Document 9Jw17YYXL4MEGgLG2XgLXqObR

ns -fee Brit.J. inJulfi.1968, 25, Si. (7/hf 1? -' */ -S, C^ Some Electrophysiological Methods for Studying the Action of Narcotic Agents in Animals, with special reference to Industrial Solvents: A Review t. HANA MIKISKOVA and ALOS MIKISKA From the Institute of Industrial Hygiene and Occupational Diseases, Prague; Czechoslovakia iur electrophysiological methods, two based on stimulation (measurement of spinal reflex excitability 'of direct excitability of the cerebral motor cortex) and nvo based on bioelectric recording (e'ectro^Jgpcphalography and electrocardiography), were used in intact guinea-pigs and rabbits for studying the nf narcotic and anaesthetic agents, especially of industrial solvents. The authors' results have been msesiewed and compared with those of other investigators in an attempt to work out experimental pro- far routine toxicity testing. s--uie-object of this paper is the description and s&hcalysis of several electrophysiological methods with respect to their application for testing the toxicity industrial poisons in animals. Only those [ues are described in detail that have proved reliable for evaluating narcotic agents, especially "Tggventsr. >ther techniques that can be directly ""tompared with ours are also discussed. We also '"'"present -same new toxicity data which illustrate ihe contribution of electrophysiological methods to yS'ffiPsblutian of toxicological problems, t Ij^The investigation of the functional state of tissues organs','as well as of their morphological and piodiemical states, is important, especially for the -central nervous system. Even very' slight and trans--**u*ry- functional nervous disturbances may have 'serious consequences in affected workers, by in creasing accidents and errors in performance. Many nervous functions, particularly the most -i--eomplicated ones, can only be studied in humans. I `--Nevertheless, animal experiments are indispensable j.-^anthis field, because in animals it is possible to test j-*~ruher concentrations of poisons, and the action of j substances recently' introduced into industry, at f, '?ell as to study the mechanisms of action in more detail. "--""A "useful addition to neurophysiological methods KUsthe electrocardiogram. It provides data on the - -"central nervous control of autonomic functions and ^allows the study of pharmacodynamic or toxic Received for publication September 4, 1967. effects on an excitable tissue whose cyclic excitabil ity changes can be estimated more simply than those of the central nervous system. For assessing the hazard of a chemical substance a quantitative comparison of its toxic effect in relation to the effects of chemically related sub stances may be of special importance: the determin ation of relative toxicity is usually more accurate and reliable than a direct estimation of a minimum effective dose or concentration. Our effort was therefore directed Towards choosing or working our electrophysiological methods which permit an exact quantitative evaluation of the toxic effect. In addition, we aimed to standardize measurement procedures to facilitate a comparison of results obtained in various laboratories. In its classical form, electropbysioiogy was divided into two large sections, dealing with problems (1) of tissue conductivity and excitability and (2) of bioelectrical potentials and currents. There are no strict boundaries between them today, but in practice excitability measurements still differ greatly from biopotential recording and analysis. When et.dtc-ihty r- messmed fo' toxici'y te,'uic one must take into account that the physiological processes evoked by the stimulation can influence the course of the poisoning ^phenomena of `electronarcosis', etc}, whereas when spontaneous bioelectrical activity is examined, the fixation of the animal and the application of the electrodes are the only factors that can affect the findings. Leading off biopotentials and recording them have no relevant effect. Consequently, biopotential recording has 8l SL 033990 im m ft! it rcP-' 82 Hana Mikiskova and Alos Mikiska become the basis of routine clinical examination techniques; and toxicological data in animals from electroencephalogram (E.E.G.), electrocardiogram (E.C.G.), and electromyogram (E.M.G.) recordings can often be compared with clinical findings in affected humans. Our experiments were performed in rabbits and guinea-pigs, because these species were found to be most suitable for evaluating narcotic industrial poisons by electrcphysiological techniques. The rabbit's size permits this animal to be used sim ultaneously for biochemical studies. Guinea-pigs, on the other hand, may be handled more easily and, as a rule, they are more quiet during recording. In guinea-pigs, all the methods discussed have been tested in control experiments as well as with some typical narcotic agents in order to compare their reliability, validity, and sensitivity. Statistical significance throughout was calculated as described by Mann and Whitney (1947). This non-parametric ranking test makes no assumptions either about the type of distribution of the popula tion from which samples are taken or about the constancy of variance in two groups, one treated, the other not. Treatment may change both dis tribution and variance. Statistical significance has been, therefore, under- rather than over-estimated in all instances. Measurement of Spinal Reflex Excitability Segmental spinal monosynaptic and polysynaptic reflexes are simple functions of the central nervous system which can be easily evaluated by quantitative techniques and which are therefore suitable for pharmacological and toxicological research of central nervous depressants. As for other types of excitability measurement, the functional state of the nervous centre can be inferred either from the latency and size of the reflex response evoked by the stimulus of a constant predetermined intensity, or from the threshold stimulus intensity, e-g., the threshold voltage or current of an electrical stimulus whose other parameters, such as duration and repetition rate, are kept constant. The latter technique, applied to a defensive reflex reaction following electrical stimulation of cutaneous re ceptors of guinea-pig hind limbs, proved to be a useful test for screening the neurotoxicity of industrial solvents and other narcotic agents. Procedure Two copper discs (about 4 cm. diameter), covered with several layers of flannel soaked in 5% aqueous sodium chloride, serve as the stimulating electrodes. The distance between the centres of the electrodes is adjusted according to the animal's size, being on an average about 8 cm. During measurement, the guinea-pig is held by the experimenter so that it touches the electrodes with its hind paws, previously wet ted by dipping into 5 % saline. A stimulator with constant current output (e.g., Multisrim-DISA, Herlev, Denmark, used in our laboratory; is necessary in order that the stimulating current should not be dependent on the resistance of the skin and electrodes. The stimulation is effected by rectangular impulses of 3 msec, duration, repeated with the frequency of 0 8 c/s. The current is increased from zero in steps of about 0-2 mA, stimulation by each current being repeated three or four times. A current that evokes a motor response to each stimulus is taken as the threshold. The response to a threshold stimulus is the dorsal flexion of the toes in rhvthm with the stimulation. When the stimulus is increased further, flexion in all three joints of the hind limbs results. In untreated guinea-pigs, the threshold current varies from about 0'4 to 1-6 mA, and is distributed approximately normally. Experiments have shown tlyt the threshold current cannot be considered as an individual character istic of each animal, stable for a long time. It should nor be measured more often than once a day, in order to prevent formation of a conditioned reflex, which is, as a rule, associated with a decrease in threshold. There fore, the following design of experiment is recommended. The threshold current should be measured simul taneously in several randomly selected groups, each consisting of at least 10 animals. The control group is injected with physiological saline (or the solvent for the test compound) or exposed to a stream of pure air in inhalation experiments. The significance level of the results can be evaluated according to statistical tests for samples of independent observations (Student's twosample t-test, Alann-Whitney's U-test, regression analysis, etc.). Results Some of the results, i.e., those con cerning the action of ethanol, trichloroethylene, trichloroethanol, and chloral hydrate, are sum marized in Table I. All were administered to guinea-pigs by intraperitoneal injection. Their depressant effects were tested at their peaks-- 20 to 30 minutes after trichloroethylene and 10 to 15 mmuces after the other compounds. An increase in the threshold current, approxi mately in direct proportion to the dose was observed for all these compounds. The linear dependence was valid only within certain dose Limits; larger doses caused complete abolition of spinal reflex excitability. The following equations were the best estimates of the straight line regression of threshold current (Y, in mA) v. dose (X, in mM./kg.): Y = 0 021X -j- 0 975 for ethanoi Y ---= 0-104X -- 0-985 for trichloroethylene Y = o*539X -- 1-184 for trichloroethanol Y = o-6nX -f- 1-090 for chloral hydrate. Si* 033991 ft Some Electrophysiological Methods for Studying the Action of Narcotic Agents in Animals Sc TABLE I 83 Action of Some Central Nervous Depressants (administered intraferitoneally) on the Threshold Current Intensity of Skin Stimulation of the Hind Limes in Guinea-pigs V- . & if - - Compound Dose No. of Threshold Current Difference front (mM.'kg.) Animals (Mean - S.E.M.) Control Group --- (tr.A) <%) Significance Ethanol 0 19 0-974 -r 0-041 6-7 19 1124 ;= O'lOO +154 13-4 19 I -53 9 074 4 IS-7 N.S. 0 005 Trichloroethylene......................................... 0 3*34 6-7 25 0 9S0 -- 0 G17 15 l-afio -- C`0S2 8 1*575 = 0*215 - 38-8 -f 60-7 o 001 O'OOI Trichloroethanol......................................... O 084 1-67 17 iiss -- 0059 17 I-6lS u 0-087 '7 2106 = 0 119 4- 36-1 4- 77'2 0`001 0-001 Chloral hydrate *1 0 0-84 1-67 20 1-085 n 0 048 20 1-660 - C-115 20 2-C7J - c 157 t 53-0 + 91 *3 0001 o-ooi <30-. >fe The slopes are a measure of relative neurotoxicity with respect to spinal reflex excitability. "7' Regression lines for trichloroethylene and tricj^kpethanol have. been presented elsewhere and Mikiska, 1966). In the present paper, Fig. 3 shows a comparison of trichloro ethylene, chloral hydrate, and dial. The large black -circles (upper right) indicate approximately equally effective doses, each compound at those doses evoking a threshold increase of about 60%, ,^,-As the three compounds represent different groups of anaesthetics and, therefore, cannot be .. compared on the basis of molar doses, we decided to use the equi-effective doses (6 7 mM./kg. of trichloroethylene, 0 84 mM./kg. of chloral hydrate, and 0-19 mM./kg. of dial) in further trials with 'other electrophysiological techniques. Discussion Experiments testing the action of drugs on spinal reflex excitability have been detisned either to study the mechanisms of the pharmaco dynamic action, especially to reveal the mechanisms of muscular relaxation in anaesthesia, or for the -quantitative estimation of toxicity. Mechanisms have mostly been studied in snrgieffiy p-encred animals, e.g., spinal, decerebrated or decapitated preparations with exposed nerve trunks or spinal roots for electrical stimulation or recording. For toxicity testing, animals with an intact nervous system are more suitable, A review dealing with both types of proolems from the pioneer work of Sherrington (1906, 1909) up to the present has been given by Aiikiskova (1962). (An extended version in English can be obtained from the author on request.) In this paper we shall limit the discussion to work related tc the methods used in, our laboratory. Rozin (1954), working with rats and mice, was probably the first to eliminate fixation of the animal in spinal reflex studies. He held the animals by hand during stimulation. The voltage was chosen in advance and the number of impulses (2/sec.) required to evoke the flexion of one limb was counted before and after exposure to toxic substances. This technique, extensively used in Russian laboratories of industrial toxicology, is referred to as `testing