Anaesthetic agents and the chemical sensitivity of cortical neurones

Neuropkrmacology, 1970,9, 31-46Pergamon Press.Printed inGt.Britain.

ANAESTHETIC AGENTS AND THE CHEMICAL SENSITIVITY OF CORTICAL NEURONES” Department

J. M. CRAWFORD of Physiology, The John Curtin School of Medical Research, Australian National University, Canberra (Accepted 5 May 1969)

Summary-Volatile and non-volatile anaesthetic agents have been administered systemically to cerveau isol6 cats while recordings were made of the frequency of the spontaneous and druginduced firing of single pericruciate cortical neurones. Upon a given cell, an anaesthetic produced essentially similar effects on both the spontaneous synaptic firing and the responses to alternate microelectrophoretic ejection of acetylcholine and excitant amino acids. Barbiturates depressed the chemical sensitivity of cortical neurones in doses well below those necessary for surgical anaesthesia, the duration of depression resembling the persistence of each agent m clinical practice. Sub-anaesthetic doses of urethane were not very effective, but full anaesthetic doses of chloralose markedly reduced chemical sensitivity of these cells for long periods. Low to moderate concentrations (“analgesic” levels) of nitrous oxide, trichlorethylene and halothane had little direct effect on the firing of cortical neurones. The effects of higher “anaesthetic” concentrations of nitrous oxide on the cells were obscured by associated hypoxia, but halothane levels exceeding 1.5% depressed excitability. Methoxyflurane seems remarkable in that it did not appreciably alter chemical sensitivity of cortical neurones, even at full anaesthetic levels. None of the agents tested appeared to prevent depression of corticai neurones by GABA. It is concluded that general anaesthetics non-specifically reduce the chemical excitability of cortical neurones, possibly by an action on postsynaptic membrane conductance changes. However, it may be concluded that neurones in various regions of the nervous system differ in their susceptibility ‘to particular anaesthetics. HITHERTO, most pharmacological studies of central neurones have been made on anaesthetised preparations, though it is well recognized that interpretation of the observed drug actions may be complicated by the presence of the anaesthetic (e.g. MARLEY and VANE, 1963 ; BISCOEand KRNJEVIC,1963 ; KRNJEVICand FHILLIS, 1963~; BAUMGARTENet al., 1963 ; SALMOIRAGHIand STEINER, 1963; SALMOIRAGHI and STEFANIS,1965; BLOOM et al., 1965; SALMOIRAGHIand WEIGHT, 1967; PHILLISand TEBBCIS,1967). Variability in the proportion of cortical cells affected by microelectrophoretic ejection of ACh in different preparations has also been attributed to the state of anaesthesia (KRNJEVICand PHILLIS, 1963b, c; RANDIC et al., 1964); although this is not the only factor involved (see also CRAWFORD and CURTIS, 1966; SALMOIRAGHI and STEFANIS, 1967). On the other hand, unanaesthetised preparations may not always be entirely suitable for particular studies, even if they are acceptable on humane grounds. The necessary surgical procedures may interrupt physiological pathways of interest; the animals as a rule require to be paralysed and artificially ventilated for long periods, altering their condition from the *This investigation was supported in part by grant NB 05774, National Institute of Neurological Diseases, U.S. Public Health Service. 31 c

32

J. M. C%A~~~RD

physiological normal; and the relatively large amount of apparently random afferent activity, both excitatory and inhibitory, can mask small drug-induced effects. It was of interest therefore to examine the effects of a series of anaesthetics upon the sensitivity of cortical neurones to acetylcholine and DL-homocysteic acid as representative excitants, and to the depressant substance y-aminobutyric acid, and to compare these effects with changes in the spontaneous synaptic firing of the cells. In this manner it was hoped to determine whether any of the commonly-used anaesthetics had specific actions upon the excitation or depression of neurones by these compounds, or whether the changes in chemical sensitivity merely reflected depression of neuronal excitability in anaesthesia; and perhaps to discover satisfactory conditions for the study of chemical sensitivity of neurones in anaesthetised cats. This work is a continuation of studies reported previously (CRAWFORDand CURTIS, 1966).

