superfused the surface of the preparations, the population e.p.s.p.s and required for nerve impulse generation. Thus, the decrease of the discharige

Transcription

1 J. Physiol. (1975), 248, pp With 9 text-figures Printed in Great Britain THE ACTION OF ETHER AND METHOXYFLURANE ON SYNAPTIC TRANSMISSION IN ISOLATED PREPARATIONS OF THE MAMMALIAN CORTEX By C. D. RICHARDS, W. J. RUSSELL* AND J. C. SMAJEt From the National Institute for Medical Research, Mill Hill, London N W7 IAA (Received 25 September 1974) SUMMARY 1. The actions of ether and methoxyflurane on the evoked potentials of in vitro preparations of the guinea-pig olfactory cortex were studied. Following stimulation of the lateral olfactory tract (l.o.t.) evoked potentials could be recorded from the cortical surface; these potentials consisted of an initial wave (the compound action potential of the l.o.t.) followed by a negative field potential which was associated with the synchronous excitation of many superficial excitatory synapses (population e.p.s.p.). Superimposed on the population e.p.s.p. was a number of positive peaks. These positive peaks reflect the synchronous discharge of many neurones and so have been called population spikes. 2. When ether or methoxyflurane was added to the gas stream that superfused the surface of the preparations, the population e.p.s.p.s and population spikes were depressed at lower concentrations than those required to depress the compound action potential of the afferent fibres. 3. The evoked activity of individual cells in the cortex was depressed by ether and methoxyflurane. However, five of the twelve cells tested in ether showed an increase in their evoked activity at concentrations below 4-5 %, but at higher concentrations these cells also became depressed. 4. Both ether and methoxyflurane depressed the sensitivity of cortical neurones to iontophoretically applied L-glutamate and may similarly depress the sensitivity of the post-synaptic membrane to the released transmitter substance. 5. Neither anaesthetic appeared to increase the threshold depolarization required for nerve impulse generation. Thus, the decrease of the discharige * Wellcome Research Fellow. Permanent address: Departinetit of Anaestlhetics, Royal Postgraduate Medical School, DuCane Road, London, W I 2 t MRC Junior Research Fellow.

2 122 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE of the post-synaptic cells was primarily caused by a depression of chemical transmission. 6. Ether caused some cells in the cortex to alter their normal pattern of synaptically evoked discharge and both anaesthetics induced similar changes during excitation by glutamate. INTRODUCTION Studies of the anaesthetic action of diethyl ether have shown that it depresses the synaptic potentials of sympathetic ganglia at lower concentrations than those required to interfere with the conduction of impulses along the afferent nerve fibres (Edwards & Larrabee, 1955; Larrabee & Posternak, 1952). Similarly, ether has been shown to depress the monosynaptic excitatory post-synaptic potentials (e.p.s.p.s) of motoneurones in the spinal cord (Somjen, 1967; Somjen & Gill, 1963). However, the action of ether on synaptic structures within the brain has not been examined in detail, and yet the brain is thought to be the main site of action of anaesthetics. Isolated preparations of the olfactory cortex of guinea-pigs have already been used to study the mode of action of pentobarbitone and halothane on synaptic transmission within the cortex (Richards, 1972b, 1973b). Such in vitro preparations are convenient for the following reasons; first, they can be studied both in the presence and absence of anaesthetic, thus ensuring a valid comparison between the control and anaesthetic-treated states; secondly, the preparations are electrically silent unless they are artificially stimulated, thus control over the electrical activity of the nerve cells they contain is possible; thirdly, the electrical potentials elicited in these preparations are remarkably stable and relatively easy to interpret; fourthly, known concentrations of anaesthetic can be administered directly to the nerve cells under investigation. Therefore it seemed worth while to examine the effects of diethyl ether ('ether') and methoxyflurane (2,2 dichloro, 1,1 difluoroethyl methyl ether) on synaptic transmission in isolated preparations of the mammalian olfactory cortex. The experiments described in this paper were undertaken to answer the following questions: first, do these anaesthetic ethers depress excitatory synaptic transmission in isolated preparations of the olfactory cortex at or below those concentrations likely to be found in the intact brain during anaesthesia? Secondly, if so, what are the mechanisms whereby the depression is brought about? Thirdly, are there any differences in the action of these anaesthetics on synaptic transmission?

