explain the differences found in effective drug concentrations. With infusions of etomidate for sedation, concentration in man approaches the presently observed threshold concentration (cf. Nimmo and Miller, 1983). As a whole, a comparison of therapeutic drug concentrations in man with those on parts of the nervous system in/n vitro studies is generally difficult. Thus, it is wellknown that the same spectra of drug effects can be exerted in different nervous systems (of the same and of different animal species) using different spectra of drug concentrations. As to the elementary mechanisms underlying hypnosis, in the buccal ganglia of the mail, Helix pomat/a, etomidate inhibited the main excitatory drive at the low concentration range. In contrast to this, another ganglionic network which was normally inactive was activated by etomidate. Concerning the
functional meaning of this second network, Croll et al. (1985) showed that in the buccal ganglia of the snail, Pleurobranchaea, there are two networks which are responsible for egestion and for ingestion. The activity of these networks resembled those found in Helix pomatia (Altrup and Speckmann, 1982; Altrup et al., 1990. Applying the data about the networks in question in the different molluscs, etomidate may block the neuronal network responsible for ingestion and activate that for egestion. This might correspond to the often observed vomiting in man after etomidate-induced narcosis (cf. Nimmo and Miller, 1983). In the higher concentration ranges (above ca. 200 gruel/l), etomidate induced typical epileptic activities. Clinically applied etomidate was reported to be accompanied by increases in amplitudes of cortically evoked potentials which can be interpreted as increases in neuronal excitability (Kochs et al., 1986; McPherson et al., 1986). Furthermore, some patients were reported to show epileptiform activities with etomidate application. Thus, Grant and Hutchinson (1983) described 4 patients with generalized or focal activities 6-28 br after the beginning of etomidate infusion. Gaucher et al. (1984) found tonic seizures in 2 patients prepared for a surgical treatment of epilepsy, and Nickel and Schrnickaly (1985) observed increased probablity of seizures in patients with infusions of etomidate for the treatment of delirium tremens. In contrast to this, there are observations described in literature concerning antiepileptic effects of etomidate (cf. Hoffmann and Sehockenhoff, 1984; Thornton et al., 1985). These different observations may be explained by the effects of etomidate in different concentrations. In the lower ranges
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etomidate blocked the predominating synaptic drives in the ganglia, an effect which is in principle able to reduce the probability of epileptic discharges and to decrease neuronal synchronization. With increased concentrations, excitability of neurons increased, leading to epileptic activities especially when there was an increased seizure susceptibility. In Fig. 9, an increased seizure susceptibility is induced by the application of the drug pentylenetetrazol in a subthreshold concentration. F r o m this the effective doses of etomidate to induce epileptic activity was reduced in fact. As a whole, as is known from other drugs like, e.g. lidocaine (Brown, 1983), etomidate may exert antiepileptic and epileptogenic effects in the lower and higher concentration ranges, respectively. Comparing the PDS induced by pentylenetetrazol (Speckmann and Caspers, 1973; Madeja et al., 1989) with those during application of etomidate, the same properties are mostly encountered. Thus, (i) the identified neurons B1 to B4 show the same order of sensitivity against both drugs in that the B3 and B4 neurons are most and least sensitive, respectively; (ii) PDS induced by both drugs are synchronized within the same intraganglionic networks; (iii) PDS are of comparable amplitudes; (iv) they cannot be blocked with "high Mg 2+, low Ca 2+'' solutions; (v) they fail abruptly with increasing membrane potential, and (vi) they are blocked by the organic "calcium-antagonist" verapamil. The main difference between the PDS induced by these drugs consists in the duration, lasting mostly in the range of min vs. sec using pentylenetetrazol and etomidate, respectively. The experiments have finally shown that etomidate is a useful tool in experimental epileptology for the induction of epileptic activities. The main advantages of etomidate in comparison with pentylenetetrazol consists in (i) the relatively low doses needed and (ii) the little effects of etomidate on resting membrane potential.