METHODS Forty-five adult cats of either sex were used. These were surgically prepared under halothane anaesthesia induced with the aid of a face mask. Anaesthesia was maintained by concentrations of 18-2.5 “/, halothane from a Fluotec vaporizer (Cyprane Ltd.), administered through a tracheal cannula. The animal’s blood pressure was continuously monitored by a strain gauge transducer (Statham P23Db) connected to a cannula placed in the femoral artery. The systolic and diastolic blood pressure readings were recorded at intervals during the experiment, while a continuous record of the mean blood pressure could also be made by a Servo-Riter connected to the monitor instrument (Statham model AM2). The pericruciate cortex was exposed by removal of the overlying calvarium as previously described (CRAWFORDand CURTIS, 1964). A small oval area of bone over the cerebellum was also removed to permit insertion of a rack of four insulated steel needles for highfrequency electrocoagulation of the brainstem (CRAWFORDand CURTIS, 1966). This rack was inserted stereotaxically by a small micromanipulator attached to the headframe, the needle track lying close under the tentorium at an angle of 48” to the Horsley-Clarke vertical. Currents of 35-40 nA at 500 kHz were passed between adjacent needles (2 mm apart) by means of a Wyss coagulator (Type OC60; J. Monti, Geneva), the deepest lesions being made at a point just behind the dorsum sellae. Successive lesions were made as the rack was withdrawn in steps of l-1.5 mm over a total range of 12 mm. Following decerebration the anaesthetic was discontinued, and in almost all animals the pupils became constricted. There was no pupillary response to painful stimuli or to light. Limb muscle tone and reflexes became exaggerated after IO-15 min, and it was often necessary to paralyse the animal with gallamine triethiodide (Flaxedil, May and Baker Ltd. ; 5-8 mg/kg intravenously, supplemented every half hour by doses of 3-4 mg/kg). Artificial respiration was commenced, its depth being adjusted by a variable-resistance outlet valve on the side arm of the tracheal cannula in order to maintain the cat’s end-tidal COz (continuously monitored by a Beckman Medical Gas Analyzer, model LB-l) at 3.6-42 % because the excitability of cortical neurones varies with the local CO, tension (KRNJEVICet al., 1965). In some experiments a small silver-ball electrode was placed on the surface of the somatosensory cortex after opening the dura, and the effectiveness of the decerebration was confirmed by the failure to elicit changes in cortical activity by strong stimuli to the paws or face. The position and extent of the coagulated areas of the brainstem were determined

Anaesthetics and cortical neurones

33

by postmortem examination, and were in every case bilateral, involving the pontine nuclei, the medial lemnisci, the medial longitudinal bundles and the brachia conjunctiva. As in the earlier investigations, after incision of the dura the cortex was kept flushed with a warm (38°C) mammalian Ringer solution equilibrated with 95 % Oa and 5 % CO,, and was covered with polyethylene sheeting except for the area into which the microelectrode was to be inserted. The pia mater was carefully teased from suitable small areas of the surface, and a small pressor foot was used to control cortical pulsation. Extracellular spike potentials from pericruciate neurones were recorded by the centre barrel (containing 4M NaCI) of five-barrel micropipettes of 4-6 pm overall tip diameter. The outer barrels as a rule contained solutions of DL-homocysteic acid (DLH; 0*2M, pH 85-g), acetylcholine chloride (ACh; O-5 or 1 M), y-aminobutyric acid (GABA; 0.2 or O-5M, pH 4.5) and either 5-hydroxytryptamine creatinine sulphate (5HT; saturated solution, approximately 0*05M) or 4M NaCI. The latter could be used to check the effects of the passage of current upon particular neurones. Diffusion from each pipette was controlled by appropriately directed retaining currents, the required potential being as a rule 0.5 or 1V. The microelectrodes were filled by centrifugation less than 24 hr before use, and were stored at 34°C. At least 13 hr elapsed from the end of halothane anaesthesia to the start of recording. The firing frequency of selected neurones was plotted directly on a rectilinear paper recorder (Recti-Riter, Texas Instruments). The indicating system, and the precautions necessary to ensure an accurate count, have been described previously (ANDERSENand CURTIS, 1964; CRAWFORDand CURTIS,1964). Non-volatile anaesthetics (pentobarbitone, diallylbarbituric acid, methylthioethyl-2-pentyl-thiobarbiturate, urethane and a-chloralose) were administered intravenously as solutions in isotonic saline, while the gaseous and volatile agents were introduced through the Palmer respiration pump. A series of vaporisers, previously calibrated for the gas flow rates used, enabled known vapour concentrations of halothane, methoxyflurane or trichlorethylene to be added to the air or nitrous oxide-oxygen mixtures supplied to the pump and thence to the cat. Gas flow was kept well in excess of the minute volume delivered to the animal, excess anaesthetic mixture passing through a mercury bubble-trap to the atmosphere outside the shielded room. Under these conditions there was minimal contamination at the pump intake between successive gas mixtures supplied to the cat, and a stable concentration of anaesthetic vapour was reached at the tracheal cannula less than 20-25 set after any alteration of the vaporiser settings. Occasionally changes in systemic blood pressure, and more rarely alterations in the recorded spike potential, were seen after administration of the anaesthetics. Where the spike altered concurrently with the blood pressure, results had to be discarded because of possible relative movement of the cell with respect to the microelectrode tip. This could affect the concentrations of electrophoretically ejected substances reaching the receptors, and thus obscure changes in the cell’s sensitivity to the drugs. Also, relatively little weight could be given to several runs in which either the blood pressure, or the cell’s chemical sensitivity failed to return substantially to the control levels after the anaesthetic had had time to be removed from the system. Nevertheless, such incomplete runs often provided confirmatory evidence of the actions of particular anaesthetic agents. Throughout each experiment the cortical surface was viewed under a dissecting microscope. The experiment was terminated when either changes in circulation or cortical swelling became marked. The animal’s body temperature, measured by a subscapular probe, was maintained at 37&0*8”C by means of a thermostatically-controlled heating element beneath its abdomen.