3 ETHER ANAESTHESIA 123 METHODS Guinea-pigs were killed by a blow on the back of the neck; the skull was opened and the brain removed after section of the olfactory bulbs. To prepare the thin slice of olfactory cortex, a block of brain was cut in such a way that the curvature of the olfactory region was reduced. This block of brain was then laid flat on a small platform, and a thin tangential slice was taken from the surface of the olfactory cortex (see Fig. 1) with the aid of a razor strip and a glass template. The slices had a nominal thickness of 410 /zm and were incubated at C in the chamber described by Dore & Richards (1974). Stimulation of the slices was achieved by placing a pair of silver ball electrodes across the anterior end of the olfactory tract. The stimulation pulses were derived from an isolated stimulator driven by a Digitimer at Hz. The evoked field potentials were recorded monopolarly from the surface of the preparations by glass micropipettes (1-2 pm tip diameter. 1-2 Mn resistance) filled with 20 % (w/v) NaCl; the indifferent electrode was placed in the saline that bathed the lower surface of the slice. (The upper surface was exposed to a humidified atmosphere containing 95 % 02, 5% CO2.) The recording electrode was coupled to a voltage follower through Ag-AgCl wires which was in turn connected to an oscilloscope and a FM tape recorder. The overall flat band width of the recording system extended from 3 Hz to beyond 2-5 khz. Saline solution. The saline solutions used to bathe the preparations had the following composition: control saline, NaCl, 134 mm; KCl, 5 mm; KH2PO4, 1 25 mm; MgSO4, 2 mm; CaC12, 1 mm; NaHCO3, 16 mm; glucose, 10 mm; high Mg saline, NaCl 134 mm; KCl, 5 mm; KH2PO4, 1 25 mm; Mg 04, 10 mm; CaCl2, 1 mm; NaHCO3, 16 mm; glucose 10 mm. The solutions were saturated with a mixture of 95 % 02 and 5% C02 before use and had a ph of Iontophoresi&. Conventional five-barrelled electrodes were used, three of which contained 20 % (w/v) NaCl, the other two contained 0 5 M Na L-glutamate (ph 7-1). One NaCl-filled barrel was used to record the spike discharges while another served to balance out the backing and driving currents through the electrode. This barrel also served as a control to check for artifacts caused by the passage of current. A backing current could be applied to both of the glutamate-containing barrels to prevent diffusion of the drug from the electrode tip. The electrodes had overall tip diameters of 3-8 pm. Conventional procedures were followed to isolate the responses of individual neurones to glutamate; thereafter glutamate pulses were applied to the cell in a fixed schedule from one barrel; the pulse duration and the ejecting current remained constant throughout each trial. Application of anae8thetic8 to the preparations. The anaesthetics were applied to the preparations in the gas phase: part of the 02: CO2 gas mixture used to superfuse the slice was passed through a Drechsel bottle containing liquid anaesthetic; the vapourladen gas was then mixed with the remaining 02: CO2 gas mixture before it reached the slice. The final concentration of anaesthetic in the superfusing gas depended on the flow rates of the two gas streams and could be varied at will by altering the flow rate of either the vapour-laden gas or the 02: CO2 gas mixture. The gas stream was warmed to 370 C and then passed over the tissue slice. The final concentration of anaesthetic applied to the preparation was determined automatically by an online gas chromatograph (see Richards, 1973b). In early experiments, the methoxyflurane was distilled from commercially available PenthraneR (Abbot Laboratories Ltd) to remove the preservative; later experience showed this precaution to be unnecessary. Measurement of wave forms. The general shape of the evoked potentials will be described in the Results and can be seen in Fig. 1. The amplitude of the l.o.t.

4 124 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE A B Stim. electrode Rec. electrode f1.os~~~~~o~. fibres A 0~o ~ Pial surface F 4400t4A 3 00rPyr. cells C Lost. API ~e.p.s.p. I ~< / 2 2 mv Pop. spikes D 2 msec Fig. 1. Diagrammatic representation of the anatomical organization of the olfactory cortex together with examples of the evoked field potentials elicited by l.o.t. stimulations. A, the ventral surface of the brain of the guinea-pig: the interrupted lines indicate the region from which the slices were taken. B, schematic drawing of a section through the prepiriform cortex to illustrate the innervation by the lateral olfactory tract and the electrode placements. C, drawing of the characteristic potential recorded from the surface of the prepiriform cortex following l.o.t. stimulation. The components of the evoked potential have been labelled to show the nomenclature and the measurements used: l.o.t. AP is the compound action potential of the lateral olfactory tract; stim, the stimulus artifact. D, an example of an actual recording consisting of ten faint superimposed sweeps to show the very small variation in the size and shape of the evoked potentials. The preparation was stimulated at 1 Hz throughout. compound action potential was measured peak-to-peak. The amplitude of the population e.p.s.p. was measured at a fixed time from the beginning of the stimulus artifact on the falling phase of the potential. This amplitude was used as an estimate of the rate of growth of the population e.p.s.p. The exact time chosen (usually 3-4 msec after the stimulus) was dependent on the conduction time of the l.o.t. fibres, but the e.p.s.p. was measured at about 80 % of its maximum negativity. With low concentrations of either anaesthetic there was no evidence to suggest that the anaesthetics increased the conduction time of the l.o.t. fibres; however, with concentrations of ether above 4-6 % or with concentrations of methoxyflurane above %, the l.o.t. compound action potential became depressed and its conduction time was then increased. The population spikes were measured by estimating their area. This was achieved by projecting the film of the evoked wave forms on to squared paper, and then counting the squares between the observed wave form and that expected had no population spike been present (see Fig. 1 C).

