J. PhyWol. (1978), 284, pp. 307-325 With 12 text-figur Printed in Geat Britain

307

FACILITATION OF SYNAPTIC TRANSMISSION BY GENERAL ANAESTHETICS BY MARY E. MORRIS From the Department of Research in Anaesthesia, McGill University, Montreal, Canada

(Received 3 February 1978) SUMMARY

1. The actions of five structurally different intravenous and inhalation anaesthetics (alphaxalone/alphadolone, halothane, ketamine, methohexitone, and pentobarbitone) have been studied on synaptic transmission through the cuneate nucleus of the dorsal column-lemniscal afferent pathway in the decerebrate cat. 2. Synaptic input and output were estimated from antidromic and orthodromic potentials, which were evoked by either afferent volleys from the periphery or micro-electrode excitation of the presynaptic fibre terminals in the cuneate and recorded at forelimb nerves and the medial lemniscus. 3. Each of the anaesthetic agents potentiated the efficiency of synaptic transmission, as shown by the elevation of input-output curves constructed from the integrals of the potentials evoked by varying intensities of either peripheral or cuneate stimulation. 4. The excitability of the afferent terminals, as measured at the peripheral nerves by the antidromic responses to micro-electrode stimulation, was depressed by the anaesthetics. Post-synaptic excitability, which was assessed from the direct lemniscal response to intra-nuclear stimulation, did not appear to change. 5. Hypotensive states (mean arterial levels < 60 torr) produced depolarization of presynaptic terminals and depression of synaptic efficiency and transmission; these changes opposed the primary effects of the general anaesthetics. 6. It is concluded that anaesthetics do not depress activity at all synapses of the central nervous system. Their facilitatory action on cuneate transmission is attributed to an enhanced release of excitatory transmitter; the underlying mechanism may be hyperpolarization of the primary afferent terminals, secondary to an increase in K+ conductance. INTRODUCTION

The mode of action of general anaesthetics has for long resisted definition. Whatever the mechanism, it will be essential in attempts to establish a unifying hypothesis not only to determine how cell membranes, mitochondria, and metabolic processes are affected, but also to fully define differential actions on the various components (cell and axons) involved in transmission in the central nervous system. There is substantial evidence that the most vulnerable site for depression of transmission in Reprint requests address: Room 4302, Medical Sciences Building, University of Toronto, 1 King's College Circle, Toronto, Canada M5S 1A8.

308 M. E. MORRIS neuronal pathways by anaesthetics is at the synapse (Larrabee & Posternak, 1952; Carpenter, 1954; Chalazonitis, 1967; Somjen, 1967; Richards, 1972, 1973; Richards, Russell & Smaje, 1975; Richards & White, 1975). In some studies it has been concluded that this action is at the post-synaptic membrane (Brooks & Eccles, 1947; Shapovolov, 1963; Sato, Austin & Yai, 1967; Barker, 1975a, b), while a presynaptic locus has been implicated in others (L0yning, Oshima & Yokota, 1964; Matthews & Quilliam, 1964; Weakly, 1969; Seeman, 1972; Staiman & Seeman, 1974). It also seems possible that the anaesthetic drugs may produce an imbalance between inhibitory and excitatory synaptic actions (Nicoll, 1972; Barker & Gainer, 1973), and that different synapses may have different susceptibilities (Mark & Steiner, 1958; Richards & White, 1975). In contrast, the experiments described in this paper, which utilize an analysis of the input-output relation of the cuneate nucleus in the decerebrate cat (Morris, 1971; Krnjevic & Morris, 1976), demonstrate that the influence which anaesthetics have on the central synapses of at least one, highly secure pathway of the central nervous system is facilitatory. This effect was first observed with pentobarbitone (Krnjevic6 & Morris, 1976), and is now shown to be produced by several structurally different anaesthetics. A brief report of the data has already been published (Morris, 1976). METHODS