34

J. M. CRAWFORD

RESULTS Sufficiently stable responses were obtained from 60 pre- and post-cruciate neurones to warrant testing the effect of anaesthetics. Between 5 and 15 min recording was needed to establish control values for the spontaneous and drug-induced firing rates of each neurone, while observations of recovery from an anaesthetic required that the cell be “held” for from 45-130 min. In only a few cases was it possible to test more than one anaesthetic agent on a given neurone. Cells were located at depths of 620-1380 pm beneath the cortical surface, and those readily excited by the microelectrophoretic ejection of both DLH and ACh were selected for further study. Characteristically they possessed an irregular spontaneous activity of some 5-25 spikes/set. No attempt was made to identify the cells physiologically, but their location, chemical sensitivity and spontaneous activity resemble those previously described for Betz cells and other acetylcholine-sensitive deep pyramidal neurones (KRNJEVIC and PHILLIS,1963b, c; KRNJEVIC,1964; CRAWFORD and CURTIS,1966). Barbiturates have previously been shown to reduce the overall rate of spontaneous activity of cortical neurones, and to alter its character from an irregular “projection” activity to one of grouped discharges or “spindles” (see KRNJEVICand PHILLIS, 1963 ; CRAWFORDand CURTIS, 1966). The reduction in firing rate presumably reflects a decrease in the amount of afferent activity or its effectiveness in producing synaptic depolarisation of the neurone, upon which excitation by micro-electrophoretically ejected drugs is superimposed. Thus it was important during the control period to find doses of ACh and DLH which elicited comparable (submaximal) firing rates. Unless this was done, removal of the background synaptic depolarization might reduce just-suprathreshold firing produced by one excitant, yet cause little change in firing due to a more potent agent, or even in that produced by larger doses of the first compound. On the basis of currents required to attain equally effective concentrations, DLH was from 1.4-9.1 times more potent than ACh (mean ratio 4.5). In other tests, ACh was approximately two-thirds as potent as L-glutamate upon cortical neurones, an estimate in keeping with the known relative potency of DLH and glutamate when directly compared (see also CRAWFORD and CURTIS,1964). 1. Pentobarbitone Doses of 2-10 mg/kg of sodium pentobarbitone (Nembutal) intravenously, reduced the sensitivity of eight cortical neurons to both ACh and DLH to a comparable extent in every case. An initial rapid decrease in sensitivity over 3-1 min was followed by a further slow fall, reaching its nadir some 5-10 min after giving the anaesthetic. Simultaneously with the fall in chemical sensitivity, the cell’s spontaneous firing was reduced or abolished, and in a few experiments typical barbiturate “spindling” was observed. Recovery of all forms of excitation was relatively slow, requiring from 20 min to over an hour, and there was little difference between the rates of recovery of firing by ACh and DLH, although the spontaneous firing rate almost always reached its control levels before the drug-induced excitation. These effects have been illustrated previously (CRAWFORDand CURTIS, 1966: Fig. 5), though it was not then realised that the synaptic firing usually recovered first, presumably because of a high safety factor for some cortical afferent pathways. As a rule, these doses of pentobarbitone also caused a fall in systemic blood pressure, maximal 15-60 set after the commencement of injection of the barbiturate, and recovering over 8-10 min. However, since there were no associated changes in the size of the extracellularly recorded action potential, and the time-course of depression of chemical sensitivity

Anaesthetics

and cortical neurones

35

differed from that of the hypotension,

it is unlikely that the effect of pentobarbitone on cell excitability was simply an artifact due to the fall in blood pressure. When ejected electrophoretically from freshly-prepared solutions (0*2M, pH 9-9.6), pentobarbitone again depressed the spontaneous activity of twelve pericruciate neurones, and both slowed the onset and reduced the peak firing rate achieved by DLH. The peak excitation due to ACh was initially almost unaffected, but the duration of the firing after ACh was somewhat curtailed. These features are illustrated in Fig. 1, in which pentobarbitone was tested twice upon a cell excited by the alternate ejection of DLH (11 nA) a Pentoborbitone DLH

b DLH 1-

II

20 nA

I

II

i ”

DLH

Pentobarbitone

./

-

20

nA DLH

FIG. 1. Firing frequency record of a spontaneously active pericruciate neurone excited by ahernate ejection of DLH (11 nA, upper markers) and ACh (38 nA, lower markers). Record b commences 5 min after the end of record a. During the periods shown by the topmost horizontal bars, pentobarbitone was ejected as an anion with currents of 20 nA. Ordinates: Firing frequency in spikes per second; Abscissa: Time in minutes.