5 ETHER ANAESTHESIA 125 RESULTS If an electrode is placed on the surface of the prepiriform cortex and a supramaximal stimulus is given to the l.o.t., a characteristic potential can be recorded which comprises the l.o.t. compound action potential followed by a negative wave (N-wave) upon which one to three positive peaks may be superimposed (see Fig. 1). The N-wave has been identified as the field potential originating from the synchronous activation of many excitatory synapses in response to the l.o.t. volley; for this reason the N-wave has been called the population e.p.s.p. The positive peaks have been shown to reflect the synchronous discharge of the cortical cells in response to the evoked e.p.s.p.s, and so are termed 'population spikes'. The evidence for this interpretation of the evoked potentials of the olfactory cortex has been discussed at length in the papers by Richards & Sercombe (1968, 1970) and by Richards & ter Keurs (1971). If a preparation was stimulated at Hz with constant pulse characteristics and was not treated with anaesthetic, the amplitude of the population e.p.s.p. and the size and number of the population spikes were characteristic of the preparation and remained steady or slowly declined over a period of several hours. Any decline in the potentials was accompanied by a slow increase in the latency of the population spikes and a decrease in their area, the later population spikes being affected first. Effects of ether and methoxyflurane on the evoked field potentials The effects of ether and methoxyflurane on the evoked field potentials were examined in sixteen preparations of the olfactory cortex, seven with ether and nine with methoxyflurane. All the experiments were conducted with concentrations of anaesthetic similar to the minimum alveolar concentrations required to maintain anaesthesia (MAC) for each anaesthetic (see Discussion). For the dog the MAC for ether is about 3 %, and that for methoxyflurane is 0 2 % (Eger, Brandstater, Saidman, Regan, Severinghaus & Munson, 1965). During treatment with either 2-4% ether or % methoxyflurane the population e.p.s.p. was depressed while the l.o.t. compound action potential was unaffected (see Figs. 2, 3). Thus at concentrations of either anaesthetic close to the MAC both anaesthetics 'selectively' depressed the population e.p.s.p. However, at higher concentrations, above 4 % ether or % methoxyflurane (i.e. above times the MAC for ether and 2-3 times the MAC for methoxyflurane) both agents progressively depressed the l.o.t. compound action potential as the concentration of anaesthetic was increased. The depression of the l.o.t. compound action

6 126 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE potential was accompanied by a slowing of its time course, a decrease in conduction velocity and an increase in the onset latency of the population e.p.s.p. Neither ether nor methoxyflurane affected the threshold of the l.o.t. fibres to electrical stimulation at concentrations which reduced the population e.p.s.p. but which did not affect the amplitude of the l.o.t. compound action potential (see Fig. 4). The depression of the population e.p.s.p. caused by methoxyflurane 120 r A E : _ 100 L0"0% ** ine 80 see"%_ 0~ '0OL ~~~ o B 6 < 100v 80'- so Time (min) Fig. 2. The time course of the action of diethyl ether on the evoked potentials of the olfactory cortex. The area of the first population spike is plotted in A, the amplitude of the l.o.t. compound action potential in B, the amplitude population e.p.s.p. in C, and the concentration of ether in the atmosphere above the tissue slice in D. The area of the population spike and the amplitudes of the l.o.t. compound action potential and population e.p.s.p. are expressed as percentages of their average amplitude during the first 5 mm of the experiment. The population e.p.s.p. and population spikes were depressed by 2-3 %/ ether without any depression of the amplitude of the l.o.t. compound action potential but 4@5 %/ ether caused a 10 %/ decrease in the compound action potential. The effects of 3.5 %/ ether were equivocal, the extent of any depression being within the normal variation of the l.o.t. action potential. (Note the suppressed zeros on the ordinates labelled A, B and C.)

7 ETHER ANAESTHESIA 127 was accompanied by a depression of the area of the population spikes and a small increase in their latency of onset. In contrast, while ether depressed the population e.p.s.p. and the first population spike, this depression was associated with an increase in the size of the last population spike in the majority ofpreparations (six out of seven preparations tested). Furthermore, four preparations that were exposed to 3-5 % ether generated an additional late population spike after those normally evoked by l.o.t. stimulation. This additional population spike was always small and variable in amplitude. Higher concentrations of ether (above 5 %) '1 40 A * t *.*.0. I 0 L0 %ft WO fl VW.*. 120 C~~~~~~~~~~~~. CL~~ 2100 * * O s0 100 Time (mini) Fig. ise plte 3. Theintimeancourse h ofehxfuaecnetain()inteupr athe action methoxyflurane on the evoked potentials of the olfactory cortex. The area of the first population spike (0) is plotted in A, the amplitude of the presynaptic L~o.t. compound action potential (V) is plotted in B, the amplitude of the population e.p.s.p. (A) is plotted in C and the methoxyflurane concentration (0) in the superfusing gas stream is plotted in D. The abscissa is common to all four ordinates. The amplitude of the population e.p.s.p. and l.o.t. compound action potential are expressed as percentages of their average amplitude during the first ten minutes of the experiment. The area of the population spike is expressed in arbitrary units. The e.p.s.p. and population spike were depressed by % methoxyflurane without any evident decrease in the amplitude of the presynaptic Lo.t. compound action potential. Above 0.5 % methoxyflurane the l-o.t. compound action potential was also slightly depressed. (Note the suppressed zero on the ordinate of B.) PHY 248

8 128 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE progressively abolished all the population spikes. These effects of ether and methoxyflurane were reversible, were dose-dependent, and were independent of the time for which the preparations were exposed to anaesthetic once equilibration had occurred. The experimental results showed that the population e.p.s.p.s were reduced in amplitude, and so, presumably, were the e.p.s.p.s of the individual cortical cells. However, the decrease in the population spike could 100 0A A 100 B x E A0 50so Stimulus (V) Stimulus (V) Fig. 4. The absence of any effect of ether or methoxyflurane on the threshold of the l.o.t. compound action potential. A, ether, key to symbols: 0, control; A, 3 % ether. B, methoxyflurane, key to symbols: 0O control; A, 0 4% methoxyflurane; *, recovery. In neither A nor B was a stronger stimulus voltage required to elicit a l.o.t. compound action potential of a given size. also reflect an increase in the threshold depolarization of the cortical neurones required for impulse generation. To test this possibility, the relationship between the population e.p.s.p. and the area of the first population spike was examined in ten preparations; five in the presence and absence of ether and five in the presence and absence of methoxyflurane. The variation of the size of the afferent volley and of the population e.p.s.p. was achieved by varying the intensity of the l.o.t. volley. Weak shocks elicited a small population e.p.s.p. but no population spike but, as the stimulating shock increased in strength, more afferent fibres were recruited and the population spike grew together with the population e.p.s.p. (see Fig. 5). If either ether or methoxyflurane had raised the electrical threshold of the cortical neurones, then a larger population e.p.s.p. would be required to elicit a population spike of a given size. Thus the relationship between the population e.p.s.p. and the first population spike shown in Fig. 5 should have been shifted along the abscissa to the right. However, in no case was the area of the population spike smaller for a given amplitude of the population e.p.s.p. when the anaesthetic was