the summation ability of the central nervous Rvstem'. For example, Oijunin (i957i iff?, ha, published results concerning threshold toxic concentrations of acetone, diethyl ether, benzene and xylene, while Korbakova, Krcmneva, Kulagina, and Ulanova (196c) have used thus technique to study rats with chromebenzene poisoning. Bartenev (190:) stood the animal on two metal bars, fixed ;r. a special stand and wrapped with wet gauze, that served as stimulating electrodes. The first ir.ovi_me:,: ' ' en. cxiremue dr--- to itir./u'ation was regarded ts a positive reaction. Speranskij (1962) used the same electrode arrangement but used a gradually increasing voltage. Kc was, therefore, testing rather the threshold voltage than the summation ability. The variant introduced since 1961 in the toxico logical Jabnratorv of Korbakova (personal com munication/ represents, in our experience, a definite SL 033992 84 Hand Mikiskova and Alos Mikiska improvement. It consists of (i) the use of a stimulator with constant current output, and (2) a slower frequency of impulse (o-S c s, and impulse duration 3 msec.). We recommend these con ditions for routine testing. For rats they give closely similar threshold currents to those found in guinea-pigs (Mikiskova and Mikiska, 1962, and this paper). Ail these responses differ physiologically from the classical flexion reflex, as defined by Sherrington (1906), and first studied in poisoned animals with intact nervous systems by Zakusov (1948, 1953). During simultaneous stimulation of both hind paws, opposite reactions would come from the afferent nerves in each limb (ipsilateral flexion t>. crossed extensor reflex), if the integrative action of the spinal cord did not inhibit one of the reactions. If all four limbs are stimulated at once, the situation is still more complicated, because the current flowing through each paw depends on the skin resistance and cannot be controlled by the experimental design. For this reason techniques based on simultaneous use of more than two stimulation electrodes cannot be recommended. There is much terminological confusion in toxicological literature. The determination of spinal reflex excitability in intact animals used for testing industrial poisons is often referred to as `determination of neuromuscular excitability', which suggests that the response to direct stimulation of muscle or its motor nerve is being studied. It seems, therefore, necessary to submit evidence that for general anaesthetics, and probably for ether narcotic compounds too, the action manifesting itself in the test results is mediated through the synapses of the central nervous system, and that the functional changes in the common efferent path can be neglected. Depression or complete abolition of motor responses elicited by electrical stimulation of skin receptors in the animal with an intact nervous system gives an over-all indication of the pharmaco dynamic effect which may be caused in many wavs. The cause may involve functional deterioration of any of the following systems: receptors, afferent nerve fibres, spinal reflex centres, the motor area of the cerebral cortex with its tracts, brain-stem activating systems, common efferent motor paths, neuromuscular transmission of excitation, and the contractility of the muscles. Excitability of receptors during anaesthesia has been studied by most authors for lung receptors only. The observation by Matthews (1933) that the excitability of muscle spindles is increased during ether anaesthesia is difficult to compare with other data. The concentrations of narcotic compounds which affect impulse transmission in peripheral nerves in vitro are usually several times greater than those blocking synaptic transmission (Larrabee and Postemak, 1952; Toman, 1952). The possibility that depression of neuromuscular transmission may play a part in muscular relaxation and reflex abolition during surgical anaesthesia has been investigated by many authors (Naess, 1950a, b, c; Secher, 1950, 1951a, b, c, d; Sabawala and Dillon, 195S). It has been found, however, only for ether, and that only in the tolerance stage, while for the other anaesthetics tested (cyclopropane, chloro form, halorhane, barbiturates, and other drags) concentrations blocking neuromuscular transmission in vitro were higher than any concentration in blood or tissues found in general anaesthesia. This also holds for inhibition of muscle contractility. It appears, therefore, that deterioration of central nervous synaptic transmission is the principal factor in the depression of spinal reflexes. .Occasionally a drug is effective in a decapitated arwnal at a con centration equal to that causing only moderate anaesthesia in the intact animal (ethanol in the cat (Kolmodin, 1953)); but the depression of brain-stem activating systems appears to be controlling in intact animals (Magoun and Rhines, 1946] Niemer and Magoun, 1947). The depression of synaptic transmission is greatly dependent on the number, and repetition rate, of afferent impulses as well as on specific features of the compound. A review on this subject, starting from the pioneer observations of Bremer and Bonnet (1948), was given by Schneider (io54). Conclusions The measurement of the threshold current for the skin stimulation of hind limbs is a simple way of estimating quantitatively spinal reflex excitability in intact animals. Its use does not need my special instruments and the measurements can be performed quickly on large groups of animals. For screening tests as well as for estimating relative toxicities these features enable one to obtain statistically significant results. On the other hand, the examination cannot be repeated within a short time interval, so that the time course of action cannot be studied. It is also impossible to study differences between animals in their sensitivity to drugs; and experimental results cannot be com pared directly with clinical observations and ex periments. We should like, however, to stress that these disadvantages are common to all tests of spinal reflex excitability, including those more elaborate and time-consuming (Zakusov, 1948, 1953; Ljublina, 1948, 1959). SL 033993 : \ ***.. '*-- ' 'V ; ,, '.. . . ...' - :;!: v . )-." v.`.'. #1 Some Electrophysiological Methods for Studying the Action of Narcotic Agents in Animals 85 Measurement of Electrical Excitability of the Cerebral Motor Cortex The excitability of neivous centres can be estimated not only with reflex stimulation via receptors or afferent nerve fibres, but also by direct electrical stimulation of the central nervous system tissue. The motor area of the cerebral cortex seems to be one of the most suitable structures for the use of direct electrical stimulation in applied research, because it may be easily exposed both for acute experiments and for implantation of electrodes, and because its stimulation results in constant, clearly defined motor responses. For drug evaluation, lucre are the following principal possibilities : (1) estimation of the response intensity to stimula tion of the motor area by electrical current of con stant parameters (Wyke, 1955); (2) estimation of rhe response latency with consrent stimulation parameters (Zakusov, 1953 j BarySnikov, Vinogradov, Nikiforov, and Sanin, 1056) i and (3) determination of the threshold voltage and or curient of the electrical stimulation, This is preferred by most other authors. Reproducible te^^^ can, however, be obtained only with rcJ^Rd stimulation (Mikiska, i960). Method For acute studies basal anaesthesia can hardly be avoided (Bohm and Petersen, 1953). For pharmacological and toxicological experiments the use of implanted electrodes is necessary for testing the threshold before administration of the drug. The following procedure of implantation is used in our labora tory (Krilova, Mikiska and Parizek, 1957; Mikiska, i960). The electrode consists of two contacts of silver wire, embedded in an electrode body made of polymethyl methacrylate (as used in dentistry). The form and size of the electrodes for rabbits and guinea-pigs are shown in Figure 1. Implantation is performed under dial anaesthesia (40 mg. kg. = 0-19 mM.,kg. irtraperiloneally). Twenty to 30 minutes after dial injection the skin is infiltrated with 1 % procaine and the surgical procedure is started. The trephining is begun on the fronto-parietal suture, about 4 mm. from the midline in rabbits and 3 mm. in guinea-pigs. When a small opening has been made to the dura mater, the exact localization of the motor area for the fore-limb is checked by electrical stimulation, and the opening is broadened to an elliptical shape. The cranio-cerebral topographic relations of the motor area are remarkably constant and are similar for all laboratory rodents (in Fig. 1 they are illustrated for the guinea-pig's skull). For electrode fixation in rabbits, the diploe is removed around the periphery of the opening to allow the electrode holders to penetrate between the lamina externa and the lamina interna of the skull bones. In guinea-pigs, the electrode holders, located on the lower edge, are inserted between the bones and the dura mater. The inserted electrode is turned to a right angle and fixed in the correct position to the bones by polymethyl methacrylate. After the electrical excitability has been re-tested with the electrode in snu, the skin is sutured. No special post-operative care is necessary, and the reliability of the surgical procedure is nearly too''... Stimulation ^ Rabbit Outnea piq Nt./ r r^ !r 1 /? (i C ( ! !, n>C'*cf arc-o 0 IO 20 3C-- C lO 20 UQn.n, r. 'A Conpcc'-c Sponqiosa I | Fieefrode body B ^9 n~iTTl 1 Di.ro mater ~3 Acrylate Fig. i. Bipolar epidural electrodes for electrical 4timuIaTion and ii.K.G. recording. To the right the ' position of the motor area in the guinea-pig is shown SL 033994 *;' v-jl f ^->y ' *t.j\l a-c. -A ">*T-7?* >-t'Vt^*' .J-:'-' ':r-' -yfay -'-Vjc* t-j^''^Hr^tgVTCT"* ,.v Ti.*''' -v .'. 'V.-^/^yr."TM:. \* VAAv-V-jM^rt-v^^. , pm *>> , >y*v+'.v;*vc^v v .-, * <; ,, .v-v-*.*.- *-T< -nJww.fc ';' t f, c'.':':rr-'V' -^'^'''^,v^'t '*-'--^l- ***!''-**-*>,.:. >.- .`Jr-r - ;/ x,H *.'.*-'>>-;* ->4-- *> 86 Ham Mikiskovd and AloS Miktska experiments can be started 5 to 8 days after implantation and continued for several weeks. For stimulation the animal is tied to a fixation table in the ventral position by tapes attached to the extremities as for routine polygraphic recording. Stimulation of the motor area is provided by sinewave alternating current, 50 c,;s, transformed from the mains and switched on every sixth second for o-6 sec. by means of an electromechanical switch. 'S's star: die stimulation with a voltage 20-30% above the expected threshold, known approximately from the earlier measurement. The voltage is then decreased in steps of about 5" , stimu lation being repeated three or four times at each voltage. The threshold is taken as that value which results in a stable flexion of the contralateral forelimb, r.ot dis appearing with repeated stimulation. The threshold is determined every 5 minutes; and the mean value of six estimates in half an hour is taken. In untreated guinea-pigs and rabbits the threshold voltage found was usually 1 to 2 volts, R.M.S., and the fluctuation of threshold within 4 to 6 hours was very small (Mikiska, i960). Changes were distributed approximately normally and did not exceed 6% (99% confidence limits). Results Intraperitoneal saline did not affect the threshold (Table II), so every significant change in the threshold following the administration of a compound is likely to be due to its pharmacological action. Reports in the literature and our own experience support the validity of a general rule rhat an increase in threshold voltage, reflecting a decrease in excitability, is a manifestation or a central nervous depressant effect, whereas a decrease is often a manifestation of an excitant or sub-convulsive action. The results obtained with some narcotic agents and solvents, administered intraperitoneally to guinea-pigs, are summarized in Table If. In the homologous series of aromatic hydrocarbons the depressant action was observed to increase from benzene to xylene. On the other hand, in some experiments with benzene, a convulsive effect could be demonstrated. This was rarely observed with toluene and xylene and then only at least 90 minutes after injection. The increase in depressant action was clear even though the hydrocarbons were TABLE II Action of Some Organic Solvents administered intraperitoneally) on the Threshold Stimulus Voltage (SINE-WAVE JO C/S FOR 0-6 SEC.) Of THE MOTOR AREA OF THE CEREBRAL CORTEX IN GUINEA-PIGS Compound 0-9",, sol. NaCl Benzene Toluene Xylene Trichloroethylene Trichloroethanol Dose 0-4 ml. i x*. oj Animals |5 6 7 m.M.'ng. (0-52 g./Kg.) 5 6 mM.kg. (0 52 g./kg.) 3 3 m.M. Ag (0-35 g./Ag.) 4 9 m.M. .eg. (0 52 g., Kt-.' 