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Drug-induced dissociation between evoked reticular potentials and the EEG
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ELECTROENCEPHALOGRAPHY AND CLINICALNEUROPHYSIOLOGY DRUG,INDUCED
BETWEEN
EVOKED
RETICULAR
DISSOCIATION POTENTIALS
AND
THE
EEG 1
RICHARD P. WHITE, HOBARTH. SEWELLJR. AND ALLANS. RUDOLPH Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tenn. (U.S.A.)
(Accepted for publication:November23, 1964) INTRODUCTION Numerous studies indicate that drugs can produce EEG or behavioral effects wilich appear unrelated to their actions on reticular potentials evoked by peripheral stimulation (see Discussion). Since many conflicting inferences have resulted from comparing the effects of a few drugs, it seemed pertinent to study a wide variety of compounds, and their antagonists, at various dose levels to ascertain whether drugs may be grouped systematically according to their actions on the reticular evoked responses and their effects on the EEG. In addition, such a study seems important because any demonstrable effect of a drug on the reticular formation should help reveal its participation in central pharmacodynamic mechanisms. To this end 16 drugs were studied in 147 animals. METHODS Adult albino rabbits weighing between 2.3 and 3.1 kg were used and all surgery was performed under local anesthesia (1% procaine). The animals were immobilized with decamethonium or d-tubocurarine (1 mg/kg) given slowly i.v. and placed under artificial respiration with tidal volumes and rate set to maintain control heart rate, blood pressure and EEG tracings. Body temperature was maintained with heating pads. EEGs were obtained from steel electrodes inserted into the cranium over the right motor and left limbic areas of the cortex (White and Daigneault 1959) and recorded with a Grass t This investigation was supported by U.S. Public Health Servicegrants K3-NB-15279and NB-04305 from the National Institute of Neurological Diseases and Blindaess. Portions of this article have been published in abstract form (Fed. Prec., 1964,23: 560; Pharmacologist, 1964,6" 170).
Polygraph. Blood pressure was recorded from one femoral artery by a Statham P23 transduc~:r connected to the polygraph. In most animals the contralateral sciatic nerve was stimulated with single shocks of near maximal strengths from 3 to 7 V with 0.1 msec duration squ~.re waves. In eleven experiments midbrain evoked responses were elicited by excitation of the bulbo-pontine reticular substance (Loeb et al. 1960). Initially the responses were picked up by bipolar concentric steel electrodes, insulated except at the tips, with the tips separated by 0.5 ram, but later unipolar electrodes were employed with the referential one located in the nasal bone. Comparable recordings of drug induced changes were obtained by either technique. The midbrain reticular electrode was positioned stereotaxically and located according to the atlas of Fitkovfi and Margale (1962). Infiltration anesthesia was performed at pressure points with the stereotaxic apparatus. The electrode position was verified by sectioning the fixed brain. In 109 animals the midbrain electrodes were placed either at AP 9, V - 2 to - 4 , L 1.5 to 3, or more com,aonly at AP 10.5, V - 1 to -3.5, L 1.5 to 3 with 97% of all electrodes being located in the nucleus reticularis tegmenti in an area adjacent to, and parallel with, the periaqueductal gray. These locations correspond to sites described by Randt et al. (1958)in cats and by Chin and Domino (1961) in dogs where reticular evoked potentials were commonly recorded. Action potentials were also recorded from the sciatic nerve central to the point of stimulation in 16 animals and from the medial lemniscus (AP 9, V --6, L 1) in 9 to ascertain whether changes in nerve excitability of conduction might account for some of the findings. In most experiments the morphology of the response rather than strictly topographic criteria Electroenceph. clin. NeurophysioL, 1965, 19:16-24
DRUG-INDUCEDDISSOCIATION was used to select the recording point. At least 20 single responses were studied during control periods and again after giving the experimental drug. The single shocks were applied at random intervals, usually with at least 20 sec separating each response. Responses produced by the single shocks were amplified (Grass preamplifier) and displayed on a Dumont dual beam oscilloscope. In approximately 25% of the animals cortical evoked responses were also recorded simultaneously. The drugs studied were: pentobarbital, parab dehyde, chiorpromazine (Thorazine), thioridazine (Mellaril), chlordiazepoxide (Librium), phenoxybenzamine (Dibenzyline), imipramine (Tofranil), scopolamine, atropine and JB-329 (l-ethyl-2-pyrrolidyl-methyl-phenyl-cyclopentyl. glycolate hyd rochloride, Ditran, Lakeside Laboratories). The last compound is a hallucinogen which exerts central atropine-like actions in animals (White and Carlton 1963). The degree of EEG depression induced by these agents was tested physiologically by noise and touch stimuli and further by high frequency (e.g., 60 c/sec) sciatic stimulation with varying voltage, t Also studied were: physostigmine, catechol, methamphetamine, d-amphetamine, methylphenidate (Ritalin), caffeine and pentetrazol (Metrazol). In addition, neostigmine was administered to ascertain whether its peripheral actions could