9 40 A ETHER ANAESTHESIA 129 i e.p.s.p *o s,-20 CL o 0. 0~~~~~~~ A~~~~ O~~~~~~ 04Z~06 0 A LAt L S Amplitude of pop. e.p.s.p. (mv) 40 B._ C~~~~~~~~~~ 30 ~~~~~~~~so 0.0 at20 _,^e,0 EU 00 o * A 0X.010 _A 0 0 i 0 O0S Amplitude of pop. e.p.s.p. (mv) Fig. 5. The action of ether and methoxyflurane on the relationship between the population e.p.s.p. and the first population spike. A, the action of ether; B, the action of methoxyflurane. The e.p.s.p. was measured at a fixed time (t) from the stimulus artifact throughout the experiment and the area of the population spike was estimated as that shown by the cross-hatching in the inset figure. Key to symbols: A, control (0); 4% ether (AL); recovery (= 1 % ether) (@). B, control (0); 0.4% methoxyflurane (A); recovery (0). In neither case was the area of the population spike smaller for a given size of the population e.p.s.p. when anaesthetic was administered. 5-2

10 130 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE given (see Fig. 5). Thus these tests provided no evidence to suggest that the firing threshold of the cortical cells was increased by ether or methoxyflurane. Effect on post-tetanic potentiation It could be argued that the depression of the population e.p.s.p. caused by ether and methoxyflurane was the result of a decrease of transmitter mobilization or synthesis which, in turn, would lead to a smaller store of transmitter available for release and so to a smaller amount of transmitter 175 X A A A X ~I A A AA A A A 150 C 15 R l o ti IA A A A 10 C 125 U I! CL oo 0'C *~~0 0I U Time after conditioning train (sec) Fig. 6. The effect of methoxyflurane on the post-tetanic potentiation of e.p.s.p.s evoked at 0-2 Hz after a tetanic train of 40 Hz for 30 sec. The amplitudes of the e.p.s.ps are expressed as a percentage of the average control amplitude during the minute preceding the tetanic train. 0O control; A, during exposure to 0 2 % methoxyflurane; 0, recovery. The average amplitudes of the unconditioned e.p.s.p.s are shown on the left-hand side of the Figure together with their 99 % confidence limits. In all cases posttetanic potentiation of the e.p.s.p.s was observed though it was more pronounced in the presence of 0-2 % methoxyflurane. being released by each nerve impulse. However, if this were so, a period of high frequency stimulation should be followed by a greater depression of the population e.p.s.p. because the release of transmitter during such stimulation would accelerate the depletion of the presynaptic stores. This point was examined in five preparations, three with ether and two with methoxyflurane. In no case was there a greater depression of the population e.p.s.p. after a period of high frequency stimulation corresponding to 100 min of normal low frequency stimulation. Indeed the period immediately after such stimulation was associated with post-tetanic

11 ETHER ANAESTHESIA 131 potentiation of the population e.p.s.p. which approached, but did not exceed, the amplitude of the unconditioned population e.p.s.p.s seen in the absence of anaesthetic (Fig. 6). The effects of methoxyflurane on evoked unit activity The decrease in the area of the population spikes with increasing concentrations of methoxyflurane implied that fewer of the post-synaptic cells discharged in response to the afferent volley. If this is the correct interpretation, the stimulus-evoked activity of individual cells in the cortex should be depressed. To see if this was so, the evoked spike activity of individual cells in the prepiriform cortex was monitored with extracellular microelectrodes before, during, and after exposure to methoxyflurane. In all, ten cells were successfully studied. None of the cells were spontaneously active. Methoxyflurane depressed the evoked activity of all ten cells tested in a dose-related manner (see Fig. 7); most cells ceased to fire when % methoxyflurane was applied to the preparations. The depression of the evoked activity of the cells was generally accompanied by an increase in their latency of discharge, this increase being greatest (ca. 1 msec) for those cells which had short latencies (4-6 msec) and which also had the greatest resistance to the anaesthetic. The evoked activity of cells with latencies of 10 msec or more was generally depressed with little change in the latencies of the peaks in their post-stimulus histograms. The effects of ether on evoked unit activity The action of ether on the evoked field potentials was distinct from that of methoxyflurane in its effects on the population spikes; the depression of the population e.p.s.p. and first population spike was accompanied in most preparations by an increase in the size of the late population spikes. These results implied that, while the short latency discharge of some cells would be depressed, some cells in the prepiriform cortex were more liable to fire at longer latencies after the stimulus than hitherto. Extracellular recordings were made from twelve cells in the prepiriform cortex; all of the cells were stimulated by l.o.t. stimulation and none were spontaneously active. The evoked activity of seven cells, including two that had been previously tested with methoxyflurane, was depressed by concentrations of ether between 3 and 6 % This depression was similar to that just described for methoxyflurane. The evoked activity of the remaining five cells was not depressed; instead, each l.o.t. volley elicited a short train of two or more spikes when the preparation was exposed to ether (see Fig. 7). This burst firing of cells was dependent on a concentration of ether within a particular range (usually 3-5 %), below this critical range no effect was seen and above it the cell's activity became depressed.