2'2 1 ll\i. Rg. (0'29 g. Kg.) I ; i 1 6 "7 m.\i (o-88 g./rg.) 2 2 mM. 'Kg. (0 34 g./kg.) 6 6 6 6 6 6 6 Time from Injection (min.) s- 30 65- 90 155-180 5-- 30 65- 90 155-180 5- 30 65- 90 155-180 5- 30 65- 90 5- 3 65- 90 5- 30 65- 90 155-180 5- 30 65- 90 155-180 5- 30 65- 90 155-180 Percent Change _S.E.M.____ of Threshold " 17 - 1*0 - I'3 ZZ O'S -- i'4 ^ i*2 - 1-8 - 1-7 - 21 24 - 3-0 5 3 -- 6 0 A 2-6 -1- 1-6 2 0 -- 27 2-8 - 8-0 - 7-7 -- 2`2 ^ 2*5 - 17-4 = 34 - 5 1 t 53 - If; 23 - 17 A t 7 - o-6 3 13 - *3 2 ^ 33 iz-5 r; 41 - 159 A 5* A 2107 A 57'3 --108-0 = 52-2 -- 245 -- 11-6 Significance 0013 0 001 0-001 0032 'OOOS 0001 _ 0*001 fr SL 033995 aw*. -- * Wm*-^ -." >> '**:.-*. . . *,-:! ? -v J,T,%-'-'.`'*t;'_,':Jr^`J -.'. /"TjS--Jfx MJJ^f.'HtWfflWaO lij.LP'Piy U?rMgf*CK"*l.`,*".***",|f--^J**LWff,MIM ' I * '* ml m ;- `<7-c -% r^* -'-I"*^V**-!>_.n*:"!&.c-*.*_!^. tti;w'- -*r4r._l^y"'4w'.-*n%f.ifhaj^flf*c.'tXlivf.; *<"*>1'**"'6-.*'4^A ~*7-"," "'" v; : -*.<>J1flu rtfr *, ~>r*` ' ">*** -' --' --*-"' 'i^" -- -;'v..."'y^ >-.iC;fi-? -.>-/. Some Elecrrophysiological Methods for Studying the Action of Narcotic Agents in Animals 87 given in equal weight doses, i.e., in molar doses inversely proportional to the molecular weight. The analysis of these results-(Mikiskov4, i960) will be reviewed in the next Section. Trichloroethylene and trichloroethanol affected the excitability of the cerebral motor cortex like typical depressants, i.e., by increasing the threshold. The relative effectiveness of both compounds, and conclusions concerning the possible role of trichloro ethanol in poisoning by trichloroethylene, have been analysed by us in detail in previous work (Mikiskovi and Mikiska, i960, 1963, 1966). Discussion Direr' electrical excitation of central nervous tissues has more complex con sequences than has excitation of peripheral tissues --nerves or muscles. The stimulus excites thousands of neurons, mutually connected by ey.citatory and inhibitory synapses, which often form closed reverberating drcui*.. Consequently, a single stimulus causes a series of neuronal discharges lasting several seconds even in the neuronally isolated neocortex (Bums, 1951, 1954). For the intact motor- cortex this repetitive discharge can eajjj^e followed by recording from the pyramidal tr^^B(Brookhart and Zanchecti, 1956; Schlag, i95of To use a threshold for testing the action of chemical compounds, it is necessary to standardize both the stimulus and conditions which could exert reflex influences on the excitability of the cerebral motor cortex. For the stimulus, voltage and current, waveform, duration and fcpetition rate of impulses, the use of unipolar or bipolar stimulation and the electrode size and shape arc probably the most important things to standardize. Among reflex influences, that of proprioreceptors in muscles participating in the test response seems to be the most important, but other factors have been studied, too, such as stimulation of acoustical and tactile receptors, interoreceptors, brain-stem and diencephalic excita tory and inhibitory ascending systems, and the establishment of conditioned reflexes. Summation effects, appearing with repeated stimulation, can sometimes re'ult ir. convulsive epileptoid rhen~ omena u" the stimulation is too strong or too longlasting. On the other hand, if stimulation is started at sub-threshold voltages or currents, and the stimulus is gradually increased, the appearance cf a clearly defined threshold can be inhibited by adaptativc processes. We have reviewed elsewhere the data on these essential factors in a paper which describes cur attempt to work out a standardized procedure for toxjfltiogical studies (Mikiska, 3960). 2 Various centrally acting drugs and poisons can be classified into three broad groups: (1) depressants (narcotics, anaesthetics), (2) stimulants (analeptics, convulsants), and (3) compounds with more com plicated mechanisms of action. The three groups will be discussed separately. (ij Depressants uniformly evoke an increase in the threshold, which increases with the depth and duration of anaesthesia (Richard (1939) for Evipan; Rascanu, Kapri, and Popovici (1939) for chloroform, Evipan, and Veronal (barbitone); BeneSova, Hor vath, and Mikiska (1956) for various intravenous barbiturates; and Kralovd etal. (1957) for trichloro ethylene). The elecrrophysiological effect paral leled the depression of reflex excitability, so determination of threshold voltage was taken to be a valid quantitative indicator of depth of anaesthesia, and was used for studying the synergism between barbiturates and phenothiazine derivatives (Votava, Benesova, and MetySovd, 1958) and for com paring the depressant action of trichloroethylene and trichloroethanol (Mikiskova and Mikiska, i960). Wyke (1955), who measured the excitability of a motor area from the strength of muscular con traction evoked by stimuli of constant intensity, also found a close parallelism between the decrease in cortical excitability and other measures of the depth of barbiturate anaesthesia. Some early workers (Fulton, Liddell, and Rioch, 1930; Fulton and Keller, 1932) claimed that the excitability of the cortex is affected 'not only by the depth of anaesthesia but also by the type of anaesthetic used'. They did not, however, measure the depth of anaesthesia quantitatively and did not know the threshold of the motor area before the anaesthetic was administered. Their observations cannot therefore be compared with those of more rcccri. cUwiors. Rohm and Petersen (1953) examined the increase in threshold for various parts of the motor area in dial anaesthesia. A larger effect was observed in hind limbs than in fore limbs. The same was also found in acute experiments with the use of basal anaesthesia. [z] Th-: inn ewe in excitch'hry threshold af:er depressant-- urny be as high as a few hundred per cent. The maximum decrease in threshold due to analeptics and convulsants is not more than 15 to 20(;. Stimulation of the motor area after higher doses of excitant compounds results in motor restlessness or transient convulsions. Data on the following compounds have been published: pentameth'. iene tetrazoie (Malkiman, 1054b); strychnine, caffeine, and amphetamine (Lev, 3956); and para th: on (Krilova ct al., 1957). SL 033996 ;( -* *- o t.&-irf. -'f-A . -^V;; i' - - v vT-1- triVam* X..oc.,T. >>^LT-I ' X'fc1 l^tt- **. m^K<* v?*r* v^^-sMt-*' ,V-:<K'^rf*f*7u'v IV^'".A*7; r-'.^ rf'i'V ^-V-^-t.r^tv;-,^ Hana Mikiskcva and Alois Mikiska (3) Schiag (1956) observed an increase in the motor area excitability after several drugs, evoking vasodilatation of brain vessels, acetylcholine, eserine, methacholine, histamine, nicotinic acid, and amyl nitrite. Both increases and decreases of excitability were observed after bulbocaprtine (Levitina, 1948; Vaiainik, 1949) and adenosintriphosphoric and monobromoacetic acid (Babskij and Malkiman, 19525 Malkiman, 1954a). The central nervous action of aromatic hydro carbons (Results above) consisted of both depres sion of cortical excitability, especially for toluene and xylene, and of an excitation, most pronounced for benzene. Other authors have also observed both effects. Lazarev (1954) reported results on rats and guinea-pigs exposed to high concentrations of aromatic hydrocarbon vapours; Ljublina (1950) evaluated the excitability for simple spinal reflex functions; in the study by Jaroslavskaja (1952) complex unconditioned reflexes were also ex amined; and Novikov (1956) studied conditioned reflexes. Furnas and Hine (1958) examined the effect of aromatic hydrocarbon vapours on the E.E.G. of rats with implanted cortical electrodes. Besides narcotic effects, like those from volatile anaesthetics and lipid solvents, a spike-like E.E.G. activity was found, which was assumed to be a manifestation of a convulsant effect. Desi and Nikolits (1967) have recently shown in cats marked facilitation, by intraperitoneal benzene and xylene, of the spike activity evoked by local application of strychnine to the neocortex, benzene being the more effective. Conclusions Measurement of the electrical excitability of the cerebral motor cortex is a method for the quantitative estimation of the direct electrical excitability of the central nervous tissue. No special instruments are necessary to ascertain the response to stimulation. Highly reproducible, quantitative results are obtained in acute experi ments lasting several hours, and individual eval uation cf each experiment is possible. Excitant and depressant actions on the central nervous system result in contrary changes of the threshold voltage, so that some broad analysis of the mechanism of action is possible. However, implantation of electrodes several days before the experiment makes the technique timeconsuming. Experimental results cannot be directly compared with clinical observations and ex periments. The methods seem to be suitable for the advanced testing of compounds, the basic toxicides of which are already known from screening or pilot experiments.- Registration and Analysis f the Electroencephalogram Less is known about the action of industrial solvents on the E.E.G. in animal experiments than about the action of depressant pharmacological agents. There are many E.E.G. reports on humans poisoned with industrial solvents, but few for the same subjects before the poisoning. For trichloro ethylene, however, carefully controlled experiments have been carried out in humans, because of its use for general anaesthesia (Schneider, X954; Couitin, 1955i Okuma, Shimazono, and Narabayashi, 1957; Martin, Faulconer, and Bickford, 1959; Faulconer and Bickford, 1961). For other solvents animal experiments may complement the clinical findings in padents by yielding quantitative toxicity data and by con tributing to the knowledge of the mechanism of action. References will be discussed-together with our experimental results. Method Our experiments were carried out mostly on guinea-pigs or rabbits with chronically implanted epidural electrodes, localized over the motor area, t'.e., with the same type as for the electrical stimulation of the motor cortex (Fig. I). Rabbits were either placed in a restraining box or tied to a table. Guinea-pigs were tied to a special fixation table. To avoid serious discom fort the head was not fixed. The animal's movements were monitored continuously by E.M.G. recording, which enabled us to discriminate slow E.E.G. waves from movement artefacts. For gross movements E.M.G, superimposed over the E.C.G. leads or special leads from both hind limbs were satisfactory, while even very small movements of the head could be clearly detected from the lead between both anim.'J's ears. The E.E.G. was recorded bipolarly in the unearthed animal. The amplified E.E.G. voltage was led off from one control grid of the fourth amplifier stage (output for oscilloscope, etc.) to an electronic E.E.G. integrator, and to the modulator of a frequency-modulation magnetic tape recording system. Both were of our own design (Mikiska, 1962, 1963, 1964). The integrator, an analog device for on-line analysis (Mikiska, 1963), operated in a manner similar to the integrators designed by Drohocki (1948, 1956) and by Bickford (1950), but zero resetting was performed with an electromagnetic relay, as in the circuit used in the equipment for servo-anaesthesia by Burei, Petxan, and Zachar (1960). The results of integration were written out on another channel of the E.E.G. as a series of impulses whose average frequency was directly propor tional to the mean E.E.G. amplitude (Fig. 2). The mean E.E.G. amplitude was then determined by counting the number of integrator impulses for a representative trac- SL 033997 Some Electrophysiological Methods for Studying the Action of Korcotic Agents in Animals 89 ing section over several minutes. The absence of move ment artefacts in tie record could be verified from the E.M.G. tracingrecorded simultaneously. The equipmmt for magnetic tape recording consisted of a conventional tape recorder for home use (half-track record, speed of tape 3/5 cm.,'sec.), a modulator, generator of an F.M.-carrier frequency 5,coo c's . cob, and of an F.M.