Preparation Experiments were carried out on seventeen cats (weighing 3-53 + 0.37 (S.D.) kg), which were initially anaesthetized during the surgical preparation with N20 and halothane (Fluothane, Ayerst Laboratories). After cannulation of the trachea and ligation of the carotid arteries, catheters were placed in a femoral artery for the measurement of arterial pressure (with a Statham P23B transducer) and in one or both femoral veins, for the injection of drugs and infusion of 150 mm-NaCl. The dorsal surface of the medulla was exposed by laminectomy of the first two cervical vertebrae and removal of part of the inferior occipital bone. The superficial radial and median nerves of the right forelimb were dissected and mounted on bipolar platinum electrodes in a bath of paraffin oil. Decerebration was carried out by transaction of the brain stem at the midcollicular level, with removal of the forebrain. Glass micro-electrodes (filled with 1 M-NaCl, I % (w/v) Agar, and having tips of 7-15 #m and resistances between 0.8 and 1-5 MO) were inserted into the cuneate through small openings in the pia mater. The exposed medulla was continuously irrigated with Ringer solution (NaCl 150 mM; KCl 2-5 mm; CaCl2 1.5 mm; MgCl2 1-3 mM; NaH2PO4 1-0 mm; NaHCO3 12 mM), maintained at 39 00 by a thermistor feed-back circuit. After decerebration anaesthesia was discontinued, and an interval of at least 4 hr ensued, prior to the administration of test anaesthetics. For the remainder of the experiment the animal was paralysed either by an infusion of 01 % (w/v) succinylcholine chloride (Anectine, Burroughs Wellcome & Co. Ltd.) or with intermittent injections of pancuronium bromide (Pavulon, Organon Canada Ltd.), and received constant volume ventilation with 100% 02 (tidal volume 20 ml.; rate 30/min) from a Palmer respirator. Arterial PC02' measured in some experiments, ranged from 25 to 30 torr; body temperature was maintained constant at 38 + 1-5 (S.D.) °C by a servo-controlled electric blanket. -

Stimulation and recording techniques Monophasic square wave pulses from a constant current source were applied at a frequency of 1/sec via either a micro-electrode inserted into the cuneate (usually to a depth of < 0-6 mm), or bipolar Pt electrodes positioned proximal to those used for recording from the most distal part of the peripheral nerve (see Fig. 1A). Currents ranged from 1 to 100 ,uA for intranuclear stimulation, and 1-10 mA for the peripheral nerve. Cathodal pulses were applied between the micro-electrode and the animal ground, which was a chlorided Ag plate attached to posterior

ANAESTHETICS AND SYNAPTIC TRANSMISSION

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muscles of the neck; during nerve stimulation the distal contact of the bipolar electrode was the cathode. Different intensities of current were repetitively cycled, each group being composed of a series of eight or sixteen stimulus strengths, incrementing by equal steps and applied every 10 or 18 sec (see upper traces, Fig. 11) or in a quasi-random, mixed order of presentation. The duration of pulses varied between 0-1 and 0-2 msec. Stimulation of the afferent terminals in the synaptic regions of the cuneate (in sixteen cats) evoked an antidromically conducted potential which, recorded from the ipsilateral peripheral forelimb nerve, allowed indirect assessment of terminal excitability (Wall, 1958; Andersen, Eccles, Oshima & Schmidt, 1964). The orthodromically conducted lemniscal response to this stimulation was recorded from the contralateral brain stem: an initial alpha (a) component, attributable to direct excitation of the cuneate relay neurones, was sometimes evoked with more deeply placed electrodes (see Fig. 8); synaptic responses to the excitation of presynaptic fibre terminals were represented by a later, usually larger beta (fi) group (see Fig. 4A between arrows marked ML, and Fig. 8). The simultaneously evoked antidromic nerve potentials and transsynaptic lemniscal potentials provided respectively estimates of synaptic input and output, and a means of assessing the efficiency of cuneate synaptic transmission (Morris, 1971; Krnjevi6 & Morris, 1976). When a peripheral nerve was stimulated (in experiments in five cats) the antidromic potential recorded at the distal nerve electrode provided a measure of afferent input, which could be matched with the centrally conducted lemniscal response. This permitted an analysis similar to that described above, in order to observe what might be considered more physiologically relevant information about the overall transfer from the periphery of the synaptically transmitted signals. Monophasic compound action potentials, evoked by either nerve or cuneate stimulation, were therefore recorded from (a) the distal end of one or more (in combination) of the forelimb nerves and (b) the contralateral medial lemniscus (by differential recording from electrodes placed at an active and remote site of the transacted brain stem). After amplification and oscilloscope display of the responses, limits for the integration of portions of each potential complex were manually set (see Figs. 1 B and 4A) for two separate methods of information storage and display: (a) with an analogue circuit and polygraph recording (see examples of Fig. 11), and (b) with a Linc-8 computer and magnetic tape, which provided on-line display of matched integral (from either one or four consecutive stimulus cycles) as input-output plots (Figs. 1, 2, 4, and 5) as well as subsequent analysis with calculation of power functions of best fit (y = axeb: full details of method and assumptions described in an earlier paper (Krnjevi6 & Morris, 1976)). TABLE 1. Summary of total numbers of tests with different anaesthetics, including mean (± S.D.) of doses and durations of inhalation or injection. Halothane dose expressed as per cent inspired concentration No. of No. of Duration Dose animals tests (min) (mg/kg) Drug Halothane 17 10 11-4 (±9-98) 0.5-2.0% 16 7 0-72 (± 0.88) 2-42 (± 1.54) Alphaxalone/alphadolone Methohexitone 18 8 2-65 (± 2-16) 3-95 (+ 3-44) 5 Ketamine 3 1-30 (±0-73) 16-29 (±8-15) 2 Pentobarbitone 3 2-70( 0-43) 10-46 (0-51)