12 132 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE A Contrl EB Control j 0-1_ 03 0rn 0-15 % methoxyflurane % ether :1 L _, 0: _a % methoxyflurane 02 [ L -Thi u ~0-4r Recovery 0*2 ~Recovery02 LL II Time after stim (msec) Time afte r stim (msec) Fig. 7. The action of ether and methoxyflurane on the stimulus-evoked activity of two cells in the prepiriform cortex. The results are expressed in the form of post-stimulus histograms compiled from the responses to 250 stimuli given at 1/sec. A, action of methoxyflurane; methoxyflurane reversibly depressed the evoked activity of the cell in a dose-related manner. B, action of ether on another cell; ether caused this cell to discharge in groups of spikes after an l.o.t. volley, thus the probability of discharge was increased at long latencies; the effect was reversible. The ordinates show the probability of cell discharge at a particular latency following a l.o.t. volley. The first 10 msec after stimulation are not represented in the histograms. The action of ether and methoxyflurane on the sensitivity of cortical neurones to L-glutamate The identity of the substances that mediate transmission between the fibres of the l.o.t. and the cells of the olfactory cortex is not known. For this reason it has not proved possible to test whether ether and methoxyflurane reduce the amount of transmitter released from the presynaptic

13 ETHER ANAESTHESIA 133 terminals for a given nerve volley, or whether they reduce the sensitivity of the cortical cells to the released transmitter substance or both. However, neurones in the olfactory cortex can be excited by iontophoretic application of L-glutamate, a method that provides a means of exciting cells chemically 60 A 0 50 _ -.* o f 40 0 * 00. U *.0 _ * _ 0, *.. oo 0 e 208 LU Time (min),, 50 X ' 30_*.%. * 0 0 -C a, 20 _ * %**0.; % v) 10_ o L. 203r 0.2 x r- 0,1,1.. I 6 % Time (min) Fig. 8. The action of ether and methoxyflurane on the sensitivity of two cells in the prepiriform cortex to iontophoretically applied L-glutamate. A, the action of ether on a cell 340 /etm deep in the cortex which was excited by a 24 na pulse of glutamate of 10 sec duration every 30 sec throughout the experiment. B, the action of methoxyflurane on a cell 270,tm deep in the cortex which was also excited by a 24 na pulse of glutamate of 10 sec duration every 30 sec. In this experiment, the preparation was bathed in saline containing 10 mm-mg to block synaptic transmission. I0

14 134 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE by a process resembling that of natural synaptic excitation (see Discussion). Thus any changes in the sensitivity of neurones to iontophoretically applied L-glutamate caused by ether or methoxyflurane may reflect similar changes in the sensitivity of the post-synaptic membrane to the actual transmitter substance. The effects of ether and methoxyflurane on the sensitivity of neurones to L-glutamate were examined on thirty-six cells in twenty-two preparations of the isolated olfactory cortex. In all cases ether and methoxyflurane depressed the responsiveness of the neurones to the iontophoretically applied glutamate. The degree of depression was related to the concentration of anaesthetic applied. For example, Fig. 8A shows that 1 % ether reduced the average firing rate of the neurone to about 85 %, and 3 % ether reduced the firing rate to about 30 % of the control values; the effects were reversible. Similar results were found with methoxyflurane. This depression could result from the depressant action of ether and methoxyflurane on the synapses of adjacent cells which have also been excited by the glutamate and which, directly or indirectly, excite the cell under observations via intracortical connexions. This possibility could be discounted if similar results could be obtained in circumstances that did not permit synaptic transmission. It has already been shown that saline solutions containing high Mg concentrations (10-13 mm) block the normal excitation of neurones by l.o.t. stimulation (Richards & Sercombe, 1970), so the actions of ether and methoxyflurane on the glutamate sensitivity of eleven neurones were tested in the presence of 10 mm-mg. The preparations were first equilibrated with the 10 mm-mg saline before the action of the anaesthetics on the glutamate sensitivity was studied, because Mg raises the threshold depolarization required for impulse generation in addition to its action on transmitter release (see Kelly, Krnjevic & Somjen, 1969; Richards & Sercombe, 1970). Of eleven neurones studied under these experimental conditions, five were treated with ether alone and six with methoxyflurane alone; all eleven cells showed a similar dose-related depression of their spike activity when they were exposed to anaesthetic (see Fig. 8B). Thus the depression of the firing rate during exposure to anaesthetic must reflect a change in the sensitivity of the individual neurones to glutamate. In addition to depressing the average firing rate of the neurones, both ether and methoxyflurane caused subtle changes in the firing pattern of a proportion of the cells. Generally, each cell had a constant firing pattern which was either a regular train of discharges or slightly grouped discharges. Analysis of the interspike intervals showed that ten of the thirtysix cells tested had an increased tendency to fire in groups during exposure