-demodulator. Diagrams and further technical data have been published elsewhere (.Mikiska, tofa). Magnetic tape recording enabled us to amuse cur I.E.G.s "hTancther"laboratory equipped witn the Petersen analyser, manufactured by Kaiser Ltd, (Copenhagen) and modified by Marousck (1967). This mstrumenr divided rhe E.E.G. signal into six frecuenc. bends, each of them being written ou: ana integmted separately, Li must r.v otir E.E.G. oxnarimentc i*i 0: - - e - rirr :ir.t omy the E.E.G. but aLo the summation E..M.G. tend E.C.G. were recorded. The E.C.G. records are des cribed later in this paper. Results In the normal E.E.G. recorded tipolailv from rhe motor area of a waking guinea-pig fast rhythms in the alpha and beta ranges usually dominate. Sometimes, bowei er, a dominant frequency in the theta range may be present (Mikiska and Mikiskova, 1964). Examples of normal tracings are presented in Fig. 2 and in a previous paper in this journal (Mikiskova and Mikiska, 1966). In seme experiments we have observed short episodes of tegular 14 c/s spindles, corresponding very probarb. to a light physiological sleep, a pattern well known for cars (Hess, Koclla, and Aren. Jpf?.' and rLcbits (GatiglofF and Mourner, 1956: 1957I. Nocirerrbe stimulation associated with mtra- peritoneal breedon of physiological saline caused, in some experiments, a transitory decrease in E.E.G. amr hredc, cr .'.rousal refer; on. Other reflex * gS hi *1.4*.'' 'j, ill an. Civ, CIdTl;',.llv 4 to 6 hours did nc; evoke any significant change in the H E,G. pattern or ampblude (Table III). The E.E.G. was very stable m many control experiments lasting up to several weeks, but the possibility of sudden changes, dee mostly to damaged electrodes, could never be completely excluded. Therefore, 90 Hana Mikiskoz'd and AloS Mikiska TABLE Ili Action of S jme Anaesthetics ^aoministehed intbatekitoneally) on the E.E.G. Amplitude in Guinea-pigs the reliability of electroencephalography in long term experiments was lower the longer the experiment. Some quantitative results (E.E.G. integration) with depressants are given in Table III. Means of control experiments and significance levels are presented only for observations after a lapse of 15 minutes or more-after the injection, when the arousal reaction due to the injection bad already disappeared. Both the change in the E.E.G. amplitude and the E.E.G. pattern in light and moderate anaesthesia differed with the type of anaesthetic. Trichloroethylene and trichloroethanol, volatile anaesthetics, induced only a moderate increase in E.E.G. amplitude. Dial, a barbiturate, caused a much larger increase in E.E.G. amplitude. Chloral hydrate was in between. Chloral hydrate influenced the E.E.G. more than txichloroethanol: 0-84 mM./kg. of chloral hydrate increased the E.E.G. amplitude more than 2-2 mM./kg, of trichloroethanol. This finding dis agrees with the view advanced by Butler (1948) and < J \ Some EJearopkysiological Methods for Studying the Action of Narcotic Agents in Animals 91 that^thloral acts as an anaesthetic only after metabolic conversion to trichloroethanoL For increasing the threshold ofthespinal defensive reflex (Fig. 3), 6-7 mM./kg. of trichloroethylene, 0-84 mM./kg. of chloral hydrate, and 019 mM./kg. of dial were equally effective. The doses are not equieffective on tire E.E.G. There are several factors that could explain the observed differences. The most important of them seems to be the relative solubility in water and lipid phases (Mikiska, 1965). The lack of correlation between the action of anaesthetic agents of various types on spinal reflex 'irritability and on E.E.G. amplitude shows that `central nervous toxicity' is not a homogeneous concept but depends on the function studied. The waveform patterns of E.E.G. were affected by these compounds, as shown in Table IV. The of the- patterns ' Observed' virth the" quantitative E.E.G. data will be discussed in the next section. As an example of the action of inhaled solvents, the effects of carbon disulphide are shown in Figure 2. The most striking feature was a marked desynchronization of the neocortical E.E.G. lasting up to several hours after the exposure; spindles of high voltage and very irregular shape, consisting of components of various frequencies, superimposed over the flattened tracing and accompanied some- times by trains of slow waves, were manifestations of a still deeper stage of central nervous depression (Mikiskova and Mikiska, 1964). Discussion The first work known to us on electroencephalography on animals experimentally exposed to industrial solvents was carried out in vJ-itv fattisitei mm FlO. Comparison of three anaesthetics of different types by means of two neurophysiological tests and E.C.G. recording, pel rijjhi: skin stimulation threshold current t\ dose* -Cl a.:-'--. o34000 su tbt' K ' .N>. ***ix.-W-` ''g^-"cn*.J-.*'' r i j-.--v:*;.:.; ' ' - ' -'*-' ; v``. "`-*r c "S*'';'**:*'-'V'-:' >-^/T.:'tr't-^-';V.:Vv,*-,,ft'.:.,*,..f^ >l4v*.,.7-:-- o'V'T' Ci ^. j-,.-- Ji3^*"* ''W\--.; ..- . g- --? -~5*-iv /faim Mikiskova and AloI Mikiska TABLE IV E.E.G. Pattern of Raimi*' Xeocortex Wacer-insotubU Compounds1 Random slow waves and/or spund'es (superimposed o-.er unchanged or `di^nehronized' T: E.G.) IVuzer-iulubU Compounds2 Random spxndles (superimposed o', er E.E.G. simitar to that of the ;Ec;- s'ltc) 'D-synchronization' of E.E.G : decrease in amplitude, especially in rhat of slower frequency components Not observed Continuous high-voltage slow wave* and/or spindles . High-voltage fast activity (14-30 c/s, particularly in the anterior regions ot the cerebral hemispheres) Not observed - Continuous spindles and highvoltage slow waves Bunts of E.E.G. waves of moderate to high voltage alternating with sections of low-voltage, predominantly slow, electrical activity Low-voltage, predominantly slow, electrical activity only Corresponding E.E.G. Stage of Anaesthesia Hypnotic effect appearing only when environment conditions favour falling asleep, but absent if onset of anaesthesia is :oo_ rapid Fast3 Rhythmic* (3-7 c/s regular E.E.G. activity) Complex5 (E.E.G. pattern similar to sleep stages Ca D, E or to E.E.G. of `cerveau isole1 preparations') Burst-suppression1 4 Suppression* 'Lipid solvents and volatile anaesthetics, barbiturates and chloral hydrate. 'Classification of Martin, Fauleoner, and Bickford (1959). our Institute and reported by Horvath and yMichalovi (195e> 1956). A decrease in the E.E.G. amplitude of the optic and motor area of the rabbit's neocortex was observed after inhalation of carbon, disulphide. After repeated exposure occasional low-voltage theta activity appeared. These changes were not accompanied by any striking behavioural ones. Later, Horvath and Mikiska (1957) stated that in rabbits with implanted electrodes in the di encephalon a regular theta rhythm followed carbon disulphide administration, and that this disappeared only after considerable overdosage, eventually result ing in the rabbit's death. In the same paper ex posure to trichloroethylene vapours was reported to evoke anaesthetic patterns comparable to those fol lowing intravenous barbiturate administration. An other early paper in this field was that by Andersen and Kaada (1953) on the action of a toluene-butyl acetate mixture on the rabbit's neocortex. A drawback common to all these early observa tions was the overestimation of the similarity between the observed E.E.G. pattern noth clinical findings in poisoned humans and even with E.E.G. abnormalities of other than toxic origin. The same is, however, true for most pharmacological studies from that period (see the reviews by Toman and Davis (1949), Greville and Heppenstall (1950), Schneider (1954), and Verdeaux and Marty (1954)). Better understanding of mechanisms of action has arisen only from the analytical electrophysiological work of, for example, French, Verzeano, and Magoun (1953), Brazier (1954), Arduir-j. and Ardnini (1954), Domino (1955), and King (1956); but these authors paid little, attention to the spontaneous E.E.G. pattern. > Many of the more recent experimental papers on the E.E.G. of animals given industrial solvents are descriptive, so that it is difficult to decide whether different E.E.G. patterns should be attributed to differences in the action of tested compounds or only to different degrees of central nervous depression. This is particularly true when the toxic vapour concentration was neither controlled nor measured. A review on trichloroethylene was published in our previous paper (Mikiskova and Mikiska, 1966). For other solvents, very thorough reviews may be found, e.g., in the monograph by Serra and Ambrosio ('961) or in the review article by Higashida (1964). Table IV represents an attempt to provide a comparison of the E.E.G. patterns observed in our experiments (having appeared, at least in some of SL 034001 'll Some Electrophysiological Methods for Studying the Action of Narcotic Agents in Animals 93 .hem, as a regular sequence of defined stages) -with are available in English. Desi and Nikolits (1967) ihe general EfE.'G. stages of anaesthesia as described have demonstrated the central nervous action of oy Martin and his- colleagues (>9S9)-asd further---benzene and xykne-by studying their action on-spike used for classification by Faulconer and Bickford discharge following local application of strychnine '1961). 'This classification seems to us to be a to the_at*s neocortex7 In another paper on the suitable basis of comparison, because of the E.E.G. of rats poisoned with benzene Desi (1967) following features: examined the effect on the desynchronization (x) It stresses the similarity between the E.E.G. response to electrical stimulation of the occipital patterns found in the various species and brain neocortex. The spontaneous E.E.G. v as analysed structures, which is the more striking the-deeper- in--these animals by multichannel spectral and the anaesthesia. autocorrelation techniques. (2) It is not confused with the stages and planes of anaesthesia according to the depression of reflexes and autonomic functions, e.g., as described by Guedel (1951). (3) It is partly based on the mean E.E.G. amplitude, a quantitative result. Conclusions The E.E.G. gives information on. the functional state of the central nervous system from its bioelectrical activity. Recorded waveform patterns change in a characteristic manner under the influence of various drugs. Besides these rather In light and moderate anaesthesia, Martin tt alls '1959) classification on its own was not completely satisfactory. Table IV attempts, therefore, to specify the difference in E.F.G. patterns for waterinsoluble and water-soluble anaesthetics. The observed differences seem to be closely related to different mean E.E.G. amplitude increases. Watersoluble anaesthetics, particularly barbiturates, are characterized by the high amplitude of the fast qualitative characteristics, E.E.G. data can be evaluated quantitatively by analysis of the mean voltage, frequency spectrum, etc. Reproducible E.E.G. changes were measured for several hours after a reflex response had elapsed. In studies of chronic toxicity, however, the reliability was lower. E.E.G. findings in animals often re semble clinical observations, suggesting that the mechanism of action is similar. The specificity of which they evoke in the rodents' neocortex, E.E.G. patterns with respect to various physio oo^^^he `fast' and `complex' stages of anaesthesia. logical conditions and toxic factors should not, Vo acceptable neurophysiological explanation of however, be overestimated. Simultaneous record his difference has been suggested, as far as we know. ing of the bioelectrical activity of other organs It is, however, certain that both high-voltage fast contributes to an effective exploitation of the ictivity in light barbiturate anaesthesia (Shimazono, necessary electronic equipment and, in many cases Dkuma, Fukuda, Hirai, and Yamamasu, 1953; also, to better understanding of the mechanisms of Cbafetz and Cadiihar, 1954) and low-voltage fast action of the compounds studied. ictivity of the E.E.G. desynchronized by volatile maesthetics (Rossi and Zirondoli, 1955 j Schlag and Brand, 1958) disappear after any suppression of Electrocardiographic Examination he activating influences ascending from the .mesencephalic reticular formation. We hope that the data in Table IV can serve as a guide for the classification of E.E.G. patterns in ight anaesthesia to those who intend to use a ecording technique similar to that described in this paper. A more detailed electroencephalographic malysis should also include data on the E.E.G. oattem of rhinencephalic and subcortical structures is well as some characteristic kinds of evoked ictivity. Evoked E.E.G. activity or analysis of spontaneous E.E.G. have been used only exceptionally for testing the action of industrial solvents in laboratory animals. Higashida (1964) studied the action on various types of activity evoked by electrical brain mmuiation by the techniques worked out by Longo (1962). Unfortunately, only the abstracts Electrocardiography is an electrophysiologicai technique that may be of interest to pharmacologists and toxicologists for several reasons: (1) It sup plements neurophysiological methods by providing data or the central nervous control of autonomic functions. (2) It enables the pharmacodynamic effects to be studied on. an excitable tissue whose cyclic excitability changes can be examined more readily than those of the central nervous system. 