REFERENCES Altrup U. (1987) Inputs and outputs of giant neurons B1 and B2 in the buccal ganglia of Helix pomatia. Brain Res. 414, 271-284. Altrup U., Lehmenkiihler A. and Speckmann E.-J. (1990) Wirkungen von Etomidat in einem Modellnervensystem (Buccalganglien, Helix pomatia ). In Epilepsie 89 (Edited by Wolf P.), pp. 149-153. Eihorn-Presse, Reinbek. Altrup U., Lehmenkiihler A., Madeja M. and Speckmann E.-J. (1990) Morphology and function of the identified neuron B3 in the buccal ganglia of Helix pomatia. ComP. Biochem. Physiol. 97A, 65-74. Altrup U., H6hn C. M., Schulze H., Speckmann E.-J., Kuhlmann D. and Nolte A. (1980) Effects of extracellularly applied proteolytic enzymes (Pronase) on electrophysiological activities of identified neurons in the buccal ganglia of Helix pomatia L. Comp. Biochem. Physiol. 67A, 1-7. Altrup U., Reith H. and Speckmann, E.-J. (1989) Wirkungen von Na-Valproat (VPA) auf Pentylentetrazolinduzierte epileptische Entladungen in einem Modellnervensystem (Helix pomatia, Buccalganglian). In Epilepsie 89 (Edited by Wolf P.), pp. 158-162. Altrup U. and Speckmann E.-J. (1982) Responses of identified neurons in the buccal ganglia of Helix pomatia to stimulation ofgangiionic nerves. Comp. Biochem. Physiol. 72A, 643-657.
Altrup U., and Speckmann E.-J. (1988) Epileptic discharges induced by pentylenetretrazol: changes of shape of dendrites. Brain Res. 456, 401-405. Altrup U., Speckmann E.-J. and Caspers H. (1979) Axonal pathways and synaptic inputs of three identified neurons in the buccal ganglion of Helix pomatia. Malacologia 18, 473-476. Benjamin P. R., Elliott C. J. H. and Ferguson G. P. (1985) Neural network analysis in the snail brain. In Model Neural Networks and Behavior (Edited by Selverston A. I.), pp. 87-108. Plenum Press, New York. Browne T. R. (1983) Paraldehyde, chlormethiazole, and lidocaine for treatment of status epilepticus. In Advances in Neurology (Edited by Delgado-Escueta A. V., Westerlain C. G., Treiman D. M. and Porter R. J.), pp. 509-517. Raven Press, New York. Chalazonitis N. and Takeuchi H. (1968) Amples oscillations du potentiel de membrane induites par le metrazol (neurons autoactifs d'Helix pomatis). C.R. Soc. Biol. 162, 1552-1556. Cohn B. F., Rejger V., Hagenouw-Taal J. C. and Voormolen J. H. (1983) Results of a feasability trial to achieve total immobilization of patients in a neurosurgical intensive care unit with etomidate. Anaesthesia 38, 47-50. Croll R. P., Davis W.J. and Kovac M. P. (1985) Neural mechanisms of motor program switching in the mollusc Pleurobranchaea. I. Central motor programs underlying ingestion, egestion and the neutral rhythm(s). J. Neurosci. 5, 48-55. Ebrahim Z. Y., DeBoer G. E., Luders H., Hahn J. F. and Lesser R. P. (1986) Effects of etomidate on the electroencephalogram of patients with epilepsy. Anesth. Analg. 65, 1004-1006. Gaucher S., Laxer K. D. and Krieger W. (1984) Activation of epileptogenic activity by etomidate. Anesthesiology 6, 616-618. Goldensohn E. S. and Purpura D. P. (1963) Intracellular potentials of cortical neurons during focal epileptogenic discharges. Science 139, 840-842. Nickel B. and Schmickaly R. (1985) Gesteigerte Anfallsbereitschaft unter Etomidatlangzeitinfusion beim Delirium tremens. Anaesthesist 34, 462-469. Grant I. S. and Hutchinson G. (1983) Epileptiform Seizures during prolonged etomidate sedation. Lancet 2, 511-512. Hoffmann P. and Schockenhoff B. (1984) Etomidate as an anti-convulsive agent. Anaesthesist 33, 142-144. Kochs E., Treede R. D. and Schulte am Esch J. (1986) Verg6Berung somatosensorisch evozierter Potentiale w/ihrend Narkoseeinleitung mit Etomidat. Anaesthesist 35, 359-364. Krieger W., Copperman J. and Laxer K. D. (1985) Seizures with etomidate anesthesia. Anesth. Analg. 64, 1226-1227. Lficke A., Speckmann E.-J., Altrup U., Lehmenkiihler A. and Walden J. (1990) Decrease of free calcium concentration at the outer surface of identified snail neurons during paroxysmal depolarization shifts. Neurosci. Lett. 112, 190-193. Madeja M., Altrup U. and Speckmann E.-J. (1989) Synchronization of epileptic discharges: temporal coupling of paroxysmal depolarizations in the buccal ganglia of Helix pomatia. Comp. Biochem. Physiol. 94C, 585-590. Matsumoto H. and Ajmone-Marsan C. (1964) Cortical cellular phenomena in experimental epilepsy: interictal manifestations. Exp. Neurol. 9, 286-304. McPherson R. W., Sell B. and Traystman R. J. (1986) Effects of thiopental, fentanyl, and etomidate on upper extremity somatosensory evoked potentials in humans. Anesthesiology 65, 584-589. Nickel B. and Schmickaly R. (1985) Increased tendency to seizures as affected by long-term infusions of etomidate in delirium tremens. Anaesthesists 24, 462-469. Nimmo W. S. and Miller M. (1983) Pharmacology of etomidate. Conterap. Anesth. Praet. 7, 83-95.