Administration of anaesthetics Halothane (Fluothane, Ayerst Laboratories) was supplied in concentrations ranging from 0-5 to 2-0 % (determined by the settings of a Mark III Fluotec vaporizer) in a 5 l./min flow of 02 to the respirator, for periods of 3-40 min. Intravenous agents were infused in dosages chosen to include the recognized values required to produce anaesthesia in the cat (Clifford & Soma, 1969; Child, Currie, Davis, Dodds, Pearce & Twissell, 1971; Mori, Kawamata, Mitani, Yamazaki & Fujita, 1971). Methohexitone sodium (Brietal, E. Lilly & Co. Ltd.) was injected as a 0-2-1-0% (w/v) solution in normal saline, in doses of 2-50 mg (0-5-15 mg/kg). The steroid mixture of alphaxalone and alphadolone acetate (0-9 %:0-3 % w/v) (Althesin, Glaxo Laboratories) was given either in its concentrated form or after 1:10 dilution with normal saline, in total steroid doses of 3-6-24 mg (1-6 mg/kg). Ketamine

M. E. MORRIS

310

hydrochloride (Ketalar, Parke, Davis & Co. Ltd.) (0-5 or 2-5 % w/v) was injected in doses of 25-75 mg (8-24 mg/kg). Pentobarbitone sodium (Nembutal, Abbott Laboratories Ltd.), prepared as a 1 % (w/v) solution, was given in doses of 32-37-5 mg (10-11 mg/kg). The effects of each of the five different anaesthetics were studied in two to ten animals (total number of tests was fifty-nine). With multiple administrations of these agents in a single experiment the order of drug tests was varied to permit each to be the first tested, and the interval between doses or drugs was usually 30-60 min (50-4+ 46-1 (S.D.) min). The long-acting barbiturate, pentobarbitone, was always injected towards the end of an experiment. Table 1 summarizes the doses and number of tests for the different anaesthetics. A

B

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Fig. 1. Stimulation and recording arrangement for studies of cuneate transmission evoked from periphery. A,J-L stimulation of one or more peripheral forelimb nerves (PN). 0 recording of the antidromic potential from distal nerve electrode, and at medial lemniscus (ML) in brain stem. B, examples of recorded potentials, as displayed by oscilloscope computer sampling system. Arrows mark limits chosen for calculation of stored integrals. C, example of display of input-output plots of integrals of responses (x (input), antidromically conducted potential recorded from peripheral nerve (PN); y (output), at medial lemniscus (ML)) simultaneously evoked from single repeating cycles of varying intensities of nerve stimulation with pulses of 0-2 msec duration. In each display heavy line is curve of best fit for control points. Effects shown at 1, 3, 4, and 6 min after start of injection of alphaxalone/alphadolone (Althesin) 9-6 mg (2-5 mg/kg); note elevation of data points above control curve, followed by recovery. RESULTS

Cuneate tran8mts8ton evoked from peripheral nerve The efficiency of transmission to the medial lemniscus of responses to stimulation of a forelimb nerve was most commonly facilitated following the administration of each of the anaesthetics tested in this study. This is demonstrated in the inputoutput plots of Fig. 1 C, which display integral values (limits marked in 1 B) of the antidromic potentials which monitored the afferent input volley from the nerve, and the synaptic lemniscal output, in response to varying intensities of stimulation. Here data for single stimulus cycles provide a typical example of the progressive and