1 Ur complex pattern of toe t C V-. rnrv hiunsiysed in thiee vays: (1) By gross examination of the regularity of the heart rhythm, and of the intrinsic cardiac pacemaker, and by counting the average heart rate. (2) By determination of the time parameters, e.g., pulse intervals (RR), atrioventricular con duction time (PQl, duration of the depolarization SL 034002 ^ ;'' 7- V`-*:ffi^':h-~v~,,'''` "v 1 ._'r- ;' -*V' ^i-*'.^ - ^,-..-'; Tvr'i^^-r'r-V;.^ -*a-,-,F erl2M&Z<Ei:~t&-: ('T'v.'.K^tc, y*!`v **':. a V^*^r-t,.-^. ~i tJ~ ^.tr_i'^;'irJiy,i^lt~JT-ttJjjif ^r^'i f r f*-' r^f-'i^'^-r^t_`^-"^ViiiV Vjri^fff"^i1 f-fflrKit ! 'i11T1 i1'^^HYi 1*'t^n^'^Vvr v_ X-Yur.-**-vi':;.a>r^iiflf fr' ;-i^^%. ,-*:..'- vrJfe>+:'-^`-A 94 Hana Mikiskovd and AloS Mikiska (QRS), and repolarization (ST) ph; of the electrical heart systole, etc. (3) By voltage measurement, i.e., by estimating the amplitude and polarity of the H.C.G. waves; and by ascertaining the elevation or depression of the ST segment. The first and second types of analysis can be performed with i high degree of reliability from any single E.C.G. lead. Aithougn the possibility that an E.C.G. wave is isoelectric in a particular lead can never be excluded, the probability of such an even: is small, especially if vae examine thoroughly several E.C.G. complexes corresponding to various phases of respiration. For voltage measurement, however, an E.C.G. lead is of little value. It is necessary to record at least three independent E.C.G. leads, e.g,, those corresponding to the axes of a Cartesian coordinate system: X, Y, and Z. For evaluation of the E.C.G., the animal's emotional stress due to the examination procedure should not be ignored. The demands of a very detailed examination and of minimal stress are often conflicting. For this reason, we have tested three techniques of E.C.G. recording. similar pattern in one lead only may not be significant. A schematic diagram of E.C.G. leads is shown in Figure 4. Three extremity electrodes, R, L, and F, correspond to those of Einthoven's triangle. The back electrode, Bpcannot be localized in animals in relation to the skeleton with the same degree of accuracy as ir humans. It is therefore positioned in the dorsal projec tion of maximum heart apex stroke (in guinea-pigs, approximately 1 cm. to the left of the midline). A small disc electrode filled with paste and fixed with co.Iodion or adhesive tape is used, e.g., a standard type for human scalp electroencephalography. Examples of three-dimensional E.C.G. records h;v. 0 been presented in a previous paper in this journal (Mikiskova and Mikiska, 1966), Recording Techniques (z) From Animals tied to a Fixation Table This technique is suitable both for screening or pilot experi ments and as 'a part of detailed examinations, including E.C.G. voltage measurement or simultaneous recording of other bioelectric data (E.E.G., E.M.G.). The animal was fixed to a fixation table. Electrodes for the leads to the extremities were made from alligator clips by soldering two silver plates to the jaws in order to reduce the pressure on the skin by increasing the contact area. Before applying them the skin was shaved and covered with electrocardiographic paste. Inter-electrode resistance below 10 kilohms ensures, as a rule, artefactfree recording, except during gross movement. To obtain data on the magnitude and direction of the electrical heart vector (dipole moment), the lead system proposed by Wilson, Johnston, and Kossmann (1947) appeared to be the most convenient. No system of leads proposed for spatial vectorcardiography is perfect, but Burch, Abildskov, and Cronvich (1953', have reported thar Wilson's system was not inferior, for determining the absolute vector direction in humans or dogs, to any other system based on fixed reference points (needing no special examination of the individual's thorax). In rodents, such evidence is lacking. For this reason, only relative changes in vector were evaluated in our experi ments, in order to discriminate changes in E.C.G. wave amplitudes due to the pharmacodynamic effect from changes due only to heart rotation. If, for example,_a ^decrease in the amplitude or inversion of the polarity of the T wave occurs in' alT leads, this result indicates a pharmacodynamic effect (suggestive, in this case, of myocardial anoxia). A Fig. 4. Schematic diagram of the three-dimensional system of E.C.G. leads. (N'- nbers in parentheses denote input plugs of the recorder.) (2) From Freely Moving Animals Electrodes, con sisting of silver plates of 8 mm. diameter embedded in plexiglass bodies, were used for recording. The contact area of skin was shaved and depilated, and the electrode was filled with paste and fixed with collodion. Cable leads were attached by nuts. (The same type of electrode is also used for radio-telemetric transmission.) In guinea-pigs, electrodes were best placed on the head vertex and on the back over the sacral bone. The E.C.G. waveform did not greatly differ frov lead aVF--a distortion by electromyographic potentials occurs only during gross movements. On rabbits, it was better to place the anterior electrode over the lower part of the c-rvical vertebral column to thwart the animal's attempts to pull the electrode down, even though it gave lower E.C.G. voltages and greater E.M.G. distortion. After the reliability of fixation and interelectrode resistance had been checked, the animal was returned to its own cage (without lid), and a two-lead, thin, flexible cable, attached to the electrodes, was fixed by SL 034003 -IT )- ,i i., ''. -y ^... .v,' * H > f*- - "* ;*-- *'*^t-~*- /L.^~*~~v^",` uSome Ehctrophysiological Methods for Studying the Action of Narcotic Agents in Animals 95 insertion into the input plugs of the recorder, placed approximately 1 m. above the animal. The cable length was adjusted so that h did not form too loose a loop. . In guinea-pigs the reliabQity-of E.C.G. recording in a six-hour experiment was more than 90%. .' (3) By Radio Telemetry The same construction and localization of electrodes as was used in (2) was suitable also for wireless radio-telemetric transmission of the E.C.G. The transmitter was fixed on the animal's back by four tapes, the anterior ones forming loops passing under the shoulders, and the hind ones joined beneath the animal's belly by a broad, very soft rubber band. - Radio telemetry has three principal advantages: (:) It provides nearly full reliability of recording; no matter where the animal moves in the laboratory, (2) It elim inates the necessity for any special precautions against mains pick-up or other external interfering potentials. {3) The E.C.G. can be telemetered from hermetically scaled exposure-chambers. In a typical toxicological experiment we exposed the animal first of all to a stream of pure air and only af-er at least the 30 minutes required for the animal to adapt to its new environment was the supply of toxic vapour switched on. When the exposure was discontinued, the animal was left in the chamber so that recovery could be followed up without any handling (Figs 5 and 6). In order to prevent radio-frequency" interference caused by sparking of motors, switches, and other heavy-current equipment, especially that included in the exposure apparatus, we chose a double-frequency modulation system of radio telemetry, using an F.M.subcarrier frequency. The schematic diagram presented in Fig. 7 shows the general arrangement of the telemetry system. The transmitter had no radiating antenna, so that its radius of action was limited to 10-15 metres. The distance to the recording site could nevertheless be substantially greater when the receiving dipole was located in the same room as the exposure equipment and the signal was led to the receiver by a coaxial cable. A conventional F.M.-broadcast timer was used for receiving, as the limitation of the signal amplitude can be easily achieved in the demodulator circuit. After passing a band-pass frequency filter and amplitude limiter, the sub-carrier frequency was (1) demodulated for recording ana,or on-line analysis, (2) amplified for auditory monitoring, and (3) led to a magnetic tape recorder, providing the possibility for further automatic signal processing. The E.C.G. transmitters were designed and built in our laboratory from the `human' model described ir. a i-A-^^577 g i j&n m $: * 1 ' .IT Vt.' A1*" V -A/u-VV/r^v; Hours FtG. 5. Effects on the "heart rate of the expos ure ef guinea-pics to carbon disulphide, measured with radio-telemetric E.C.G. transmission from within the exposure chamber. O mean rates in controls; mean rates during exposure to carbon disulphide. ------~ S.E.M. n,, n._ -- numoers of animals a - level of significance at the maximum toxic effect. SL 034004 f'i..' - * <r cs'?':.~i*t\.- fc.v ^`''i-v.'1' v: v-,^^i-i^;'-1CT^.'' -:- * -' j "?Tv''i --* .v.vrJbfc' fr>---$r** ** :***, ' `iT.t * 96 Ha/w Alikiskovd and Alos Mikiska tOhOOm. (345mirf1) * 13hOOm (155mm;') 750p.p.m.CS2 (10h.0 5m.-13h.20m) ~UJJJJJJA Fig. 6. Examples of guinea-pig's E.C.G. telemetered from within the exposure chamber. i ! 1----- j1-~ ---------1-- Preamplifier 1 l Transmitter VHFoJCiHator h Submodutator J CD rJt.'iL'* '"``V1-v! U-\,4-Tiiy " .7 y."^ Tope re*ploynq recei vi nq - reeordinq ------- ^ Demodulotor Bond-pas; frequency filter Logarithmic vo' tmeter AF Ampl ifier !_______________ i Amplitude limiter i_______________ : Diode inteqra tor Fig. 7- Schematic diagram of the radio telemetry--magnetic tape recording system. SL 034005 111 11 Bin niy . ->- 7 11 nif~'in '1 n ii ^`-f.fci'm 11 pi'1- IbVW'h yw.-^, d** **-"^T> 1^3;: ***& . . Some Eleetrophysiobgical Methodsfor Studying the Action of Narcotic Agents in Animals previous1 paper (Mikitka, 1967). They differ only in regression of rate against weight. Weight was used that no shunting and coupling capacitors larger than instead of age because the age was not always known zo pF were used and that two mercury cells served-as - accurately, as the guinea-pigs were supplied from the power supply. breeding farms. __ Figure 8 also shows how individual values were Results For the quantitative estimation of distributed about their means, and car. be used to pharmacodynamic action the most important `standardize' the animal population with respect to characteristics are the presence or absence of gross the heart rate. If one aims to reduce interindividual irregularities of rhythm in the intrinsic cardiac variability, animals with heart rates near the mean pacemaker and the average heart rate (beats/min.). can be chosen, c.g., those whose heart rates lie The heart rate of a guinea-pig can be determined between the 25% and 75% percentage points. from a one-minute count with an accuracy greater In female guinea-pigs tied to a fixation table, than 0-5%. The duration of the QRS complex, heart rates consistent with those in Fig. 8 were., 0-025 to 0-050 sec., recorded at a paper speed of found. Heart rates of young animals weighing 60 mm./sec., represents a length of only 1-5 to more than 300 g. also fit the nomogram. 3-0 mm. and is a semiquantitative rather than a In less than 5% of the guinea-pigs tied to a quantitative E.C.G. characteristic. Other E.C.G. fixation table a marked cardiac arrhythmia was parameters should be regarded as supplementary found. Mostly it was only a sinus arrhythmia, data, although-they-can.