Effects of etomidate on buccal ganglia Peters M. and Altrup U. (1984) Motor organization in pharynx of Helix pomatia. J. Neurophysiol. 52, 389-409. Schulze H., Speckmann E.-J,, Kuhlmann D. and Caspers H. (1975) Topography and bioclectrical properties of identifiable neurons in the buccal ganglion of Helix pomatia. Neurosci. Lett. 1, 277-281. Speckmann E.-J. and Caspers H. (1973) Paroxysmal depolarization and changes in action potentials induced by pentylenetetrazol in isolated neurons of Helix pomatia. Epilepsia 14, 397-408. Spcckman E.-J., Schulze H. and Walden J. (1986) Epilepsy and Calcium. Urban und Schwarzenberg, Miinchen. Thornton C., Heneghan C. P. H., Navaratnarajah M., Batement P. E. and Jones J. G. (I 985) Effect of etomidate
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on the auditory evoked response in man. Br. J. Anaesth. 57, 554-561. Walden J., Speckmann E.-J. and Witte O. (1988) Membrane currents induced by pentylenetetrazol in identified neurons of Helix pomatia. Brain Res. 473, 294-205. Witte O., Speckmann E.-J. and Walden J. (1985) Acetylcholine responses of identified neurons in Helix pomatia. I. Interaction between acetylcholine-induced and potential-dependent membrane conductances. Comp. Biochem. Physiol. 80C, 15-23. Yaari Y., Konnert A. and Heinemann U. (1986) Nonsynaptic epileptogenesis in the mammalian hippocampus /n vitro. II. Role of extracellular potassium. J. Neurophysiol. 56, 424-438.
0306-4492/91 $3.00+ 0.00 © 1991 PergamonPresspie
EFFECTS OF THE HYPNOTIC D R U G ETOMIDATE IN A MODEL NERVOUS SYSTEM (BUCCAL GANGLIA,
HELIX POMATIA) U. ALTRUP,* A. LEHMENKOHLER'~and E.-J. SPECKMANN*t *Institut fiir Experimentelle Epilepsieforschung, Hfffferstr. 68, 4400 Mfinster, F.R.G.; and ~'Institut fiir Physiologie, Robert-Koch-Str. 27a, 4400 Miinster, F.R.G. (Telephone: (0251) 83-5530; Fax: (0251) 83-5551) (Received 3 December 1990)
Abstract--1. Effects of the hypnotic drug etomidate were studied with intracellular recordings of the identified neurons BI to B4 of the buecal ganglia of Helix pomatia. 2. At threshold doses of 10#mol/l, etomidate mainly affected interneuronal networks. 3. In concentrations above 200pmol/l, the drug induced typical epileptic activities (paroxysmal depolarization shifts, PDS). Neurons BI to B4 generated epileptic aetivites in differential concentration ranges. PDS were synchronized via electrical contacts. PDS could be blocked by the "calcium antagonist" verapamil but not by a block of chemical synaptic transmission. 4. In comparison with the epileptogenic drug pentylenetetrazol, effective doses of etomidat[ to induce PDS were about 100 times lower.