and ACh (38 nA). At the first trial, the firing due to DLH was virtually abolished within 45 set by pentobarbitone (20 nA), but recovered very rapidly after ejection of the barbiturate ceased. Firing due to ACh was briefer, but only slightly less rapid, even after more than 44 min administration of pentobarbitone (Fig. lb). Substantially larger amounts (currents of 50-100 nA) and longer ejection times would however frequently depress or abolish excitation by ACh as well as that by DLH. Both the spontaneous and drug-evoked firing recovered in 15-30 set, even after the largest doses of pentobarbitone. Changes in the shape of the extracellular spike potential were rarely seen during ejection of this barbiturate. These findings confirm the report by KRNJEVIC (1965) of potent but short-lasting depression of cortical neurones by micro-electrophoretic administration of pentobarbitone (and diallylbarbituric acid). The more rapid and more potent depression of DLH-induced firing than of excitation by ACh when pentobarbitone is given microelectrophoretically, contrasted with the almost equal effect of intravenous administration on the two firing rates, has suggested that the receptors activated by DLH may receive a higher concentration of the barbiturate during

36

J. M.

CRAWFQRD

microelectrophoresis. This in turn presumably reflects differences in the spatial distribution of amino acid and ACh-receptors upon cortical neurons (see CRAWFORD and CURTIS,1966). 2. Other barbiturates One short-acting barbiturate (Thiogenal, Merck A.G.; sodium methylthioethyl-2-pentyl thiobarbiturate) and another intermediate to long-duration derivative (Dial, Ciba Ltd.; sodium diallylbarbiturate) have been given systemically in doses of 5-20 mg/kg. Both compounds depressed the spontaneous and ACh- and DLH-induced firing in a qualitatively similar manner to pentobarbitone, the most marked difference being in their time-course of action. Apart from one cat in which two doses of 5 mg/kg of Dial each reduced the spontaneous activity and the sensitivity of a pericruciate neurone to ACh but apparently had little effect on its response to DLH, neither barbiturate specifically antagonised either of the excitants. Typical results from one experiment with Thiogenal are shown in Fig. 2. The cell was relatively sensitive to both ACh and DLH, the points on the graphs representing the peak Thiogenol 5

160-

IQmg/kg

IOmg/kg

I

I

I

I

I

I

I

I

140-

+

DLH

25

.

ACh

100

nA nA

min

FIG. 2. Peak firing frequency record of a pericruciate neurone in response to alternate microelectrophoreticejection of DLH (25 nA, +) and ACh (100 nA, 0). Successive doses of Thiogenal (5, 10 and 10 mg/kg, i.v.) were given at the times shown by the arrows. Further description in

text. Abscissa: Time in minutes.

37

Anaestheticsand corticalneurones

firing frequencies elicited by alternate ejections of each excitant. The first dose (5 mg/kg) of Thiogenal produced no apparent change in these peak firing rates, but a subsequent dose of 10 mg/kg rapidly reduced both responses. Some 16 min later the responses had recovered, and both ACh and DLH fired the cell at rates well above the control values, presumably because of slight relative movement of the electrode with respect to the cell. A second dose of 10 mg/kg Thiogenal again reduced the sensitivity to both excitants and converted the spontaneous firing (not shown in Fig. 2) into a typical pattern of barbiturate “spindles”. The cell was unfortunately impaled some 9 min later, but it was already evident that substantial recovery of chemical sensitivity had occurred. The time course of depression by Thiogenal was thus much closer to that of the brief hypotension due to its injection than was the case with either pentobarbitone or Dial. The depressant action of Dial upon chemical excitation was very little longer than that of pentobarbitone, requiring 5-10 min to reach its peak and lasting up to an hour. Phenobarbitone was ejected with currents of 30-60 nA near three cells, and reduced both spontaneous activity and chemical excitability in each case. Firing by DLH was slightly more susceptible to depression by microelectrophoretic phenobarbitone than was synaptic firing or ACh-induced excitation, and concentrations of phenobarbitone adequate to depress sensitivity to ACh also reduced the size of the extracellular spike potential. All responses recovered over 4-6 min after termination of the phenobarbitone ejection. 3. Urethane (ethyl carbamate, British Drug Houses Ltd.) Urethane appeared to depress chemical sensitivity of cortical neurones less readily than the barbiturates. Although only four cells were tested, doses of 50 and 100 mg/kg of urethane seemed to have no effect on either the synaptic or drug-evoked firing. Even 200 mg/kg (roughly 5-k of the usual anaesthetic dose in a cat) produced only a slight decrease in the background activity of two cells, results from one of which are shown in Fig. 3, and Urethane

200mgIkg + DLH IlnA ACh 14nA 0 ACh 94nA

l

IO-

0

I

1 I IO

I

I 20

I

I 30

I

I 40

J 45

min

FIG. 3. Peak firing frequency record of a pericruciate neurone excited by DLH (11 nA, +) and ACh (14 nA, 0). The mean spontaneous firing rate just before the ejection of each excitant is also indicated (V), together with the apparently maximal responses evoked by ejection of ACh with currents of 66 nA (m and 94 nA (0). 200 mg/kg of urethane were given intravenously at the time shown by the horizontal bar. Abscissa: Time in minutes.

38

J.M.