15 ETHER ANAESTHESIA 135 to ether or methoxyflurane; the number of short inter-spike intervals increased compared to the control and this occurred even while the mean firing rate was falling (Fig. 9). This tendency for discharges of individual cells to be more tightly grouped was dose-related, for example one cell showed a depression of firing in the presence of 1-5 % ether but showed no Control Mean rate 7/sec C % ether Mean rate 4-5/sec S 10 Li L5 E Z Recovery Mean rate 6-9/sec Inter-spike intervals (msec) Fig. 9. The effect of ether on the distribution of interspike intervals of a cell in the prepiriform cortex that had been excited by iontophoretic application of L-glutamate. The cell was 290,Im deep in the cortex and was excited by a 15 na, 10 sec pulse of glutamate every 30 sec. Note that even though the mean discharge rate fell from 7 to 4.5/sec when 1'7 % ether was administered there was an increased tendency for the spikes to occur in groups as shown by the increase in the number of short inter-spike intervals. This effect was reversed when the ether was removed. The numbers above the arrows on the right show the number of intervals greater than 150 msec (not including the periods between the pulses of glutamate). Each histogram was compiled from the intervals between 250 spikes.

16 136 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE grouping of spikes, whereas in 3 % ether it always discharged in pairs or triplets. This effect appeared to be more persistent with ether; four of the sixteen cells examined in ether showed a clear increase in the number of short inter-spike intervals; with methoxyflurane three of the twenty cells examined maintained a firing pattern in which the nerve impulses were grouped. Three other cells showed a transient grouping of nerve impulses during the onset of the exposure to methoxyflurane. The effects of ether and methoxyflurane on spike height None of the twenty-two neurones excited by electrical stimulation of the l.o.t. showed any clear and consistent decrease in the amplitude of their evoked spike discharges when ether or methoxyflurane was applied. Fifteen of the thirty-six neurones excited by application of glutamate were examined for changes in spike height during exposure to anaesthetic. Three cells showed a depression of spike height when they were exposed to ether or methoxyflurane. The effects were related to the concentration of anaesthetic applied; in agreement with the results obtained from the neurones excited by an afferent volley, less than 0-2 % methoxyflurane or 3 % ether had no significant effect. One of two cells examined in 0 2 % methoxyflurane showed a 20 % depression of spike height, the other was unaffected. One cell exposed to 0 3 % methoxyflurane showed a 10% reduction in spike height. One cell, of seven examined, showed a clear depression of spike height in ether, a 25 % reduction was seen in the presence of 5-5 % ether and a 40 % reduction in the presence of 8 % ether. However, as the spikes are small in amplitude (ca TV) it is neither easy to see small changes in spike height that may be caused by low concentrations of anaesthetic, nor is it possible to exclude movement artifact as the cause of small changes in the height of spikes. DISCUSSION In any study of the mechanism of action of inhalational anaesthetics the choice of appropriate concentrations is difficult, particularly when isolated preparations are being used, because of the large range of concentrations employed in clinical practice. During the induction of anaesthesia, high concentrations of anaesthetic are administered to cause a rapid accumulation of anaesthetic in the brain; thereafter, the concentration administered is reduced to a level sufficient to maintain the patient at an appropriate depth of anaesthesia. There are, therefore, two phases: that of induction, which is characterized by the administration of high concentrations of anaesthetic and which necessarily must be associated with concentration gradients; and that of maintenance, which is characterized by the

17 ETHER ANAESTHESIA 137 administration of low concentrations of anaesthetic sufficient only to maintain a steady state. For the experiments described in this paper we have attempted to mimic the maintenance phase of anaesthesia. One simple approach to the problem is to apply a given concentration of anaesthetic for a sufficient time to allow the tissue's responses to reach a new steady level; when this level is reached, the anaesthetic will be in equilibrium with the various phases of the preparation. Thus, under steadystate conditions, a defined concentration of anaesthetic in the gas phase will be equivalent to a specific (though unknown) tissue concentration, and this will be true both for isolated preparations and intact animals (see Ferguson, 1939, 1951), provided that both the animal and isolated preparation are fully equilibrated with the anaesthetic. One convenient standard is the minimum alveolar concentration (MAC) of an inhalational anaesthetic required to maintain a given level of anaesthesia, arbitrarily defined as that concentration which abolishes all motor response to a strong stimulus in half of a test group of animals. The stimulus usually chosen is a scalpel cut in the body wall. For ether and methoxyflurane these values are 3 % and 0O2 % respectively for the dog (Eger et al. 1965) and similar values have been found for a number of other species including man. Throughout the present experiments, the concentrations of ether and methoxyflurane have been maintained at, or near, these levels; 3-4 % ether or 02 % methoxyflurane causes a 25 % reduction in transmission across the synapses of the olfactory cortex (see Figs. 2, 3). Thus the effects of these substances on the synaptic activity of the neurones in the olfactory cortex are likely to be among the central actions of these substances during anaesthesia. Mode of action of ether and methoxyfturane As there is good evidence that transmission between the fibres of the l.o.t. and the neurones of the olfactory cortex is chemical in nature (see Richards, 1972 a), the depression of the population e.p.s.p. by anaesthetics could result from one or more of the following processes: (a) a block of nerve impulse conduction in the afferent axons or in their terminal branches; (b) a decrease in the amount of transmitter liberated from the nerve terminals by each afferent impulse; (c) a decrease in the sensitivity of the post-synaptic receptors to the related transmitter; (d) an increase in the threshold depolarization of the post-synaptic cells required for nerve impulse generation; (e) a decrease in the electrotonic propagation of the e.p.s.p.s along the dendrites to the axon hillock. (a) It has already been mentioned that neither ether nor methoxyflurane block nerve impulse conduction along the fibres of the l.o.t. at