INTRODUCTION The hypnotic drug etomidate (ethyl-l-(alpha-methylbenzyl) imidazole-5-carboxylate) is often used in man for the induction of short-term narcosis. However, basic mechanisms of action of the drug are widely unknown. Elementary mechanisms underlying its effects are of special interest since it has been shown several times that etomidate-induced hypnosis can be accompanied by epileptic activities (Cohn et al., 1983; Krieger et al., 1985). Thus, epileptiform activities are often observed with long-term application of etomidate as a sedative (Grant and Hutchinson, 1983; Nickel and Schmickaly, 1985) and with treatment with etomidate for epilepsy surgery (Ebrahim et al., 1986). The present paper describes mechanisms of action of etomidate in the isolated buccal ganglia of Helix pomatia. The ganglia are used as a model nervous system since (i) they can be studied easily on a cellular level using microelectrodes, (ii) the ganglia contain identified cell individuals, neurons B l, B2, B3 and B4, and (iii) the known principles of function of nervous systems are also realized in these relatively "simple" ganglia. Thus the ganglia offer the opportunity of recording electrical activities from neurons which have known general and special properties as, for example, characteristics or synaptic inputs of known origin and meaning (Schulze et al., 1974; Altrup and Speckmann, 1982; Peters and Altrup, 1984; Altrup, 1987; Altrup et al., 1990). Furthermore, the ganglia have proved useful in experimental epileptology (Speckmann and Caspers, 1973; Altrup and Speckmann, 1988; Walden et at., 1988; Altrup et al., 1989; Madeja et al., 1990). *Please address correspondence to Dr U. Altrup.
The aim of the present investigation was to analyze the effects of etomidate especially as far as the hypnotic and epileptogenic effects are concerned. To this end the following were studied: (i) the spontaneous activity of neuronal networks ("feeding" depolarizations, synchronization of activities) as well as membrane properties of single neurons (resting membrane potential, membrane resistance, action potentials), and (ii) induction of epileptic activities (changes of the ionic microenvironment, comparison with known epileptogenic drugs). With these investigations the actions of different concentrations of the drug were especially taken into account. MATERIALSAND METHODS The buccal ganglia of Helix pomatia including its nerves and cerebrobuccal connectives were separated from the animal and mounted in an experimental chamber. The connective tissue covering the isolated ganglia was dissected, except for the rigid inner sheath which maintained ganglionic integrity. No proteolytic enzymes were used (Altrup et al., 1980). Several neurons were recorded simultaneously using conventional intracellular mieroelectrodes. Membrane potential was shifted by current injections via a second intracellular electrode. The experimental chamber was continuously perfused (ca. 3 mi/min, volume of the chamber: 2 ml) with a solution containing NaCI: 130; KCh 4.5; CaCI2: 9; Triw-Ch 5 mmol/1. Temperature was adjusted to 20°C and pH to 7.4. Etomidate was added to the control bath solution in concentrations of 1-1000/~mol/1. In some experiments C.aCI2 was reduced to 2 mmol/l and MgCI2 was added to give 9 mmol/l ("high Mg2+, low Ca2+ solution"). The organic "calciumantagonist" verapamil ( 10-100/~ mol/l) or the epileptogenic drug pentylenetetrazol (10-60 mmol/1) were added to the solutions without osmotic compensation. Extracellular potassium concentration was measured with ion sensitive microelectrodes based on the potassium ionophore
579
580
U.
ALTRUP
I-cocktail B from Fluka (No. 60398). The results are based on a total number of ca. 200 intracellularly recorded cells treated with etomidate.
RESULTS With application of etomidate to the bath solution several effects were observed in the neurons depending on the concentration of the drug (Figs l and 3). (i) The normal activity of intraganglionic networks and membrane properties of the studied neurons changed with a threshold concentration of the drug of about l0/tmol/l. (ii) Epileptic discharges occurred with drug concentrations of above 200 gmol/1. These differential effects are described subsequently (cf. also Altrup et al., 1990). Changes o f normal network activities and membrane properties
Interneuronal networks in the buccal ganglia of different snails are well known (cf. Benjamin et al., 1985). In Helix pomatia there are at least two spontaneously active networks. They evoke typical synaptic responses in the neurons BI through B4 (Altrup et al., 1979; 1990; Altrup and Speckmann, 1982; 1984; Peters and Altrup, 1984; Altrup, 1987). Consequently, the activity of the networks can be monitored through the membrane potential of the identified neurons mentioned. The first network depolarizes neurons Bl through B4 nearly simultaneously (of. Altrup et al., 1990). In the isolated ganglia the depolarizations occurred at a rate of about 1/min (Fig. l, first line). These spontaneous inputs to neurons B1 through B4 were de-
et
al.