-sometimes contribute apparendynot related terrespiratory rhythm. More greatly to an analysis of the mechanism of action. rarely there were randomly occurring ventricular An example-is shown in Figure 6. extrasystoles. Other types of rhythm irregularities '. It is difficult to characterize the `normal' heart were observed only exceptionally and can be re rate in a given species by one number or by minimal garded as pathological (transient complete atrio and maximal values only. Among factors affecting ventricular block, grouped ventricular extrasystoles, the heart rate in untreated laboratory animals, age etc.). an^motional state seem to be the most important. The E.C.G. waveform observed in guinea-pigs nomogram in Fig. 8 summarizes heart rates bore a striking resemblance to human records, in^Hea-pigs tied to a fixation table, determined in unlike the E.C.G. of smaller laboratory rodents 53 males of different ages. Each animal received such as mice, rats or hamsters. The QRS complex two prior training sessions to accustom it to was usually diphasic and can be designated `RS'- handling. For most animals.mean values from three tvpe, the first deflection being of higher voltage in recording sessions were used to compute the linear most leads. In the extremity leads, as a rule, the Wciqht (9.) <*> f IIOO 1000 --900 1 800 too ,0'3l5 = O 187 loq Heort rate (fnifl.-1) (t) 500 rcentooe points + O Q-`u * 1 Am 4 50 - - 30i - 009^905 2 0- OS75 " -0950 0900 I O- -0-750 .. 400 43 50-- O '3QJO - - -- -02 so -10- l ^ 'CO -ic- Cx.'i 5 L rx7io -------- . J-OQC5 :co -1 Fig. 8. Nomogram of `normal1 heart rate values in fixed male guinea-pigs. Vjt -yi-st**-' v:;3 wswpwtjis.'r.vaar-i vr; * :,-T-- *v v; is?:; . 5jf - !t Hana Mikiskova and AIos Mikiska polarity of deflections corresponded to the standard position of the heart axis in the human E.C.G. _-A distinct isoelectric ST interval of at least 0-05 sec. duration, and elevated or depresses usually by not more than 0 05 mV, was an outstanding feature of the guinea-pig's E.C.G. which was absent in smaller rodents. On the other harm,, the T wave was often of low voltage, even lower than 0-1 mV, and sometimes diphasic, the first phase being of opposite polarity to the R wave. A monophasic T wave of a polarity opposite to that of the R wave, occurring in all E.C.G. leads at once, was, however, exceptional in untreated animals. The rabbit's E.C.G., too, resembled the human's in the same ways, but the ST interval often deviated, from the isoelectric line by more than 01 mV. The effects of handling are illustrated by the following examples. In two groups of animals the results of three experimental procedures were compared*. (l) recording in animals tied to a fixation table, first continuously for 4 hours, then only for the several minutes necessary to record the E.C.G. at. the end of the fifth and sixth hours; (2) E.C.G. radio telemetry from within a quiet laboratory; and (3) E.C.G. recording by means of a wire connexion with recorder input, the animal being unrestrained in its own cage and the experi menter absent. Table V presents mean heart rates and estimates of the standard error of the mean. Values were converted to logarithms before process ing, so the means are the geometric ones, for comparison with the nomogram in Figure 8, While in the tied guinea-pig the heart rate remained remarkably constant during a six-hour experiment, the heart rate in freely moving animals gradually slowed, especially in those with wire connexions, which obviously impose less stress than the. carrying _ of a transmitter. However, in un restrained guinea-pigs in a quiet environment the incidence of sinus arrhythmias greatly increased. Arrhythmias of other types remained rare. In rabbits under similar conditions the decrease in heart rate was less regular, but cardiac arrhythmias, including those originating from atrioventricular block, were observed more often than in guinea-pigs. Experiments with injection of physiological saline showed that intraperitoneal administration caused only very small changes in the heart, rate even as early as 5 minutes after the injection (Table VI). The results with some anaesthetics are sum marized in Table VI. The drugs were administered in doses causing light or moderate anaesthesia. A significant decrease in the heart rate was observed after all the compounds tested- A comparison of the results with those of tests of ^central nervous depressants (Tables I, II, and III) shows that for all the compounds tested the measurement of the heart rate was about as sensitive as the neurophysiological tests. Other E.C.G. results were similar to those described for trichloroethylene and trichloroethanol (Mikiskova and Mikiska, 1966)--slowing of the atrioventricular transmission of excitation, lengthening of the PQ interval, and lengthen ing of the repolarization phase of the electrical systole of the ventricles, t'.e., of the ST interval. More complicated E.C.G. patterns have been observed in guinea-pigs after exposure to carbon disulphide. Their general features were published in a pilot study (Mikiskova and Mikiska, 1964). In order to assess the statistical significance of the observations, a second experimental series was TABLE V Heart Rate of Male Guinea-pigs i.n Different Test Situations Te*r SituaEw Younger animals1 Tiea co fixation table Carrrung transmitter Unrestrained wire connexion Older siiiiaZs2 Tied to fixation table - Carrying transmitter Unrestrained wire connexion Time from Starting Experiment {hours) 01 2345 6 362 366 360 357 355 354 355 36* 352 333 325 320 320 343 290 2-0 265 251 249 243 323 341 316 309 309 311 304 304 275 -276 270 - 272 266 -264 297 274 255 251 244 242 234 Rclothe (%) No. of Experiments No. of Animals iM 2-6 A20 is 7 IK 6 7 6 -- I`S -**3-22 18 18 t3 6 8 6 'Age 2-4 months, weight 330-580 g. 'Age 12-1S months, weight 8oo-i,tco g. SL 034007 Some Elecrrophysiological Methods for Studying the Action of Narcotic Agents in Animals TABLE VI AcTton-eP-SoME Anaesthetics (administered intr iperitoneaiay) on Heart Rate in Guis'EA-itgs tied to a Fixation Table 99 Compound (dose) 0-9% sol. NaCl (0-4 ml.) Dial (019 mM./kg.) ^^Phloral hydrate (0 S4 mM./kg.) v . Trjchloroetbnol(2-2niM./kg.) t i ^ -- Trichloroethylene (67 mM /kg ) - ^~* -- - Ictraci.ioroeihyiene ('v-y Tims from Injection (min.) 5 10 15 30 45 6O 75 165 5 10 15 30 45 60 75 165 5 JO 15 30 45 60 75 5 JO 15 30 45 60 75 165 5 30 15 30 45 60 75 165 5 10 ie 3* 45 Co 75 165 Change in Rale <co) -r o*5 05 00 -- 0-9 -- 17 - 2-1 - 3 - 4-9 SijjHiJicauctf i-4 - 95 -14-4 -lS'2 -17 9 -16 0 -14-3 -10`9 0 002 0001 0001 O'OOI 0*002 -- 23 1 "25 I -22-2 -12 4 - 92 - 8-7 - 83 - 7-2 0 001 0 001 0 001 C 001 0001 O'OOI 0 001 -187 -20'i -- 201X -194 -- 18*9 -17-0 -15-2 -ii-8 0 001 0 001 O'OOI 0 002 0*001 0*001 0*002 0-050 - 11 -4 - 11 `3 - 12 5 -- JO 7 - 9-4 ~ 9-6 " T9 - A3 0-005 0*005 0*010 0025 0-025 0025 0 050 -20 3 0 001 -I&-7 O'OOI 0 or. 1 - -4 9 -- 126 0-0L3 0 001 - 12-5 O'OOI '-12-8 0 001 --12 0 1 0 025 Ko. of Amnials 10 S.D. 3-ti S.E.M. 0-99 IO S.D. 10-17 S.E.M. 3-2* S.D. 3-54 S.E.M. 177 , 6 S.D. 1322 S.E.M. - 5-40 6 S.D. 8-67 * S.E.M* 3 54 :r S.D. 0 9; S.E.M. v.4 S.D. - standard deviation; S.E.M. = standard error of mean. 100 Haita Alikiskovd and Alos Mikiska - - TABLE VII Action of Ca?3on Disulphide on the Heart Rati in Guinea-pigs Exposure Concentration (p.p.m.) {mg./I.) 375 fa Controls 375 fa Controls 750 2'3 Controls ! Durar.^n Hsjri Raid* jVItjn S.E.Af. 3 92'4 3 J 95'5 6 I 85 9 6 | 955 ` 3j 3j 78 8 957 =5'3 ;e2-3 u=i-8 -33 =47 --1'9 AY cf Animals s s 16 16 6 Significance N.S. 0*050 0 002 1Hean rate after exposure is expressed as percentage of the value before exposure. performed (Table VII), in which groups of control animals were exposed simultaneously to pure air. The heart rate was measured both before the exposure and with the shortest possible delay (approximately 5 minutes) after removal from the exposure chamber. A third series of experiments followed the time course of poisoning and recovery during and after exposure to carbon disulphide vapour (750 p.p.m. for 3 hours) (Fig. 5). The heart rate only began to slow 90 minutes after the start of carbon disulphide administration. Recovery was very slow, too. The E.C.3. showed both the signs common to other anaesthetics and sometimes also the displacement of the ST interval, increased duration of P, R, and T waves, and higher degrees of atrioventricular block (Wenckebach's periods or even atrioventricular dissociation) (see e.g., Fig. 6). Sometimes both the amplitude end duration of the T wave were increased, while the ST interval was shortened or absent. These changes were probably caused by irritation of the respiratory mucosae and dis appeared soon after the animal breathed pure air, although recovery of the heart rate to pre-exposure values was only gradual. Unlike the other com pounds tested, carbon disulphide affected the E.C-G. while causing only a slight decree of central nervous depression. After 3 hours' exposure to 750 p.p.m. the animals remained responsive and were able to walk; and the E.E.G. pattern cor responded to the desynchronization stage, but sometimes with random spindles and or slow waves superimposed, i.e., to a light stage of anaesthesia (Fig. 2). Discussion The information on heart action obtained from a short section of the E.C.G. tracing may be invalid and not representative. For this reason, probably, electrocardiography has not yet beiome a popular test of toxicity, and is used, if at all, only once on a patient, usually after the termination of subacute or chronic exposure. From this procedure only gross abnormalities of the E.C.G. can be observed. Moreover, current opinion is that E.C.G. recording in laboratory animals is tedious. We think, however, that with adequate recording, electrocardiography may help to solve many toxicological and pharmacological problems. The literature concerning E.C.G. recording in guinea-pigs will be briefly reviewed. The use of anaesthesia for recording the E.C.G. (Lombard, 1952), drastic restraint of the animal (Pratt, 1938) or keeping it in an unnatural posture (Bartmann and Reinert, 1952) seem only to be necessary for special purposes, such as the examination of all the standard chest leads. The technique of Lukoschek and Thiesen (1934), who fastened the electrodes to the paws of manually restrained guinea-pigs, is quite suitable for short-term recording. The apparatus described by Richtarik, Woolsey, and Valdivia (1965) permits both short- and long-term recording of the E.C.G. from an unanaesthetized guinea-pig in the normal standing position; movements are restricted by a removable box, on the base of v.hich are the electrodes (R, L, F, and earth). We found no references to E.C.G. recor-iing in freely moving guinea-pigs by means of either wire or ladio-telemetric transmission and have therefore devoted our attention to working out suitable tech niques useful, for example, for studying the inter action between drugs and physiological sleep, for which emotional stress of the animal due to the examination must be reduced to a minimum. By using radio telemetry we could also record SL 034009 SomeElectrophysiological Methods for Studying the Action of Narcotic Agents in Animals ioi from within, sealed exposure-chambers, which was important: (x) for studying the action of low concentrations of poison during which the animal should not be handled, as this may interfere with the manifestation of intoxication; (2) during exposure to high concentrations or to compounds for which there was inadequate inform ation on toxicity. In the latter case, continuous monitoring enabled us to prevent death from heart failure and to carry out further examination during its recovery or to attempt experimental therapy. The mean heart-ratc-of guinea-pigs is repor.ed to be 245 to 327 beats per minute. Richtarik and his colleagues (1965) have reviewed other time para meters. The agreement between authors on the heart-rate is quite good. The differences may be attributed to differences in age, weight, and handling during recording, to which most authors paid no attention. There is little agreement, however, on the E.C.G. wave duration. To measure this with satisfactory accuracy recording techniques capable of handling frequencies of sa^M hundred c/s would be required. ^^our experiments interindividual variability was reduced by taking into account the handling associated with recording (Table V) and the animal's weight (Fig. 8). The gradual slowing of the heart rate in freely moving guinea-pigs alone in a quiet environment clearly demonstrated that the heart rate of a fixed animal, althoijgh reproducible, does not represent its `basal' value. -Further experiments (to be pub lished) have confirmed that tying prevented the animal's heart rate from slowing. Similar con clusions were drawn by Essler and Folk (1961,1962) from their telemetered heart rare data, obtained in animals (dog, cat, and rabbit) isolated in a sound proof chamber for 48-ro 72 hours. For restrained rabbits very complete data on the heart rate and other E.C.G. characteristics have recently been published by Haberiand and Regoeczi (1963)- 7'he pharmacodynamic action causing changes of electrical heart activity may be very complex. Direct action on the myocardium is probable for volatile anaesthetics and chemically related com pounds, as the blood concentrations of these in general anaesthesia are also effective in isolated heart preparations (Pirringer and Kcasling, 1939). Interaction with the metabolism of mediators is probable, e.g., for chloral hydrate, which possesses a cholinergic and anticholinesterase activity (Dybing andJJybing, 1955; Krivucova, 1957). Thus depression (or sometimes stimulation) of central nervous control of the heart and the effects of metabolic shifts due to the anaesthetic state cannot easily be distinguished from a direct action on the myocardium in the intact animal. For this reason, E.C.G. recording may often reveal a toxic effect, but gives little indication of the mechanism which causes it. Heart activity is related to the functioning of the whole animal. Changes in it due to drugs can there fore contribute to an understanding of other toxic effects. For example, the heart depression due to" carbon disulphide probably contributes to the fact that no deeper electroencephalographic stages of anaesthesia could be detected for this compound (Horvath and Michalovd, 1952, 19561 Mikiskovi and Mikiska, 1964). It is more difficult to extrapolate results on guinea-pigs and rabbits to other species and to man than is the case for tests of neurotoxicity. In our previous paper (Mikiskovi and Mikiska, 1966) we stated that while the E.E.G. patterns after trichloro ethylene are strikingly similar in rodents, dog, and man, the incidence of cardiac arrhythmias is largely species-dependent (c/. also Atkinson (i960) and Defalque (1961)), and likewise for other compounds. Whereas in the experiments by Desi and Nikolits (1967), 6 7 mM./kg. of benzene, administered intraperitoneally, did not affect the E.C.G. in cats, in our experiments the same dose decreased the heart rate of guinea-pigs by about 10 to 150 and caused changes similar to other lipid sol vents. Inter-spedes differences are encountered also during chronic poisoning. For example, carbon disulphide was reported to alter profoundly the E.C.G. of dogs (Lewey, 1941) but not to affect that of rabbits (Cohen, Scheel, Kopp, Stockell, Keenan, Mountain, and Paulus, 1959; Bneger, De Meio. and Friedman, 19^9) in spite of similar signs o-f central nervous involvement. Conclusions Electrocardiography is the most convenient technique of monitoring the heart action in laboratory animals. Although the E.C.G. is not inertly related so the blood flow, it provides intormanon on the origin and propagation of excitation within the heart. , For screening or pilot experiments, very simple recording techniques and standard equipment are satisfactory. For more derailed examination, suit able elecLropolygraphs arc commercially available. Radio telemetry allows continuous monitoring during exposure to toxic vapours and contributes to an understanding of the course and mechanism of toxic action. SL 034010 102 Hand Mikiskovd and Alos Mikiska TABLE VIII Comparison of the Four Electrophysiological Techniques for Assessing the Toxicity of Industrial Solvents Testing the Action of Narcotic {anaesthetic) Agents ... (ind. -industrial salients). _ . Acute toxicity Proof of presence (screening experiments) Quantitive (c relative) toxicity data Time course of intoxication . . Analysis of mechanism of action Comparability with clinical observations Chronic toxicity Spinal Reflex Excitability Electrical Excitability of Motor Cortex j- j. -r 0 0 c T -r -- -f + 0 0 E.E.G. 0 r -14 E.C.G. -L- 4- - -r ++ -r - very suitable (method of choice): statistically highly significant results can easily be obtained; -f suitable; ----- ' - . relevant data may be found for some compounds but cannot be expected in advance; Cnot suitable (for technical difficulties or lack of stability of the studied response). The normal E.C.G. of guinea-pigs and rabbits is closely similar to that of the human. Experimental results may therefore be compared with clinical observations, although possible inter-species differ ences in response cannot be ignored. By allowing for the variation of the heart rate with the animal's age or weight and due to handling interindividual variability can be substantially reduced, and absolute heart rates obtained at various laboratories can be compared. Table VIII summarizes the strengths and weak nesses of the four electrophysiological techniques for assessing the toxicity of industrial solvents with known or suspected narcotic properties. References Andersen, P., and Kaada, B. R. (1953). The electroencephalo gram in poisoning by lacquer thinner (butyl acetate and toluene). Acta Pharmacol. (Kbh.), 9, 123-130. Arduini, A., and Arduini, M. G. (1934). Effect of drugs and metabolic alterations on brain stem arousal mech anisms. J. Pharmacol, exp. Thtr., no, 76-83. Atkinson, R. S. (i960). Trichloretb lene anaesthesia. Anesthesiology, 21, 67-77. Babskij, A. N., and Malkiman, I. I. (1952). The action of adenosin-triphosphoric acid on the chronaxy of the motor cone of cerebral cortex. (In Russian) Dohl. Akad. Nauk SSSR Otd. Bwkh., 84, 1135-1138, Bartenev, V. D. (1961). Toxicological data on hexafluoropropylene. (In Russian) In Pro.. Scientific Canf. Toxicology oj Alacromalecular Compounds, pp. 21-22. Leningrad. Bartmann, K., and Reinert, H. (1932). Das Exrremitatenund Bruscwand-elektrokardiogramm des nichtnarkotisierten Meerschweinchens. Ncunyn-Schmiedeberg's Arch. exp. Path. Pharmak., ai6, 258-267. Barylnikm, I. I., Vinogradov, V. M., Nikiforov, M. I., and banin, I. N. (1956). The action of chlorptomazine on some functions of the central nervous system. (In Russian) Zh. vys. sh. tterv. Deyat. Pavlova, 6, 885-890. Ber.eiovd, O., Korvdth, M., and -Mikiska, A. (1936). Bewertung von Tiefc und Lange dcr Narkose nach den elektrophysiologischcn Eigenschaften dcr Grosshirnrinde. Physiol, bohemoslov., 5, 188-194. Bickford, R. G. (1950). Automatic elcctrocnccpbaJogiaphic control of general anesthesia. Electroenceph. die. NcurophysioL, a, 93-96. Bohm, and Petersen, I. (1953). Differences in sensitivity to dial of motor effects elicited by stimulation of fore- and hindhmb areas of the cat's motor cortex. Acta physiol, scand., 2), Suppl, 106, 143-149. Brazier, M. A. B. (1954). The action of anaesthetics on the nervous system (With special reference to the brain stem reticular system). In Brain Mechanisms and Consciousness. Ed. DeUftcsnaye, J. F., pp. 163-193. Blackwell, Oxford. Bremer, F., and Bonnet, V. (194S). Action particulidre des barbiruriques sur la transmission synaptique centrale. Arch. tiu. Physiol., 56, 100-102. Brieger, H., DeMeio, R. H., and Friedman, M. H. F. (1949). On the theory and pathology of carbon disulfide poisoning. J. industr. Hyg., 31, 93-105. Brookhart, J. M., and Zanchetd, A. (1956). The relation between electrocortical waves and responsiveness of the corticospinal system. EUctrotnctph. din. Neuro- physiol., 8,427*444- -- Burch, G. E., Abildskov, J. A., and Cronvich, J. A. (1933). Spatial Vectorcardiography. Philadelphia. Lea and Febiger, - .3) Bures, J., PetrfA, M., and Zachar, J. (i960). Electro- physiological Methods in Biological Research. Publish-' mg House of the Czechoslovak Academy of Science, Prague. Bums, B. D. (1951). Some properties of isolated cerebral cortex in the unanaesthetized cat. J. Physiol. (Load,), ill, IJ6-I75------- (1954)- The production of after-bursts in isolated' unanaesthetized cerebral cortex. Ibid., 12$, 427-446. Butler, T. C. (1948). The metabolic fete of chloral hydrate. J. Pharmacol, exp. Ther., 92, 49-38. Chafeta, M. E., and Cadffhac, J. (1954). A new procedure for - a study of barbiturate effect and evoked potentials in _ -the. EEG.____ Electroenceph. din. Nntrophysiol., 6, 565-572- i Some Electrophysiological Methods for Studying the Action of Narcotic Agents in Animals 103 Cohen, A. E., Srticcl, L. D., Kopp, J. F., Stockell, F. R., Jr,, Keenan, R. G., Mountain, J. T., and Paulus, H, J. (1959)- Biochemical mechamsms in chronic carnon disulfide poisoning. Amcr. indtmr. Hyg. Ass. J,, *0, 303-323Counin, R. F. (195s)- Electroencephalographic and clinical observations with trichloroethylene and nitrous oxide anesthesia. Dallas mid. J., 41, 613-61S. Defalque, R. J. (1961). Pharmacology and toxicology of trichloroethylene. A critical review of the world literature. C7m, Pharmacol. Thor,. 2, 665-68S. Deri, 1. (1967). Funfctlonsstffrungen des zentralen Ncrvensysrems bei experimenteller Benzolvcrgiftung. In;. Arch. Getvcrbepath. Geucochyg., 23, 58-71. -------. and Nikolits, I, (1967). Inference of aromatic hydro carbons on conical strychnine spikes. Atfiv. nero, sup. {Praha), 9, 152-159Domino, E. F, .1955). A pharmacological analysis of the Functional relationship between the brain stem arousal and diffuse thalamic projection systems. J. Pharmacol, exp. Ther., 115, 449-463Drohocki, Z. (1948). L'integrateur de l'electroproduction rtrebrale pour l'ilectroenctphalographie quantitative. Rev. natrol., 80, 619. ----- (1956). An electronic integrator for the automatic measurement of average tension in the EEG, Electroenceph. clin. Neurophysiol., 8, 706-707. Dybing F.,and Dybing, O. (1955)- Anticholinesterase and anticurare effects of chloral hydrate and trichloroethanol. Acta pharmacol. (Kbh.), 11, 398-404. ussier, O., and Folk, G. E., Jr. (1961). Determination of ^physiological rhythms of unrestrained animals by ^^wlio telemetry. A'diure {land.), 190, No. 4770, ^V-91. -- ---- (1961). A method of determining true resting heart rates of unrestrained mammals by radio telemetry. Aston. Behav., o, 168-170. -aulconer. A., and Bickford, R. (1961). The Electroencephalo gram in Anesthesiology. Thomas, Springfield, Illinois, -rench, J. D,, Verteano, M., and Magoun, H. 5T. (1953). A neural basis of the anesthetic state. Arch. Neurol. Psyehiat. (CWe.), 69, 519-529. -ulton, J, F., antLKeller, A. D. (1932). Observations on the response of the same chimpanzee to dial, amytal and nembutal, used as surgical anaesthetics. Surg. Gynec. Obiter., 54, 764*770. ------, Liddell, E. G. T., and Rioch, D. Mc.K. (1930). `Dial' as a -surgical anaesthetic for neurological operations: with observations on the nature of its ardor.. J. Pharmacol, exp. Ther., 40, 413-432. -umas,D. W ,and Hine, C. H. (1958 . Neurotoxicity of some selected hydrocarbons. Arch. 1 natistr. Htth. js, 9-15. jangtoff. H., and Munnier, Al. (:ys6,. Electrograpliic aspects of an arousal or attention reaction induced in the uitanaestbctizcd rabbit by the presence of a human being. Electroenceph die. Nturophytiol., S. 623-620. ----- , and------- (i957k Topic action of teserpine, serotonin and chlnrp-omazine on the t-yinnaesiherized rrbhn fcia'.u. Her.', physyf.O'-",,.A... . ; j, s':-:-r -reville. G. D., and Heppenstall, M. E ` 1950!. Chapter VI: Pharmacology. In Electroencephalography. A Fympos;im op its Pattons Aster... hr Hill, D., re..`. Parr, G. Macdonald, London. suede;. A. E. (1951). Inhalation Anesthesia. 2nd ed , ASacmiilan, New York, laberland, G., and Regoeczi, E. (1963). Ut her die Konstanz der Herzaktion des Kaninchcr.;. Umersuchungrn mit Hilfe synchron rettistriertcr Herz-Lrom- und He.-zschallkurven. Z. gcs. exp. AUJ., 137, 123-143. Hess, R., Jr., Koella, YT. P., and Ahert, K. (1953). Cortical and subcortical recording in natural and artificially induced sleep in cats. Elecvpencepfl. din; Neurophysiol, 5, 75-90. Higashida, T. (1964:. Electrophysiological stud es on the r.curotoxic action of industrial solvents. P-oc. 1atk int. Cimgr. Quay. Hrn, Madrid, 1965,3, pp. 1131-1133. Excerpts rnedjec Foundation, Amsterdam. (Int. Congr. Ser. No. 62}, Horvath, M., and .Michalova, C. (1552). Changes of EEG in experimental exposure to carbon disuihee in the light of Vvedensky's theory of pamoiosis. Abstracts of Communication'- of ist Czechoslovak Physiol, Congre's, Prague (In Czech) Cs. Fyr.ol., 1, 262. -------, and ------- (19561. Veranderungen des EEG bei experimenteller Exposition m SchwefelkohJensroff im Lichte der Lehie Wedenskv's von der Parabiose. Arch. Gca.'rbcpaili. Gccaerbchyg., 15, 131-136. -------, and Aiikiski, A. (1057). The effect of carbon disulfide on the central nervous system of the rabbit (an EEG study). (In Czech, Pracoi'. Lck.. 9. 