reproduce any central effects of physostigmine (White 1963). All drugs were injected through a cannulated saphenous vein and dosage was computed from the active base. Fifty-seven animals were given a single drug in graded doses and the The authors and four volunteers assessed the sensations derived from supramaximal single shock and repetitive (60 c/see) stimuli by applying skin electrodes treated with cardiopaste and firmly pressed against the ulnar nerve. The latter stimuli produced an unpleasant, but not painful, paresthesia. Submaximal stimuli as used in these animal experiments produced a "tingle" sensation. Nevertheless, in the animals care was taken to apply stimuli only long enough to obtain an effect, to avoid any unnecessary discomfort. Single shock stimuli applied to somatic nerves seem to be ignored by unanesthetized free moving cats (Fessard 1962). To compare this neuropharmacologic with physiologic studies (e.g., French et aL 1952), where similar stimuli were used, general anesthetics were not employed. That residual amounts may significantly affect the actions of the drugs studied herein is indicated in many of our findings.
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effects of low and high doses were compared in the same animal. Fifteen to 20 min separated each injection. Results obtained with the highest dosage were verified in at least 2 separate animals by giving the drug in a single injection. A total of 59 animals received the drugs as a single injection. In 74 of these 116 animals the drug was permitted to wear off, in the remaining a cross-over design was employed where a second drug was administered 15-20 min later to ascertain whether it could alter the effects of the first. In addition, 31 animals were given drug combinations where the time interval between injections was less than 10 min. Each drug was administered at a rate which produced minimal effects on blood pressure. Experiments in which the blood pressure decreased more than 25 % of control were arbitrarily discarded. RESULTS Effects of EEG "'activators" The results are shown in Table I and Fig. 1. Physostigmine and catechol abolished midbrain reticular evoked responses and induced EEG activation. This effect on the reticular evoked potential lasted 4-16 min and recovery was slow (3-9 min), the EEG effect outlasting the abolition of the reticular response. In some animals an ephemeral enhanced evoked potential occurred during the administration of these drugs. Neostigmine or saline injections had none of these effects. Methamphetamine would also produce abolition of the single shock responses but only at higher doses than that required to induce EEG activation. Otherwise, this drug closely resembled physostigmine. Conduction was not impaired by any of these drugs. Residual effects of either physostigmi~,e or methamphetamine were still demonstrable after recovery of the midbrain evoked response
were
administered
to 2
separate animals in which the midbrain eveked response was elicited by intrareticular pontine excitation with identical results, i.e., EEG activation and abolition of the single shock evoked potential. Significantly, repetitive stimulation of Electroenceph. clin. Neurophysiol., 1965, 19:16-24
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R.P. WroTEet aL TABLE I Drug effects on reticular evoked responses and on the EEG
(Plus refers to augmentation, zero to no change, minus to decrease and an x to abolition of evoked responses. Ratio represents number per total of rabbits receiving the drug indicated*) Drug
A. EEG deactivators Pentobarbital
Paraldehyde Chlorpromazine Thioridazine Phenoxybenzamine Chlordiazepoxide Imipramine Atropine Scopolamine JB-329 B. EEG activators Physostigmine Catechol Methamphetamine
Amphetamine Methylphenidate Caffeine Pentetrazol
Dose mg/kg
EEG effects
3-6 8-10 15-30 40- 100 !20-200 I-I0 0.5-5 I-5 1-20 I-5 0.5-20 0.2-20 0.5-2
synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony
0. I 6--I 2 0.5-I 2 3-5 0.5-I 2-3
activation activation activation activation activation activation activation activation
I-I0 5 10=15 2.5-5 5- I0 5-15
Effects on evoked responses +
0
6/17 1/4
10/17 2/4
2/6
3/6
8/10 4/9 6/9 9/14 4/11 3/16 4/23 2/5
2/10 5/9 3/9 5/14 7/11 12/16 19/23 3/5
~ 1/17 I/4 3/8 1/6 4/7
Main effect on amplitude of evoked responses ×
5/8 3/7
1/16
3/12
7/12
1/12 I/3
3/14
2/14 3/7
2/9
9/14 I/7 6/9
activation
I/2
I/2
activation activation activation seizure
I/6 4/5 I/4
2/6 I/5 I/4
27/27 16/I 6 1/12 2/3 6/6 3/7
I/9 I/6
2/6
2/4 6/6
no change to greater variable decreased to abolished variable decreased to abolished increased no change to greater increased increased no change to greater no change to greater no change to greater no change to greater abolished abolished variable decreased to abolished abolished no change decreased to abolished no change no change variable increased variable abolished
~"Data obtained from 116 animals with 59 receiving by single injection the drug indicated and 57 given the drug in graded doses. Data were grouped when effects on evoked potentials were similar regardless of dose and when a single injection of a high dose verified the results obtained with graded doses. The number of animals given physostigmine or catechol appears excessive because they represent 5 control experiments plus others where prior administration of CNS depressants failed to block their actions.