pressed by etomidate. At threshold concentrations (ca. 10 #mol/1) this depression reversed within some minutes of washing. It outlasted the application for more than 1 hr when higher concentrations (above 100 #mol/l) were applied. The second intraganglionic network evokes a typical long-lasting hyperpolarization in neuron B1, depolarization in neuron B2, depolarization mostly followed by a hyperpolarization in neuron B3 and a suppression of action potentials in neuron B4 (Altrup and Speckmann, 1982; 1984; Altrup, 1987; Altrup et al., 1990). These simultaneously appearing fluctuations in neurons Bl through B4 were found only occasionally under control conditions. During etomidate application they occurred regularly (Fig. 2). With increasing etomidate concentration, frequency of occurrence of fluctuations increased. In the higher concentration range the fluctuations in neurons BI through B3 became intermingled with epileptic discharges. Thus, the described depolarizations in neurons B2 and B3 could trigger epileptic discharges and the hyperpolarizations in neuron BI could prevent them. As a whole, with increasing concentration of etomidate, the first effects consisted of a conversion of network activities in that the main excitatory synaptic inputs were depressed and the normally silent ones were activated. In neurons B1 through B4 which are efferent with respect to the aforementioned intraganglionic networks, changes in membrane properties became apparent at etomidate concentrations of higher than 10#mol/l. Generally, in a dose-dependent manner, membrane potential depolarized transiently for up to 5 mV at first. Then it turned into a permanent and
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Effects of etomidate on buccal ganglia A
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Fig. 2. Effects of etomidate (100/imol/l) on neuronal networks in the buccal ganglia of Helix pomatia. Simultaneous recordings of the membrane potential of neurons B1 to B4. Numbers and arrows in the inkwriter recordings in A point to fluctuations of membrane potential shown in an extended time scale in B. Bar and dotted lines mark duration of etomidate application in A. small depolarization or hypcrpolarization of up to 5 mV (Fig. 1). These membrane potential changes did not depend upon the initial membrane potential nor on the type of neuron. They also appeared in
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Fig. 3. Fluctuations of membrane potential of neurons BI to B4 in the buccal ganglia of Helix pomatia under control conditions and during etomidate application. Concentration of the substance are indicated. The 4 recordings in each line are simultaneously recorded. Action potentials are truncated by the recording system.
582
U. ALTRUPet al. control
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Fig. 4. Effects of "high Mg, low Ca" solution on synaptically evoked spontaneous depolarizations under control conditions (control) and during etomidate induced epileptic activity (etomidate, 700 #mol/l) in neuron B3 of the buccal ganglia of Helix pomatia. date for up to 50% of its initial value. It re-decreased with washing, showing a slight undershoot. With higher etomidate concentration (above 100 #mol/l), width of action potentials increased with the amplitude being hardly changed and after-hyperpolarizations decreased. Appearance o f epileptic activity With concentration of etomidate of above 200 #mol/l epileptic discharges occurred. They were intermingled with the effects of etomidate described for lower concentrations in the above chapter. Concerning the induction of epileptic activities, neurons BI to 134 were differentially sensitive to etomidate (Fig. 3). Neuron B3 generated typical paroxysmal depolarization shifts (PDS; Goldenson and Purpura, 1963, Matsumoto and Ajmone Marsan, 1964, Chalazonitis and Takeuchi, 1968) at concentrations of above 200 #mol/l and neurons B1 and B2 at concentrations of above 400 #mol/l. In neuron B4 epileptiform activities were recorded in the higher concentration range of ca. 1000 #mol/1. They consisted of small depolarizations as compared with those of the other neurons. In all neurons, the
A
1
2
epileptogenic effects of etomidate declined over ca. 30 min after washing with control solution. The epileptic activities induced by etomidate were studied in several respects: (a) elementary mechanisms underlying PDS, (b) synchronization of epileptic activities in the ganglia and (c) comparison of the effects of etomidate with those of the well-known epileptogenic substance pentylenetetrazol. (a) Elementary mechanisms underlying etomidate induced PDS. As already mentioned above, PDS in neurons B1 through B3 were sometimes found to be triggered by synaptic inputs from the intraganglionic networks (cf. Fig. 3, B3, 300 #tool/l). These typical synaptic inputs have been shown elsewhere to be chemically mediated since they can be blocked by the application of a "high Mg, low Ca solution" (Fig. 4, upper line; Altrup and Speckmann, 1984). Therefore, in the present study it was tested whether or not a "high Mg, low Ca solution" was able to block PDS. As shown in Fig. 4 (lower line) the blockade of chemical synaptic transmission failed to suppress the appearance of PDS. In these cases frequency of PDS often increased especially with higher concentrations of etomidate. This effect may
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5s Fig. 5. Effects of increased membrane potential on epileptic discharges in neuron B3 of the buccal ganglia of Helix pomatia. Hyperpolarization by current injection via a second intracellular microelectrode. Low (A) and high (B) resolution of time base. The tracings in A and B are related to each other by numbers.