18 138 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE concentrations corresponding to those required for the maintenance of anaesthesia. However, at higher concentrations there can be no doubt that depression of the l.o.t. compound action potential does contribute to the depression of the population e.p.s.p., this being particularly important with ether (see Fig. 2). Furthermore, it is possible that action potential conduction in the terminal branches of the afferent fibres could be preferentially blocked by lower concentrations of anaesthetics, as proposed by Seeman (1972). However, we have not been able to test this postulate in the olfactory cortex, because there is no certain way of recording nerve impulses in the terminal arborizations of nerve fibres within the C.N.s. Thus, present evidence does not enable us to exclude the possibility that ether or methoxyflurane depresses the population e.p.s.p. by causing a failure of conduction in the nerve terminals; equally, positive evidence in favour of Seeman's hypothesis has yet to be provided. (b) Although transmission across the synapses of the olfactory cortex is probably chemical in nature, the transmitter substance is unknown so we cannot directly measure the action of anaesthetics on transmitter output. It is clear that PTP was increased during exposure to ether and methoxyflurane (see Fig. 6). As PTP probably results from an increase in the amount of transmitter released by each nerve impulse (Eccles, 1964; Richards, 1972a), it is unlikely that ether and methoxyflurane significantly affect the process of transmitter synthesis and mobilization within the nerve terminals. The observation that PTP was more marked in the presence of methoxyflurane than in its absence is in accord with the results of similar experiments with halothane (Richards, 1973b) but is probably not of particular significance, for the smaller the initial e.p.s.p. caused by afferent stimulation, the greater will be the increase in the e.p.s.p. caused by a given additional amount of transmitter released (see Martin, 1955). The depression of the l.o.t. compound action potential by concentrations of ether above 4 % and of methoxyflurane above 04 % would be likely to be associated with a decreased transmitter output. Whether the depression of the population e.p.s.p. caused by lower concentrations of these anaesthetics is produced in part by a decrease in transmitter output remains unsettled. (c) It has already been noted that the transmitter substances of the olfactory cortex are unknown. For this reason it is not possible to test directly whether ether and methoxyflurane depress the sensitivity of the post-synaptic membrane to the released transmitter substance. However, the neurones of the olfactory cortex can be excited by iontophoretic application of a number of substances including L-glutamate and, for the following reasons, we believe that the excitation produced by local application of L-glutamate resembles natural synaptic excitation: Iontophoretic

19 ETHER ANAESTHESIA 139 application of L-glutamate and other amino acids to neurones has shown a clear relationship between the structure of amino acids and their ability ct excite or inhibit nerve cells, presumably by interaction with specific receptors on the neuronal membrane (Curtis & Watkins, 1960). The onset of the glutamate excitation of neurones is rapid. Glutamate causes an increased Na flux which is associated with an increase of Na and a decrease of K in slices of the olfactory cortex (Harvey & McIlwain, 1968). Thus any changes found in the sensitivity of the neuronal membrane to glutamate may reflect similar changes in the sensitivity of the post-synaptic membrane to the natural transmitter. Ether and methoxyflurane clearly depressed the sensitivity of the neurones to L-glutamate (Fig. 8): as we found no evidence to suggest that either anaesthetic increased the threshold depolarization for nerve impulse generation (Fig. 5), and as the depression of glutamate-induced spike activity could be clearly demonstrated in preparations poisoned with 10 mm-mg (in which synaptic transmission is blocked) (see Fig. 8B), the depression of the sensitivity of the neurones to glutamate must reflect a decrease in the ability of the neuronal membrane to respond to the drug. Consequently, these results are quite compatible with the idea that both ether and methoxyflurane depress synaptic transmission, at least in part, by reducing the sensitivity of the post-synaptic membrane to the released transmitter substance. A given concentration of ether or methoxyflurane depressed the glutamate excitation of the cortical neurones more than the population e.p.s.p. (compare Figs. 2, 3 with Fig. 8). This would be expected because a small decrease in the sensitivity of the post-synaptic membrane to glutamate would result in a proportional decrease in the firing rate of the cortical cells similar to that produced by a decrease in the dose of glutamate. A similar small decrease in sensitivity to the transmitter would not be so readily detected by field potential recording because a decrease in sensitivity to transmitter (or a decrease in transmitter output) does not produce a proportional decrease in synaptic current. This is a consequence of the non-linear summation of synaptic current with increasing transmitter output (see Martin, 1955) and leads to an underestimate of the extent of the depression of the post-synaptic membrane to the released transmitter (or of the depression of transmitter output). The depressant actions of ether and methoxyflurane on the population e.p.s.p. and population spikes appear to be similar to those of halothane or pentobarbitone (Richards, 1972b, 1973b). However, while the sensitivity of neurones in the olfactory cortex is depressed by pentobarbitone, just as it is by ether or methoxyflurane, halothane does not cause a similar depression (Richards & Smaje, 1974). If the sensitivity of the