CRAWFORD

a parallel but more marked reduction in their sensitivity to both ACh and DLH. This cell was particularly sensitive to ACh, maximal excitation with this substance occurring with ejecting currents of 50 nA, while ejection of ACh with only 14 nA caused almost the same rate of firing as did DLH ejected with 11 nA. No detailed dose-response curves were obtained but it will be noted that the maximal ACh-response (to 66 and 94 nA) was reduced after urethane. Depression of both ACh and DLH response was greatest 6-10 min after the anaesthetic, and they recovered together over about 40 min. 4. a-Chloralose (British Drug Houses Ltd.; Fluka A.G.)

Commercial chloralose was dissolved in hot water, allowed to cool, and filtered before intravenous injection in doses of 30 and 50 mg/kg to two preparations. Over the next 3-5 min the spontaneous firing of the three cells tested was greatly reduced, and their responses to the test doses of ACh (60-200 nA) and DLH (15 nA) were abolished. No recovery of firing by these doses of either excitant was evident over the next 30 min, although a larger amount of DLH (30 or 40 nA) could still excite the cells. Subsequent exploration of the pericruciate cortex in these animals over 2-3 hr yielded a smaller proportion of cells fired by ACh than had been found prior to the administration of chloralose, but too few cells were excited even by DLH for the results to be significant. Several cells which were not directly fired by ACh showed facilitation of the amino acid-induced firing by the more potent cholinergic substances acetyl-b-methylcholine or carbamylcholine. 5. Procaine Procaine was ejected electrophoretically (current of IO-20 nA from 0.1 M solutions of the hydrochloride) near three cholinoceptive neurones. Their sensitivity to both DLH and ACh was progressively reduced over some ten minutes, and concurrently their spontaneous activity was almost abolished. Because the spike height also decreased during the ejection of procaine, accurate estimates of the firing-rate were feasible only when the voltagethreshold of the indicating system (ANDERSENand CURTIS, 1964) could be lowered, i.e. when the spike under observation was initially much larger than those of neighbouring cells. Results from one such neurone are illustrated in Fig. 4. Recovery of the amino acid sensitivity followed within a few minutes of ceasing the ejection of procaine, and was as a rule accompanied by a substantial recovery of spontaneous firing. Recovery of the ACh responses was somewhat slower, perhaps indicating some specific antagonism by procaine at the ACh-receptors like that shown at the neuro-muscular junction by DEL CASTILLO and KATZ (1957). Previous reports have indicated mainly non-specific actions of procaine upon spinal (CURTIS and PHILLIS, 1960) and cortical neurones (KRNJEVIC and PHILLIS, 1963a, c; KRNJEVIC,1965), but these studies did not attempt to observe actions upon different excitants of the same cell. It was not possible to show any effect of intravenous procaine (4-4.5 mg/kg) or procaine amide (2 and 5 mg/kg) upon either ACh- or DLH-induced excitation of four cortical neurones in two cats. Sixty mg/kg of procaine i.v. has also been reported not to alter cortical surface inhibition (KRNJEVICet al., 1966). 6. Nitrous oxide-oxygen mixtures Concentrations of SO-SO% of nitrous oxide had variable but small effects upon the chemical sensitivity of cortical neurones. The overall spontaneous firing rate was usually almost unaltered, though occasionally the cell’s discharge pattern changed and bursts of

Anaesthetics and cortical neurones Procaine I

I

IOnA

i

20nA

60% c 0 r 6

40-

min FIG.

4.

Peak firing frequency record of a pericruciate neurone in response to DLH (15 nA, t) and ACh (30 nA, 0). The mean spontaneous firing rate prior to ejection of each excitant is also shown (0). During the period shown, procaine was ejected as a cation with currents of 10 and 20 nA. Further description in text. Abscissa: Time in minutes. I

110

I I I N,O

20

0

I

5

I 60%

I

!

60%

I

1 ,

90%

I

I

I

/

I

I

1

+DLH25nA

I I

I I

I I

I I

l

I

I

I

I

I IO

--

-

I 15

-

Bursts

20

I

-

-

25

-

ACh

70nA

30

I 32

-,

FIG. 5. Effects of various concentrations of nitrous oxide in the mixture breathed by an artificially-ventilated cerveau isole’ cat. The uppermost curve indicates the mean femoral arterial blood pressure (mm Hg), in relation to an arbitrary datum line at 130 mm Hg. The lower graphs indicate the peak firing frequencies elicited by ejection of DLH (24 nA, +) and ACh (70 nA, 0). The animal was respired initially with room air, and during the period of the horizontal bar with nitrous oxide mixtures in oxygen, the concentration of N,O increasing from 60 to 80 and then 90 % at the broken vertical lines. The cat was again ventilated with room air at the end of N,O administration. During the period indicated, high frequency “bursts” of firing were noted (see text). Abscissa: Time in minutes.