Effects of etomidate on buccal ganglia be interpreted as to be due to a suppression of synaptic inhibition or to a predominance of non-inactivating depolarizing currents with respect to calcium dependent repolarizing currents (Walden et al., 1988, Lficke et al., 1990). The dependence of the occurrence and shape of PDS on the actual resting membrane potential was studied in a further series of experiments. When membrane potential of a neuron was shifted to higher levels by current injection into the soma via a second intracellular electrode, frequency of occurrence of PDS decreased (Fig. 5, 1-3). During the first hyperpolarizing steps the amplitude of PDS was increased. Apparently, the increase in amplitude mainly resulted from the fact that the initial membrane potential increased due to the current injection whereas the plateau of the PDS remained at the same ceiling level of membrane potential (cf. Witte et al., 1985). With further hyperpolarizing current injection (Fig. 5, 4-6) the PDS failed abruptly. Steady-state depolarization of the neurons exerted the inverse effects. In further experiments it was tested whether the organic "calcium-antagonist" verapamil can block etomidate-induced PDS as has been shown for PDS induced by other epileptogenic drugs in vertebrate and in invertebrate neurons (cf. Speckman et al., 1986). A typical recording is shown in Fig. 6. The bath solution contained 500/zmol/1 etomidate resulting in PDS in neuron B3 (upper recordings in Fig. 6A) whereas the spontaneous control activity of neuron B4 was only slightly changed. The addition of
A
B
control
1
3
2
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583
verapamil to the bath solution decreased the PDS duration until only action potentials remained whereas no effects were encountered in the B4 neuron which showed no PDS. The verapamil-induced depression of PDS could be washed out within several hours. (b) Synchronization o f epileptic activities. Simultaneous recordings from several neurons showed that the induced PDS appeared synchronized in typical neuronal aggregates. Thus, in the neurons B3, which have been shown to be electrically coupled via interneurons (Madeja et al., 1989), the PDS occurred locked in time. As has already been shown for pentylenetratrazol-induced PDS (Madeja et al., 1989), the coupling in time of etomidate-induced PDS was based on a small "coupling depolarization" induced in the "follower-neuron" by the PDS in a "leader-neuron" (Fig. 7A). Within the electrically coupled network the most depolarized neuron was the "leader-neuron". PDS were also synchronized in both neurons B2 (Fig. 7B) known to be directly coupled electrically (Altrup, 1987). Simultaneous recordings from several motoneurons revealed, furthermore, that motoneurons also generated PDS coupled in time. However, recordings from a motoneuron, a neuron B2, and a neuron B3 showed that there was no synchronization of PDS among these networks beside the above-mentioned occasional triggering of PDS by the intraganglionic networks. Different neuronal networks within the buccal ganglia were found to generate PDS synchronized only within the proper network. Therefore, some
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B4r ~ Fig. 6. Simultaneous recordings of membrane potential of neurons B3 and B4 of the right (r) buccal ganglion of Helix pomatia during etomidate appfication (A, C; control) and during application of etomidate and verapamil (B). Etomidate: 500 pmol/1, verapamil: 50 pmol/l. The recordings in A and B are related to each other by numbers. The inkwriter recordings (upper lines) are interrupted for I0 min, 5 min and 3 hr.