22-2S. Jaroslavskaja, P. I. (1952). The action of benzene on some functions of the central nervous system. (In Russian) In Proc. 2nd Nat. Conf. Probl Indunr. Toxicol., Charkov. King, E. E. (1956). Differential action of anesthetics and intemeuron depressants upon EEG arousal and recruitment responses. J. Pharmacol exp. Ther., 116, 404-417. Kolffiodic, G. M. (1953). The action of ethyl alrohol or, the monosynaptic extensoi reflex and the muki'vnartiC reflex. Acta physiol teams., 29, Suppl. ipfi, 530-537 Korbakova, A. I., Krcmnevj, S. N., Kulagina, N. K., and Ulanova, I. P. (i960). Impairment of the central nervous system functions in chronic benzene poisoning. (In Russian; In Industrial Toxicology, pp. 232-238. Mcdgiz, Moscow. Krdlovf. H , A,, and Fah'/sk. O, ('.057'. The measurement of the electrical excitaruhty of cotduil motor centres: application in industrial toxtcologv. (In Czecoj Praate. Lilt., 9, 44-50. Kiivucovi, M. (t957)- Inhibition of biological oxidations by trichloroethylene and other compounds. Thesis, Charles Univ., Prague. (In Czech). Larrabee. M, G.. c-.d rov.ernak. J. M. (1952). Selective action of are-t'.-ucs or. synapse, and axons ,n mam malian sympatnev.a ganglia. \ Neuropiystc!., 15, 91-214. Lazarc., N. V. 19 r. a ' Noxious Compound; the I'.dusir . Vol. I (i.. Ku star., Afodair. .Moscow. Lev, A. A, (155C. EEG changes during increased excuabiltty of tiiu cerebral cortex. (In Russian, Ffci-i'. Zh. (Mask:,, 42. iczi-i03t. Levitin.--, G. A. I194S;, The state of subordination and d-ronexy er "hr. cerebral cortex during bulbocapnute catalepsy. (In Ru-sian) In Subrd.r.ctici. r'.-n ihe 7 V-r 1 Pi. sir'.->.L or I rr trie; \ r-rrr. .v. . Fe I.ewey, F. II (154:,. rr.p,-tmental chronic carbon disulfide potvoung .c cocs. J. snJustr. H;;., 23, 415-436. Ljublmi. 1. 'lyjS). Deirrtrtmng various charac.eristics of the flexor rerlex as a method experiments trudv of the action ti industrial petsc-s on the nervous system. ;ir. }' -::i..n. In Sr.et.tNc Pcp.is of tic In--.re of led --'rial Hyri.i.L u`.d (Ncurational Diseases. \ ol. :r. No. 5, pp. 5t-'o. Lcmngrad. (tQ;e\ (In Ru: qmed ttom Lazaiw. ,954.) Fain,oho! 1 T-.e.'/so,' , '3, 3. 53 SL 034012 ..-Vv s ^^-v'c:v* >, #*$gttel M:* * *.-v *>' 'fL '' v .`; A:_- i IOd Harm Mikiskova and Abs Alikiska Ljublina, (1959). Various methods used in establishing the max.mam allowable concentrations. Praccv. Lik., zi, Suppl. t, 26-28. Lombard, E. A. (193a). Electrocardiograms of small mam mals. Antet. J. Physiol., 171, 1'19-193. Longo, V. G. {1962). Electroerucephalogrcphh Atlas for Pharmacological Research. Effect of Drug* on the Electrical Activity of the Rabbit -Brain.- Elsevier, Amsterdam. Lukoschek, P., and Thiesen, J. (1954) Das nornale Meer- schweinchcn-Ekg. Z. Kreisl.-Forsch... 43, 172-180. Magoun, H. W., and Rhines, R. (1946 . An inhibitory mechanism in the bulbar reticular formation. J. Neurophystoi. 9, 165-17:. Malkiman, I. 1. (1954a). On the action of adenosintri- phosphoric acid on the excitability of cerebral cortex. (In Russian) Bull. exp. Biol. Med., 37, 14-17------- (1954b). The dynamics of variations of cerebral cortex excitability before and after a convulsive paroxysm. (In Russian) Ibid., 38, 47-30. Mann, H. B., and Whitney, D. R. (1947). On a test of whether one of two random variables is stochastically larger than the other, Ann. math. Statist., tS, 50-60. Marshall, E.K., Jr., and Owens, A. H., Jr. (1954). Absorption, excretion and metabolic fate of chloral hydrate and trichioroerhanol. Bull. Johns Hopk. Hasp., 95, r-:8. Martin, J. T., Faulconer, A,, Jr., and Bickford, R. G. (r959). Electroencephalography in anesthesiology. Anesthes iology, jo, 339-376Matouiek, M. (1967). Automatic Analysis in Clinical Electroencephalography. Research Report 9. Psych iatric Research Institute, Prague. Matthews, B. H. C. (1933). Nerve endings in mammalian muscles. J. Physiol. (Land,), 78, t-53. Mikiska, A. (i960). Bestimmung der elekrrischen Erregbarkeit der motorischtn Grosshimrinde und ihre Verwendung in der Pharmakologie und Toxikologie. I. Methodik, Kontrollversuche ur.d physiologische - Grundlagen. Arch. Getoerbepath. Gerwerbehyg., 18, 286-299. -------(1962). Recording of physiological data on the magnetic tape: application in work physiology and hygiene. Z. Prav.-Med., 7, 283-300. ------- (1963)- Automatic integration of EEG and EMG. (In Czech) Cs. Fysiol., 12, 151-152. -------(1964). Beitrage zur elektrophysiologischen Registrier- ungstechnik: Integration-Tonoandaufnahme, Proc. int. Symp. Corticovisceral Physiol. Path. Ther., pp. 423-424. Akademie-Verlag, Berlin, ------- (I96J). Analysis of some electrophysiological methods with respect to their application in industrial toxi cology. Thesis, Institute of Industrial Hygiene and Occupational Diseases, Prague, (In Czech). . -------(1967). Short-distance radio teleme. 7 of biopotentials in occupational medicine. Z. Prav.-Med., 12, 48-62. -------, and Mikiskova, H. (1964). Determining neurotoxicity by some eiectrographic methods (EEG, EKG, EMG) in guinea-pigs. Actk. ntre. sup. (Praha), , 56-59. Mikiskova, H. (i960). Bestimmung der clektrischen Erregbarkeit der motorischen Grosshirnrinne und ihre Verwendung in der Pharmakologie und Tox ikologie. II. Wirkung von Benzol, Toluol und Xylol bei Meerschweinchen. Arch. Ge-cerbrpjth. Gctcsr- bekyg., 18, 300-359. ------- (1962). Reitrag zur Verwendung der Abwehrreakiion auf die elektrische Hautreizung in der Toxikologie der Sio fe mit zentralnervdser Wirkung. I. Bestimmung der Latenz des Beugereftexes bei den Meerschwein- chen. Ibid., 19, 51-67. -------, and Mikislca, A. (i960). Bestimmung der elektrischen Erregbarkeit der motorischen Grosshimrinde und ihre Verwendung in der Pharmakologie und Tox ikologie. III. Vtrgleich der narkorischen Wirkung von Trichlorathylen und Trichlorathanol bei den Meerschweinchen. Ibid., 18, 310-316. ------- , and------- (1962). Eeitrag zur Verwendung der Abwehrreaktion auf die elektrische Hautreizung in der Toxikologie der Stoffe mit zentralnervbser Wirkttng. II. Bestimmung der Reizschwellenintensitkt bei den Meerschweinchen, Ibid., 19. 68-75. ------- , and ------- (1963). Evaluation by electrophysiological methods of neurotonic action of trichloroethylene and - some related substances. In 2nd ini. Pharmcol Meeting; Prague; Abstracts Biochemical Pharmacology, Con ference Issue, p. 157. ------- , and------- (1964). Elektrophyslologisches Studitim der zentralnervbsen Wirkung von Schwefelkoltlenstoff und anderen Losungsmitteln Proc. int. Symp. Corricovisceral Physiol. Path. Ther., pp, 425-417. AkademieVerlag, Berlin. ------- , and------- (19661. Trichloroethanol in trichloroethylene poisoning. Bnt.J. industr. Med., 23, 116-125. Naess, K. (1950a). An investigation into the excitability of the motor nerve of rabbits under ether anaesthesia. Acta pharmacol. (Kbh.), 6, 81-91. ------- (1950b). Changes in the excitability curve of the rabbit's motor nerve during ether anaesthesia (with special reference to the mechanism of the stimulating effect of ether). Ibid., 6, 123-126. ------- (1950c). Comparison of effect of ether and curare on neuromuscular transmission, investigated by means of prostiginine. Acta physiol. stand.,cm, It 7-124. Niemer, W. T., and Magoun, H. W. (1947). Reticulo-spmal tracts influencing motor activity, j. comp. Neurol., S7, 367-379. Novikov, J. V. (1956). The action of low benzene concentra tions in the chronic experiment on higher nervous activity of animals. (In Russian) Gig. i Sonic., 21, No. 2, 20-25. Okuma, T., Shimazono, Y., and Narabayashi, H. (1957). Cortical and subcorneal electrograms in anesthesia and anoxia in man. Electroenceph. din. Neurophysiol., 9, 609-622. Oljunin, I. V. (1957)- Changes in the activity of nervous system and autonomic functions in animals during adaptation to chronically administered ov-ax solvents. (In Russian) In Proc. Sth Cvnf. on Problems of Industrial Toxicology, pp. 37-38. Institute of Industrial Hygiene and Occupational Diseases, Leningrad. ------- ([95R\ Adaptation of animals to the narcotics (organic solvents) of two different types of action. (In Russian) pp. 206-210. In Scientific Papers of the Institute of Indust-ial Hygiene and Occupational Diseases, Leningrad. Palatnik, S. A. (1949). The summation of subdiresliold stimuli in the motor zone of cerebral cortex in poikilothermic animals d'trinv catalepsy. (In Russian) Ficiol. Zh. {Mask.), 35 27-33- Plttingcr, C. B., and Keasling, H. H. (1959). Theories of narcosis. Anesthesiology, 20, 204-213. Pratt, C. L. G. (1938;. The electrocardiogram of the guineapig. J. Physiol. (Land.), 92, 268-272. Rascanu, V., Kupri, M., and Popovici, G. (1939). _L'action du chloroforms, de l'evipan et du vironal sur l'activtti des centres inotcurs corticaux. C.R. Sac. Biol. (Paris), 130, 1602-1605. I2L2**13\ .-.' -. '.- . -, .`I ". ,,'. .,., 'V_'H^. >r*r,* :;r.|. -e.-e^ .--.i . .-.'.-e .i:-.''i. i ^.v-.sVA- ii'i'V -lastsi* An"** Stone Eleetrophysiological Methodsfar Studying the Action of Narcotic Agents in Animals 105 Richard, .A. (1939). Sur la narcose i1`dvipan. Ibid., 130, Ilt-IOl9. Riduarik, A., Woolley, T. A., and Valdivia, E. (1965). Method for recording ECGs in unanesthttized guinea-pig*. J. appi. PhyjtoL, ao, 1091-1093. Rossi, G. F., and Zirondoli, A. (19;;). On the mechanism of the cortical desynchronization elicited by volatile anesthetic*. Electsotneeph. elm, Neserophysiol,, 7, 383-390. Rozin, M. A. (1954). Determining tome time character istics of hind-limb flexion reflex in animals. (In Russian) In Producing Pathological States itt Animats for Studying Experimental Therapy, pp. . 183-189. Leningrad.. Sabawall, P. B., and Dilkm, J. B. (1958). Action of volatile anesthetics on human muscle preparations. Anesthesi- fr*y> *9* 587-594Sehlag, J, D. A- (193d). Hffert of drugs on pyramidal dis charges evoked, by cortical stimulation. Eiearoenceph. din. Neserophysiol., 8, 4x1-435. -------, and Brand, H. (1958). An analysis of electrophysiological events in cerebral structures during ether anesthesia. Ibid., 10, 305-324 Schneider, J. (1954). Etude dearogiaphique et dlcctrophysiologique des mdcanismes d'acuon des agents anesthdsiques USucls. Rev. neural., 91, 428-444 Sechcr, O. (1950). The peripheral action of ether estimated ^on isolated nerve-muscle preparation. I. Direct and ' direct Stimulation. Acta pharmaeol. (Kbh6, ^l-38j. --^TrSS1*)- The peripheral action of ether estimated on isolated nerve-muscle preparation.' II. Synergism of ether and curarixing substances. Ibid., 7,83-93. ----- - (1951b), The peripheral action of ether estimated on isolated nerve-muscle preparation. III. Antagonistic and synergistic actions of ether and neostigmine. Ibid., 7, 103-118. ------r (i95Jc). The peripheral action of ether estimated on isolated nerve-musdc preparation. IV. Measure ments of action potentials in nerve. Ibid., 7, 119-131. -- (rpsrd). Pilot-investigation into the periphe -afaction of anaesthetics. Ibid., 7,231-242. Sena, C., and Amhrosio, L. (1961). L'elettroencefaJogramma in Medidna del Lavoro (Quademi di Acta neuro logies XXI), Maples. Sherrington, C. (1906). The Integrative Action of the Nervous System. Cambridge. -------(1909). A mammalian spinal preparation, J. Physiol. (.Land.), 38, 375-383. Shimazono, Y., Okuma, T., Fukuda, T., Hirai, T-, and Yamimasu, E. (1953). An elcctroenccphalograpbic . study of barbiturate anesthesia. Eleclrotracph, din.' Neurophysiol., 5, 525-532, Speranskij, S. V (1962). Mew and modified methods for studying the relative toxicity of metals. In Industrial Toxicology and Clime 0/ Occupational Diseases of Chemical Etiology, pp. 3-64. Moscow. (In Russian) Toman, ). E. P. (1952). Neuropharmacology of peripheral nerve. Pharmacol. Rev., 4,168-218. --, and Davis, J. P. (1949). The effect of drugs upon the electrical actH-y of the brain. Ibid., 1, 425-492. Verdeaux, G., and Marty, R. (1954). Action sur l'dlectroencdphalogramme de substances pharmacodynamiques d'intdret dinique. Rev. Neurol., 91,405-427. Votava, Z., Beneiovi, O., and Metyiovi, J. (1958). L'action potentialsatrice de eertains derivds de la phenothiazine sur la durde et la profondeur de la narcose au Thiopental. J. Physiol. {Paris), 50, 563-565. Wilson. F. N., Johnston, F. D., and Kossmann, C. E. (1947). The substitution of a tetrahedron for the Einthovtn triangle. Atncr. Heart J., 33, 594-603. Wyke, B. D. (1955). Electrographic monitoring of barbiturate narcosis. Anaesthesia, 10, 247-259. Zakusov. V. V. (1948). Experimental Data on Pharmacology of Central Nervous System. (In Russian) Meagiz, Leningrad. / ------- <'953)- Pharmacology of the Nervous System. (In Russian) Mcdgiz, Moscow-Leningrad. rjT`W."^>r^Sr SL 03401* m