A NAESTHETICS AND SYNAPTIC TRANSMISSION 311 reversible effect of an injection of alphaxalone/alphadolone (Althesin) 9-6 mg (2.5 mg/kg) to increase output above the curve of best fit for the control relation. Similar potentiations of afferent transmission from the periphery were found for other anaesthetics, as can be seen in the plots of Fig. 2. In each case the comparison of grouped data from four consecutive stimulus cycles shows how administrations of halothane, ketamine, and methohexitone reversibly increased the lemniscal outA

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Fig. 2. Effects of three different anaesthetics to augment lemniscal transmission from peripheral nerve. Each plot displays input (x)-output (y) relation of integrals of potentials (as described in Fig. 1) in response to four consecutive cycles of varying intensities of peripheral nerve stimulation, and curve of best fit for control data. A, 2 % halothane inhaled for 6-5 min; B and C, ketamine 25 mg (7 mg/kg) and methohexitone 10 mg (3 mg/kg) I-V respectively (note: B and C are data from same animal; displays at times after start of injection).

put for unchanged peripheral input (i.e. there were no changes in excitability threshold and the stimulus-response relationship at the peripheral nerve). Clear-cut facilitation, such as seen here was found in fourteen of sixteen different administrations of these four different anaesthetics to five cats. On two occasions there was a

312 M. E. MORRIS depression of output which was associated with a fall in blood pressure; in one other experiment there was a brief decline in output, followed by a sustained increase. Synaptic transmission evoked by cuneate stimulation The effects of anaesthetics on cuneate synaptic transmission were also studied by observing changes in synaptically mediated lemniscal responses to direct microelectrode stimulation of the cuneate afferent fibre terminals (see Fig. 3A), while recording the antidromically conducted potential at a peripheral nerve. This provided not only an estimate of synaptic input but also additional information about the state of excitability of the presynaptic terminals. Although input is likely to be only partially measured with this technique, the cuneate input-output relation so obtained showed considerable resemblance to that found when afferent volleys were TABLE 2. Comparison of the mean values of a and b from best-fit equation y = aXb for recording input (x) and output (y) potentials evoked by cuneate and peripheral nerve stimulation. Group 1, control data from three cats only, where both types of stimulation used in same preparation (nine and thirteen separate runs respectively). Group 2, control data from twelve cats (thirtytwo separate runs) during cuneate stimulation, and from five cats (seventeen runs) during peripheral nerve stimulation (incorporates results from Group 1). Group 3, ninety-two control runs from fourteen experiments during cuneate stimulation (data from Krnjevic & Morris, 1976). In each case data (mean+ S.D., with n = number of separate runs) derived from responses of four consecutive stimulus cycles Cuneate stimulation