40

J. M. CRAWFORD

relatively high frequency firing (at rates of up to 40-60 spikes/set) were seen every few seconds. In 4 out of 7 cells tested, the responses to ACh and DLH were slightly enhanced by these concentrations of nitrous oxide, but in the remaining cells chemical sensitivity was unaltered. The increase in drug-evoked firing was less than 15-20% of the control values, and was unaccompanied by changes in the spike potential shape. Changes in blood pressure and cortical circulation with these concentrations were slight. Mixtures of 90% nitrous oxide and 10% oxygen usually increased the systemic blood pressure by 10-20 mm Hg, but had unpredictable effects on the chemical sensitivity of cortical neurones. Both cells tested with this concentration showed an initial slight enhancement of chemical sensitivity, followed by depression. Spontaneous firing was little changed, although the high-frequency bursts became somewhat more common. Recovery of blood pressure, spontaneous firing and chemical excitability occurred within 2-5 min of the end of nitrous oxide administration. A typical experiment, in which concentrations of 60, 80 and 90 % of N,O were tested, is illustrated in Fig. 5. The variable excitatory effects seen, particularly with over 80% N,O probably result from systemic hypoxia (cf. HELLERand WATSON,1962) and the fluctuations in blood pressure which occurred with this anaesthetic. Irregular bursts of firing were noted in cortical neurones of a cat deliberately subjected to brief periods of anoxia (CRAWFORD,1965), and other studies have shown depolarization of hypoxic spinal motoneurones (KOLMODINand SKOGLUND,1959; LBYNING,1965; but see NELSONand FRANK, 1963). 7. Trichlorethylene (Trilene, I.C.I.A.N.Z.) This was administered at concentrations of 0.15-0.6 % v/v in air to three preparations. In one cell, administration of 0.4 “/;:trichlorethylene was accompanied by a decrease in the extra-cellular spike size and a reduction in the responses to both ACh and DLH which lasted about half an hour after the anaesthetic was discontinued. The remaining tests showed no effect of this range of trichlorethylene concentrations upon either the spike potentials or the chemical sensitivity of pericruciate neurones. The systemic blood pressure was little affected by this anaesthetic. 8. Halothane (],I, I-triJluoro-2-bromo-2-chloroethane; jkothane, I.C.Z. A. N.Z.) This was administered in concentrations of 0.5-2.5 % v/v in air or oxygen using a Fluotec vaporizer (Cyprane Ltd.). Prolonged administration, or concentrations exceeding about 1a5% halothane, caused moderate to severe hypotension and a fall in pulse pressure. Recovery of the blood pressure required 20-30 min after halothane was discontinued. These changes sometimes complicated observations of the chemical sensitivity of neurones by causing movement of the cell relative to the micropipette, but a total of eleven cells in eight cats have been studied satisfactorily under halothane. A typical experiment is illustrated as Fig. 6a and shows that O-5 and 1.0% halothane had relatively little effect upon the spontaneous or chemically evoked firing levels despite a slow fall in systemic blood pressure. Raising the anaesthetic concentration to 1.5% accelerated the decline in blood pressure and slightly reduced the spontaneous firing and the DLH-response of this cell. Two per cent halothane decreased the blood pressure to 40/20 (mm Hg) within 24 min, and virtually abolished spontaneous and chemical firing of the cell. The size of the extracellular spike (when present) was not affected by the anaesthetic. Residual depression of the amino acid excitation was evident 22 min after halothane was discontinued, but thereafter recovery of both chemical and spontaneous firing occurred

41

Anaesthetics and cortical neurones (1 ;

05%

1.0%

1.5%

/

12.0%

Halothane

min b

I”

I

I

I

min FIG. 6.

Effects of halothane and methoxyflurane mixtures upon an artificially-ventilated cerveau isol6 cat. Records a and b show portions of the same experiment. In each case the vertical bars indicate the femoral arterial blood pressure (systolic/diastolic, mm Hg), while the lower graphs plot the peak firing frequencies elicited by alternate ejection of DLH (12 nA, +) and ACh (50 nA, l), as well as the mean spontaneous firing rate (V). The vapour concentrations in air of the anaesthetics were increased at the broken vertical lines as shown. Further description in text. Abscissa: Time in minutes.

gradually

over about ten minutes.

The blood pressure required 40 min to return to its control

levels. Nine other cells tested with halothane levels adequate to maintain clinical anaesthesia showed slight to moderate depression of both ACh- and DLH-induced firing and spontaneous activity once the inspired halothane concentration reached 1.5 %. In the absence of movement artefacts, however, halothane did not significantly reduce the neuronal spike size, even at concentrations of 2.5 % v/v in the inspired gas. In one experiment 2.5 % halothane apparently increased the spike from 600 to 750 ,uV, but a concurrent increase in the responses to ACh and DLH suggested that the effects were secondary to relative displacement of the microelectrode toward the cell. 9. Methoxyflurane (2,Zdichloro-I,

I-diJluoroethy1 methyl ether; penthrane, Abbott Ltd.)

This was administered in concentrations of 0*2- l-5 % v/v in air or oxygen by means of a Pentec vaporizer (Cyprane Ltd.). Slight to moderate hypotension was noted with this anaesthetic in each of five preparations, but the fall in systolic pressure seldom exceeded 20 mm Hg.