the sciatic nerve also abolished the intrareticular single shock response in control experiments, thus mimicking this drug action. Caffeine, methylphenidate and especially damphetamine induced electrographic changes similar to methamphetamine but in our experience manifested greater dose-~esponse variation (Table l). Indeed, such drags (in contrast to physostigmine) we originally believed were unable to abolish midbrain evoked responses. Pentetrazol in subconvulsive doses usually increased the amplitude of the reticular evoked re-
sponses but during seizures produced by this drug, evoked potentials were unobtainable. Abolition of the evoked response was strikingly evident during the post-ictal period (Fig. 1) where apparently brain depression is marked since the EEG is synchronous and high frequency excitation with supramaximal (e.g., 9 V, 60/see) stimuli failed to causc EEG activation. Conduction was not impaired in these animals. Return of the EEG alerting response and of the single shock response occurred about 20 rain after all traces of seizure activity subsided. Electroe:)ceph. clin. NeurophysioL, 1965, 19:16--24
DRUG-INDUCED DISSOCIATION
EE~I ~p.V A
EVO.EO i,oc~
RESPONSE,,.l.- - 50 NSEC
2 .~EC
CONTROL
/
I MG METHAMPHETAMINE /
/
I
CONTROL
19
l
10MIN LATER
O.I MG PHYSOSTIGMINE
CONTROL
T I
0o...o
I ,..o
2 MG M[THAMPHETAMINE
G PI,IHE[NOXYBENZAMINE T l
I
EVOI
EVOKED RECOVERY
,0 MG CATECHOL
t
Fig. I Dissociation between the EEG (right motor) and single shock reticular responses induced by drugs. Control records for each experimental animal are shown on the left. A (from left to right): methamphetamine (I mg/kg) induces EEG activation but evoked response remains unaltered; additionally 2 mg/kg abolishes the reticular evoked potential; recovery of the evoked response which precedes return of the control EEG sleep pattern. B: physostigmine causes EEG activation and abolishes evoked response for protracted period; recovery of evoked potential. C: phenoxybcnzamine induces EEG synchrony and slightly enhances evoked response; catechol exerts an activating effect similar to physostigmine and methamphetamine (third excerpts above) despite presence of phenoxybenzamin©; recovery. D: pentobarbital (18 mg/kg) produces EEG synchrony and abolishes reticular evoked potential (contrast with phenoxybenzamine in above tracing); physostigmin¢ significantly alters EEG but not the evoked record; recovery. E: pentetrazol (5 mg/kg) causes only EEG activation (similar to I mg/kg of methamphetamine, A); EEG synchrony and abolished evoked potential 9 rain after a seizure episode; recovery. Shock artifacts m~de visible by Zip-a-tone line (exception: stimulus artifact in tracings D corresponds to left edge of panel).