Peripheral nerve stimulation

~~~-A

,

A

-

a b 9 0-633 (±0-222) 0-434 (±0-156) 32 0-648 (±0-215) 0-479 (±0-150) 92 0-898 (±0-280) 0-498 (±0-168)

n

Group 1 Group 2 Group 3

n

b

a

13 0-783 (±0-206) 0-458 (+0-173) 17 0-769 (±0-228) 0-529 (+0-208)

TABLE 3. Mean values (± S.D.) for a and b of best-fit equations y = aXb for both 'random' and non-random presentation of 1/sec stimuli, either in the cuneate or at the periphery. A, data from one cat (n = number of stimulus cycles). B, data represent initial control runs from different experiments (n = number of animals)

Cuneate stimulation ~~~~A

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n

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b

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,

n

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A

b

0-626 (±0-015) 0-473 (±0-018) 0-641 (± 0-030) 0-475 (± 0-032) 0-777 (±0-259) 0-447 (±0-136) 3 0-928 (± 0-182) 0-468 (± 0-059) 0-686 (±0-222) 0-516 (±0-125) 1 0-629 0-653

evoked from the periphery. Table 2 compares the parameters a and b of the best-fit power function y = axb, calculated for data collected by these two techniques. Groups 1 and 2 are from the experiments of this paper, while for further comparison, data from earlier experiments (Krnjevi6 & Morris, 1976) are included as Group 3. Although the mean a and b values within Group 1 (where both techniques are used) were higher with peripheral nerve stimulation than with cuneate stimulation, they were not significantly different (P > 0-05). There was also no significant difference when the b data from Group 3 were compared with those of Group 2; the significant

313 ANAESTHETICS AND SYNAPTIC TRANSMISSION difference (P < 0.001) observed for a comparison of the a coefficients in these same experiments is related to differences in the selection of amplifier recording gains, and does not carry the same weight as would hold for the exponent, b. Table 3 shows the a and b values for input-output data generated by random and non-random presentations of the varying stimulus intensities (see Methods section). Comparisons showed no significant differences (t test, P > 0. 1) both within and between Group A (where paired observations were made in individual animals) and Group B (where different stimulation techniques were used in different animals). BImin

A ML

ML .

JVPN9SA

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Methohexitone 12 mg

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5 msec Fig. 3. Effect of methohexitone on potentials evoked by cuneate stimulation. A, diagram of stimulation and recording arrangement for studies of cuneate synaptic efficiency. Potentials recorded from medial lemniscus (ML) at transacted brain stem, and a peripheral forelimb nerve (PN) in response to repetitive cycles of varying intensities of micro-electrode stimulation in cuneate. B, oscilloscope traces of potentials recorded at medial lemniscus (ML) and the superficial radial nerve (PN) in response to intranuclear stimulation at a rate of 1/sec. Dotted lines at level of control peak potential amplitudes are reproduced on records at 1, 3, and 20 min after injection of methohexitone 12 mg (3.2 mg/kg).

Fig. 3B shows examples from one experiment of the changes produced by methohexitone 12 mg (3.2 mg/kg) in the evoked potentials (trans-synaptic (ML) responses above, peripheral nerve (PN) responses below). These responses to a single current value (which were selected from repeating series such as those of Fig. 4A) show the typical depressant effect of the anaesthetic on the afferent terminal excitability (represented by the antidromic nerve potentials) - both the initial direct response, and the indirect repetitive activity, which follows and is known as the dorsal column reflex (Therman, 1941; Andersen et al. 1964). At the same time there was no obvious decrease in the trans-synaptic lemniscal response. The plotted integrals of the relevant portions of these potentials, chosen so as to exclude the later reflex activity and represent the related responses to presynaptic terminal excitation (see Fig. 4A between arrows), show in another experiment with a single stimulus series above, and the superimposition of four consecutive cycles below, that data points were shifted above control curves following the injection of methohexitone 10 mg (2.9 mg/kg). This facilitation of synaptic output was accompanied by a small,

314 M. E. MORRIS distinct diminution in input (X values shifted to left; note the difference in endpoints for control curve and that obtained with anaesthetic.) Similar increases in output, in association with and in spite of a decreased input, were seen in thirty-five of forty tests in data from seventeen animals, using each of the five different anaesthetics. The examples of Fig. 5 illustrate these changes during separate administrations of three different agents (halothane, alphaxalone/ alphadolone (Althesin), and methohexitone) to a single animal. Differences in the A

B

ML

PN

E>K

Control

>

H

Methohexitone mg ~~~~10 a

Recovery

.-..

0. 5 msec

Fig. 4. Effect of methohexitone on input-output data, generated by cuneate stimulation. A, oscilloscope traces of potentials evoked by cuneate stimuli (eight examples of responses, selected from those produced by 16 different intensities of one cycle). Chosen limits of integral values for computer storage are marked by arrows. B, examples of computer display of input-output plots, constructed from integrals of PN (input) and ML (output) responses. Above: responses to a single cycle (with curve of best fit for control points). Below: displays of four consecutive stimulus cycles; lines of best fit for data and control points. Centre plots demonstrate effect of methohexitone 10 mg (3 mg/kg) to shift points and their curve to left and upwards. extent of facilitation of responses to different intensities of stimulation (cf. the curve of 5A with the flatter ones of B and C where the degree of enhancement tapered off at the higher current values) are likely to depend largely on the range of stimulus values chosen, which allow greater or less recruitment of the post-synaptic neurones. In four out of five tests in which there was instead a depression of the input-output curves (always in combination with an increase in the antidromic potential, i.e. in terminal excitability) this was associated with a marked hypotension, where mean arterial levels were

Facilitation of synaptic transmission by general anaesthetics.

J. PhyWol. (1978), 284, pp. 307-325 With 12 text-figur Printed in Geat Britain 307 FACILITATION OF SYNAPTIC TRANSMISSION BY GENERAL ANAESTHETICS BY...
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