42

J. M.

CRAWFORD

The experiment shown in Fig. 6b is a continuation of the same record as Fig. 6a, beginning after complete recovery from the effects of halothane. The chemical sensitivity of this cell, and its spontaneous firing pattern, remained unaffected by 0.2-1.2 % of methoxyflurane in air, despite a slight fall in the systemic blood pressure. In all, six cells were observed during administration of this agent, and in no case was there any significant change in their chemical sensitivity or spontaneous activity. Methoxyflurane was also apparently without affect on the spike potential size. 10. Anaesthetics and the depressant effect of GABA GABA has been shown to produce a rapid, shortlasting depression of spontaneous or chemically-induced firing of cortical neurones (KRNJEVICand PHILLIS, 1963a; CRAWFORD and CURTIS,1964), and to hyperpolarize these cells and reduce their membrane resistance in the same manner as the natural inhibitory synaptic transmitter (KRNJEVIC and SCHWARTZ,1966; 1967; 1968). It was thus of interest to see whether any of the anaesthetics reduced the action of GABA, though cortical inhibition has been reported to be fully developed in the presence of a variety of anaesthetic agents (KRNJEVICet al., 1966). The test-method adopted was to observe depressant actions of GABA on a “background” of ACh and DLH excitation, the dose of GABA being just less that that required to abolish this level of activity. When anaesthetic agents such as pentobarbitone (six cells) or halothane (five cells) were administered, this “background” firing rate fell. Increased ejection of the excitant could sometimes restore firing to the pre-anaesthetic rate, but this was not always possible because of the noise associated with the passage of high electrophoretic currents. However,it was evident that as long as any firing remained, it was rapidly depressed or abolished by the ejection of GABA. Figure 7 illustrates the failure of methoxyflurane to affect the action of GABA upon one of five pericruciate neurones tested. Since this anaesthetic did not reduce the cell’s firing rate in response to the continuous ejection of ACh (45 nA) during the period of

I GABA;

min

FIG. 7. Depression of ACh-induced firing of a pericruciate cortical neurone by GABA (pH 4.5 ; cationic current of 15 nA). Successive tracings of the portions of the firing frequency record were made, (A) during the initial control period, (B) during administration of 15 % methoxytlurane vapour in air, and (C) 49 min after the discontinuation of the anaesthetic. The high background firing rate in each tracing was elicited by the continuous ejection of acetylcholine (45 nA). Further description in text. Abscissa: Time in minutes.

Anaestheticsand cortical neurones

43

observation, conditions were satisfactory for the detection of any specific effect on the depression due to GABA (15 nA). The tracings were made just prior to the administration of methoxyflurane, after 84 min exposure to increasing concentrations (05, 1-Oand I*5 %) of the anaesthetic, and 44 min after it was discontinued. There was evidently no change in the response to the amino acid under methoxyflurane.

DISCUSSION The present results confirm reports that the sensitivity of cortical neurones to excitants is affected by a range of anaesthetic agents (KRNJEVIC and PHILLIS,1963c; KRNJEVIC,1965; KRNJEVICet al., 1966; CRAWFORD and CURTIS,1966; ROBSON,1967). When any particular anaesthetic is given systemically, the duration of its action on chemical sensitivity appears to be related to the persistence of clinical anaesthesia it produces, but there seems to be no simple relation between its potency as an anaesthetic and as a depressant of neuronal excitability in the cortex. Thus, depression of chemical sensitivity by Thiogenal was always much briefer than that due to pentobarbitone or diallylbarbituric acid, and the recovery of excitability after 1*5-2.5% halothane required similar times as were taken for substantial clinical improvement. On the other hand, all the barbiturates reduced chemical sensitivity and synaptic bring of cortical neurones with doses much less than the 30-40 mg/kg required to anaesthetise a cat for surgery, and at adequate anaesthetic concentrations both chloralose and halothane markedly depressed these cells. Yet urethane (at doses of 200 mg/kg) was not very effective in reducing neuronal excitability, and with methoxyflurane satisfactory anaesthesia could be attained without any decrease in the chemical sensitivity of cortical neurones. Methoxyflurane has also been reported to have relatively little depressant action on the sensitivity of thalamic neurones (PHILLISand TEBBCIS,1967). The most marked drawback to the routine use of methoxyflurane appears to be its respiratory depression, and to a lesser extent the hypotension seen particularly above about 0.8 % v/v concentrations. The present experiments overcame the respiratory effects by the administration of the vapour mixture through the pump which continuously ventilated the animals. Neither nitrous oxide nor trichlorethylene appeared as satisfactory an anaesthetic for pharmacological investigations as did methoxyflurane. Although trichlorethylene was tested only at “analgesic” concentrations (below O-6% v/v), it produced spike changes and depression of one of three cells, and would be expected to have more pronounced actions at higher concentrations. The use of nitrous oxide-oxygen mixtures containing 80% or more of nitrous oxide was always associated with effects attributable to hypoxia and secondary circulatory changes. Furthermore, it has been suggested that in species other than man, nitrous oxide mixtures at atmospheric pressure require the presence of hypoxia to produce adequate surgical anaesthesia (BROWNet al., 1927; see also PRICE and DRIPPS, 1965). In the present study, the direct indication of each cell’s firing frequency has greatly facilitated adjustment of experimental conditions. Provided tests were made on comparable firing rates due to each excitant, all anaesthetics tested affected the responses to ACh and the excitant amino acids (and the synaptic firing of the cell) in essentially parallel fashion. Thus it has not been possible to confirm earlier reports of a more profound depression of firing by ACh than of L-glutamate excitation of cortical neurones after Dial, pentobarbitone or chloralose (KRNJEVICand PHILLIS, 1963c). However, it was noted that if the ACh-ejecting current was reduced during the control period so that ACh excited the cell at only about