20 140 C. D. RICHARDS, W. J. RUSSELL AND J. C. SMAJE post-synaptic membrane to the actual transmitter is affected similarly, these results suggest that halothane depresses the e.p.s.p.s primarily by reducing the amount of transmitter released, whereas ether, methoxyflurane and pentobarbitone depress the e.p.s.p.s, at least in part, by reducing the sensitivity of the post-synaptic membrane to the released transmitter. (d) and (e) Ether and methoxyflurane could also have reduced synaptic transmission by interfering with the electrical coupling between the population e.p.s.p. and the discharge of the cortical cells, either by increasing the threshold depolarization required for nerve impulse generation (d), or by decreasing the electrotonic propagation of the synaptic potentials (e). These possibilities were investigated by examining the relationship between the population e.p.s.p. and the first population spike (see Fig. 6). No evidence was found to suggest that either anaesthetic affected the threshold of the post-synaptic cells or the electrotonic propagation of the synaptic potentials to the neurone soma. The depression of the evoked population e.p.s.p.s by ether and methoxyflurane was generally accompanied by a decrease in the area of the first population spike - indicating that fewer cells were discharging in response to the synaptic excitation. This was confirmed by the studies of the stimulus-evoked neuronal discharge of cells in the olfactory cortex. However, a high proportion of cells exposed to ether (five out of the twelve neurones successfully examined) showed an increase in their evoked activity (Fig. 7) which could be correlated with the additional population spike seen during exposure to ether. Thus this anaesthetic had the property of altering the temporal pattern of nerve impulses, a property which it shares with trichloroethylene (Richards, 1973a, 1974). It must be said that the stimulus-evoked units tended to be those with long latencies (about 10 msec) compared to the first population spike (about 3-5 msec) because of the difficulty of isolating single unit discharges from the massed discharge of the cortical cells. It is therefore likely that those neurones which showed an increased cell discharge during exposure to anaesthetic would have been influenced by secondary synaptic contacts in addition to those of the lateral olfactory tract. An increase in the grouping of nerve impulses was also seen during exposure to ether or methoxyflurane in cells excited by L-glutamate and was once observed in saline containing 10 mm-mg (out of seven neurones examined). Thus although the grouping of nerve impulses during treatment with anaesthetic cannot be attributed to the influence of structures such as the reticular formation or the ventrobasal thalamus (for these are not connected to the tissue slices), the phenomenon may still require the integrity of local neural connexions or it may be a property of the individual neurone. Similar changes in the pattern of cell discharge have been recorded in

21 ETHER ANAESTHESIA vivo during the administration of a number of anaesthetics. For example, Robson (1967) observed an increase in the number of short inter-spike intervals recorded from neurones in the cat parietal cortex during the administration of trichloroethylene. Similar changes have been seen with pentobarbitone (Noda & Adey, 1973) and with halothane (B. Delisle Burns & A. C. Webb, personal communication). Goodman & Mann (1967) found grouping of nerve impulses recorded from the thalamus and reticular formation during anaesthesia induced with a variety of anaesthetics including diethyl ether; these authors recorded multi-unit activity. A similar study by Baker (1971) of single unit activity in the ventrobasal thalamus showed similar results. Thus it appears that an increase in the grouping of action potentials normally occurs during anaesthesia and may indeed be an essential feature of the anaesthetic state. REFERENCES 141 BAKER, M. A. (1971). Spontaneous and evoked activity of neurones in the somatosensory thalamus of the waking cat. J. Physiol. 217, CURTIS, D. R. & WATKINS, J. C. (1960). The excitation and depression of spinal neurones by structurally related amino acids. J. Neurochem. 6, DORAS, C. F. & RICHARDS, C. D. (1974). An improved chamber for maintaining mammalian brain tissue slices for electrical recording. J. Physiol. 239, 83-84P. ECCLES, J. C. (1964). The Physiology of Synapses, pp Berlin: Springer. EDWARDS, C. & LARRABEE, M. G. (1955). Effects of anaesthetics on metabolism and on transmission in sympathetic ganglia of rats: measurement of glucose in microgram quantities using glucose oxidase. J. Physiol. 130, EGER, E. I., BRANDSTATER, B., SAIDMAN, L. J., REGAN, M. J., SEVERINGHAUS, J. W. & MUNSON, E. S. (1965). Equipotent alveolar concentrations of methoxyflurane, halothane, diethyl ether, fluroxene, cyclopropane, xenon and nitrous oxide in the dog. Anesthesiology 26, FERGUSON, J. (1939). The use of chemical potentials as indices of toxicity. Proc. R. Soc. B 127, FERGUSON, J. (1951). Relations between thermodynamic indices of narcotic potency and the molecular structure of narcotics. In Colloques Internationaux du Centre de la Recherche Scientifique, chap. xxvi: Mechanisme de la Narcose, pp GOODMAN, S. J. & MANN, P. E. G. (1967). Reticular and thalamic multiple unit activity during wakefulness, sleep and anesthesia. Expl Neurol. 19, HARVEY, J. A. & McILwAIN, H. (1968). Excitatory acidic amino acids and the cation content and sodium ion flux of isolated tissues from the brain. Biochem. J. 108, KELLY, J. S., KRNJEVI6, K. & SOMJEN, G. (1969). Divalent cations and electrical properties of cortical cells. J. Neurobiol. 2, LARRABEE, M. G. & POSTERNAK, J. M. (1952). Selective action of anaesthetics on synapses and axons in mammalian sympathetic ganglia. J. Neurophysiol. 15, MARTIN, A. R. (1955). A further study of the statistical composition of the endplate potential. J. Physiol. 130, NODA, H. & ADEY, W. R. (1973). Neuronal activity in the association cortex of the cat during sleep, wakefulness and anaesthesia. Brain Res. 54,

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