584
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5s Fig. 7. Simultaneously recorded epileptic discharges in several neurons of the buccal ganglion of Helix pomatia during application of etomidate (600 #mol/1). A: neurons B3 and neuron B2 from the left (1) and right (r) ganglion. B: neurons B2. Arrows and dotted lines indicate coupling in time of discharges. experiments were done to clarify the question whether or not the intraganglionic K+-concentration contributes to synchronization (cf. Yaari et al., 1986). For this purpose, intraganglionic K+-concen tration was measured via ion selective microelectrodes during etomidate induced epileptic activities. Measurements showed that, following PDS in the motoneurons, there was an increase in intraganglionic K+-concentration which, however, was mostly unable to synchronize the non-motoric neurons BI, B2 or B3 (Fig. 8A). In these cells the endogenous rhythms of PDS generation seemed to override the K+-induced depolarizations. To estimate the contribution of PDS in a single neuron to the increase of extracellular K+-concentration, K + was measured at the surface of the soma of the non-motoric neuron B3 (Fig. 8B, upper recording). The K+-changes were found to be below 0.1 mmol/l. This suggests that many motoneurons contributed to the extracellular K+-changes in the inner part of the ganglion (Fig. 8B, lower recording). As a whole, the K+-measurements led one to assume (i) that there was highly synchronized epileptic activity in the motoneuronal pools and (ii) that the synchronized PDS in the non-motoric B2 and B3 networks are not synchronized to the "mass-activity" in the ganglion via extracellular K+-changes.
A
(c) Comparision o f actions o f etomidate and pentylenetetrazol. The experiments have shown that etomidate is obviously a valuable tool in experimental epileptology. Thus, it is of interest to compare the effects of etomidate with those of the well-known and commonly used epileptogenic drug pentylenetetrazol. Some differences in the effects of both drugs have been observed. (i) The effective doses of etomidate with respect to the induction of epileptic activities, were about 100 times lower than those for pentylenetetrazol. As can be derived from Fig. 3, concentrations of etomidate of between 0.2 and 0.5 mmol/l already induced PDS in a neuron B3 whereas with pentylenetetrazol ca. 40 mmol/1 of the drug were needed (Fig. 9). (ii) With the use of etomidate, only slight changes of membrane potential occurred whereas pentylenetetrazol application is generally accompanied by an enduring depolarization (Speckmann and Caspers, 1973). (iii) With the use of pentylenetetrazol, the activity changes of the intraganglionic networks appear to be less pronounced. (iv) The duration of PDS evoked by etomidate was shorter than the duration of PDS evoked by pentylenetetrazol (typically in the range of sec vs. min; Fig. 9). A mixture of the half-effective doses of both drugs induced PDS of unchanged amplitudes but of about medial duration.
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10s Fig. 8. Extracellular potassium concentration (K+)0 during etomidate-induced epileptic discharges of neurons of the buccal ganglia of Helix pomatia. Etomidate: 600 #mol/l. A: changes of 0K+)0 in the extracellular space of the ganglion simultaneously recorded to one unidentified motoneuron (MN) and to neuron B3. B: changes of (K+)0 on the surface of the soma of neuron B3 (upper recording) and in the ganglionic extracellular space (lower recording) simultaneously recorded to epileptic discharges in neuron B3 (middle recording).