44

J. M. CRAWFORD

half the rate due to DLH or L-glutamate, administration of an anaesthetic would abolish direct firing by ACh while merely reducing that due to the amino acid. As noted in the experiments described by KRNJEVICand PHILLIS(1963c), ACh-excitation was then usually still evident as a facilitation of the amino acid-induced firing. The major limitations of the present method of studying anaesthetic actions arise from the extracellular position of the recording electrode. Changes in spike threshold, membrane potential and conductance, and the size of postsynaptic potentials remain undetected except by inference from alterations in the shape, size and frequency of the extracellular spikes. The postsynaptic receptors involved in excitation of cortical neurones by ACh and the acidic amino acids are presumably distinct, since atropine can specifically block the action of ACh on these cells (KRNJEVICand PHILLIS,1963~; CRAWFORD and CURTIS,1966). Anaesthetics, by contrast, decrease the effectiveness of both electrophoretically-ejected excitants (and the natural synaptic transmitters) and hence appear to act non-specifically on the postsynaptic neuronal membrane to reduce its excitability. On the other hand, the present experiments confirm earlier reports (KRNJEVICet al., 1966; KRNJEVICand SCHWARTZ,1967) that the action of GABA and cortical postsynaptic inhibition are little altered by anaesthetics, which thus possibly affect the ion-channels for K+ and Cl- less than they do those for Na+. A decrease in excitability of the cell under observation would account for its reduced synaptic firing rate, even of the afferent activity impinging on the cell were not itself reduced by anaesthesia. However additional presynaptic actions of the anaesthetics, or actions at earlier sites in the afferent pathways to the cell studied, cannot be excluded. Previous studies on autonomic ganglia (LARRABEE and POSTERNAK,1952) and the spinal monosynaptic reflex pathways (AUSTINand PASK, 1952; SOMJENand GILL, 1963 ; SOMJEN, 1963; IBYNING et al., 1964) have indicated that the synapse may be particularly vulnerable to depression by anaesthetics, acting either on the presynaptic transmitter release mechanism, or on the action of the released transmitter at its postsynaptic receptor sites (see also reviews by PATONand SPEDEN,1965; SALMOIRAGHI and WEIGHT, 1967). Intracellular recording from spinal motoneurones has shown both pre- and post-synaptic effects to occur with various anaesthetics, excitatory post-synaptic potentials being reduced in size (SOMJEN and GILL, 1963; SHAPOVALOV, 1964) while the threshold for excitation is raised (SASAKIand OTANI, 1962; SOMJENand GILL, 1963; SHAFQVALOV, 1964) or unchanged (LIZIYNING et al., 1964). Barbiturates, chloralose, ether and urethane alter the time course of spinal dorsal root potentials and their amplitude relative to integrated ventral root reflexes (ECCLESet al., 1963; SCHMIDT,1963), again suggesting actions at primary afferent terminals. With the possible exception of chloralose, from which adequate recovery was not observed, none of the anaesthetics tested showed specific antagonism towards the natural transmitters at cortical neurones. It is notable that chloralose alone amongst the anaesthetics blocks the release of ACh from the cortex (MITCHELL,1963). It is also interesting that the compounds which are clinically more potent as analgesics amongst those tested had relatively less depressant action on the sensitivity of cortical neurones. Thus analgesia (and at least in the case of methoxyIlurane, anaesthesia) may result from an action of these compounds at subcortical sites. A number of reviews have indicated the probable role of the diffuse ascending reticular formation in the maintenance of consciousness, and have suggested that anaesthetics act preferentially upon some portion of this system (e.g. BRAZIER,1961; CLUTTON-BROCK, 1961; PATONand SPEDEN,1965). The present findings would be compatible with such an hypothesis. Barbiturates, which have relatively profound depressant properties upon many neurones, are in fact antagonists to

Anaesthetics and cortical neurones

45

analgesia in small doses (DUNDEE,1960), and probably selectively depress some inhibitory sub-system (BRAZIER,1961; CLUTTON-BROCK, 1961). Thus different neurones may vary in their susceptibility to an anaesthetic agent, although there is little evidence that particular agents have specific actions upon different excitants of any given neurone. Acknowledgements-The author wishes to thank Dr. A. W. DUCGAN for his valuable assistance with a number of the experiments. Grateful acknowledgement is also due to Abbott Ltd. for the generous loan of the Pentec vaporizer used in this investigation.

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