Effects of etomidate on buccal ganglia
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(600 ~.ol/l)
2rain (3oo ~ol/i) Fig. 9. Induction of epileptic discharges by pentylenetetrazol and etomidate in neuron B3 of the buceal ganglia of Helix pomatia. Effects of pentylenetetrazol and etomidate in effective doses (40 mmol/l, 600 #reel/l, respectively;left recordings), half-effectivedoses (20 retool/l, 300 #reel/l, respectively;middle recordings), and in a mixture of both half-effectivedoses (20 mmol/l plus 300/~mol/l; right recording). Inkwriter recordings. DISCUSSION
Bolus injections of the hypnotic drug etomidate are often used for the induction of narcosis in man. In these cases, etomidate concentrations are ca. 1-2#mol/kg body weight which is below the presently found threshold concentration of 10 #mol/I in neurons of the buccal ganglia of Helix pomatia. It should be mentioned, however, that with increasing etomidate concentration, the first observed effects concerned changes in the activities of networks of internenrons. Thus, etomidate may exert either effects on endogeneous properties of these neurons (of. Benjamin et al., 1985) or slight changes of, for example, membrane potential. These effects might accumulate to distinct changes in network activities and may explain the differences found in effective drug concentrations. With infusions of etomidate for sedation, concentration in man approaches the presently observed threshold concentration (cf. Nimmo and Miller, 1983). As a whole, a comparison of therapeutic drug concentrations in man with those on parts of the nervous system in/n vitro studies is generally difficult. Thus, it is wellknown that the same spectra of drug effects can be exerted in different nervous systems (of the same and of different animal species) using different spectra of drug concentrations. As to the elementary mechanisms underlying hypnosis, in the buccal ganglia of the mail, Helix pomat/a, etomidate inhibited the main excitatory drive at the low concentration range. In contrast to this, another ganglionic network which was normally inactive was activated by etomidate. Concerning the
functional meaning of this second network, Croll et al. (1985) showed that in the buccal ganglia of the snail, Pleurobranchaea, there are two networks which are responsible for egestion and for ingestion. The activity of these networks resembled those found in Helix pomatia (Altrup and Speckmann, 1982; Altrup et al., 1990. Applying the data about the networks in question in the different molluscs, etomidate may block the neuronal network responsible for ingestion and activate that for egestion. This might correspond to the often observed vomiting in man after etomidate-induced narcosis (cf. Nimmo and Miller, 1983). In the higher concentration ranges (above ca. 200 gruel/l), etomidate induced typical epileptic activities. Clinically applied etomidate was reported to be accompanied by increases in amplitudes of cortically evoked potentials which can be interpreted as increases in neuronal excitability (Kochs et al., 1986; McPherson et al., 1986). Furthermore, some patients were reported to show epileptiform activities with etomidate application. Thus, Grant and Hutchinson (1983) described 4 patients with generalized or focal activities 6-28 br after the beginning of etomidate infusion. Gaucher et al. (1984) found tonic seizures in 2 patients prepared for a surgical treatment of epilepsy, and Nickel and Schrnickaly (1985) observed increased probablity of seizures in patients with infusions of etomidate for the treatment of delirium tremens. In contrast to this, there are observations described in literature concerning antiepileptic effects of etomidate (cf. Hoffmann and Sehockenhoff, 1984; Thornton et al., 1985). These different observations may be explained by the effects of etomidate in different concentrations. In the lower ranges
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etomidate blocked the predominating synaptic drives in the ganglia, an effect which is in principle able to reduce the probability of epileptic discharges and to decrease neuronal synchronization. With increased concentrations, excitability of neurons increased, leading to epileptic activities especially when there was an increased seizure susceptibility. In Fig. 9, an increased seizure susceptibility is induced by the application of the drug pentylenetetrazol in a subthreshold concentration. F r o m this the effective doses of etomidate to induce epileptic activity was reduced in fact. As a whole, as is known from other drugs like, e.g. lidocaine (Brown, 1983), etomidate may exert antiepileptic and epileptogenic effects in the lower and higher concentration ranges, respectively. Comparing the PDS induced by pentylenetetrazol (Speckmann and Caspers, 1973; Madeja et al., 1989) with those during application of etomidate, the same properties are mostly encountered. Thus, (i) the identified neurons B1 to B4 show the same order of sensitivity against both drugs in that the B3 and B4 neurons are most and least sensitive, respectively; (ii) PDS induced by both drugs are synchronized within the same intraganglionic networks; (iii) PDS are of comparable amplitudes; (iv) they cannot be blocked with "high Mg 2+, low Ca 2+'' solutions; (v) they fail abruptly with increasing membrane potential, and (vi) they are blocked by the organic "calcium-antagonist" verapamil. The main difference between the PDS induced by these drugs consists in the duration, lasting mostly in the range of min vs. sec using pentylenetetrazol and etomidate, respectively. The experiments have finally shown that etomidate is a useful tool in experimental epileptology for the induction of epileptic activities. The main advantages of etomidate in comparison with pentylenetetrazol consists in (i) the relatively low doses needed and (ii) the little effects of etomidate on resting membrane potential.
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