Effects of EEG "synchronizing" agents These drugs could be grouped into two categories. One, consisting of pentobarbital and paraldehyde, produced EEG synchrony in lower doses and often enhanced evoked responses. In higher doses these drugs further depressed the EEG pattern and depressed or abolished the evoked potential (Table I, Fig. 2). The second category, consisting of chior-
promazine, thioridazine, phenoxybenzamine, chlordiazepoxide, imipramine, JB-329, atropine and scopolamine all produced a similar degree of EEG synchrony within a wide dose range, often augmented the evoked response and rarely depressed this response even in high doses (Table 1), Chlordiazepoxide (2 mg/kg), phenoxybenzamine (2 mg/kg) and scopolamine (1-2 mg/kg) produced identical effects when the midbrain E/ectroenceph. clin. Neurophysiol., 1965, 19:16-24
R.P. WHITE et
20
EEG17~V
EVOKEOL~.~V
RESPONSE 5OMSEC
CONTROL
T
CONTROL
D
al.
l
IOMG CHLOROIAZEPOXIOE 'T
5 MG
O.IMG PHYSOSTIGMINE
T
0.2 MG SCOPOLAMINE
0.1MG PHYSOS'~IGM~NE
T
20 MG SCOPOLAMINE
I MG METHAMPHETAMINE l
25 MG PENTOSARBITAL
i CONTRC'..
I
CONTROL
I
/'
5 MG PHENOXYBENZAMINE
2 MG METh'AMPHETAMINE
T
0,1MG PHYSOSTIGMINE
0.1MG PHYSOST;GMINL"
I MG SCOPOLAMINE
I
0
E
"
Fig. 2 Illustrates antagonism and fundamental differences among drugs on the EEG (right motor) and reticular evoked responds. Control tracirlgs for each experimental animal are shown on the left. A (from left to right): chlordiazepoxide (10 mg/kg) produces EEG synchrony and enhances evoked potential; physostigmine actions are not blocked by chlordiazepoxide; low dose of scopolamine reverses physostigmine effects. B: thioridai, ine has effects like chlordiazepoxide (above A); physostigmine actions are not blocked by thiorida;,ine; scopolamine (20 mg/kg) reverses the physostigmine effects but even this enormous dose fails t~ impair the evoked response. C: pentobarbital (5 mg/kg) produces efl'ects similar to above ataraxics; methamphetamine (I mg/kg) antagonizes EEG effects of pcntobarbital but evoked response persist,J; anesthetic dose of pentobarbital (25 mg/kg) produces changes not seen with either of the previous drug injections. D: phenoxybcnzamine (5 mg/kg) blocks actions of methamphetamine (2 mg/kg) but not those of physostigmine. E: evoked responses obtained by intrareticular stimulation. Scopolamine (I mg/kg) reverses the effects of physostigmine (0. I mg/kg) and blocks a 0.2 mg/kg dose, Shock artifacts made visible by Zip-a-tone lines.
evoked response was elicited by intrareticular stimulation. Furthermore, only scopolamine blocked the usual actions of physostigmine in these animals (Fig. 2).
Effects of drug combination Scopolamine (0.2-2 mg/kg), atropine (2 mg/kg) or JB-329 (0.5-2 mg/kg) blocked completely and consistently both of the electrophysiological actions of O.1 mg/kg of physostigmine.
In some experiments when the antagonistic dose was minimal (e.g., 0.5 mg/kg atropine) a physostigmine-induced EEG activation occurred without a concomitant suppression of the evoked potential--another example of drug-induced dissociation. Imipramine in 3-5 mg/kg doses markedly shortened in 3 animals and abolished the actions of physostigmine (0.1 mg/kg) in another. In contrast, pentobarbital (5-10 mg/kg), Electroenceph. clin. Neurophysiol., 1965, 19:16-24
DRUG-INDUCED DISSOCIATION
chlorpromazine (1-10 mg/kg), thioridazine (1-5 mg/kg), phenoxybenzamine (2-4 mg/kg) and chlordiazepoxide (1-20 mg/kg) failed to block the usual actions of 0.1 mg/kg of physostigmine (Fig. 2), but any of these CNS depressants elevated the dose of methamphetamine necessary to produce EEG activation and to abolish the evoked potential. Lower doses of pentobarbital (2 mg/kg) or chlordiazepoxide (2 mg/kg) promptly restored single shock responses following high doses (3 mg/kg) of methamphetamine in 7 of 8 animals. The usual actions of catechol (6-12 mg/kg) were not blocked by 5 mg/kg of phenoxybenzamine (Fig. 1), 5-10 mg/kg of pentobarbital, or 1 mg/kg doses of scopolamine. Also, there was no obvious synergism between the EEG activators in abolishing the evoked potential (e.g., 5 mg/kg of caffeine followed by 0.5 mg/kg of methamphetamine) but too few experiments were performed to make definite inferences. Another case of dissociation occurred in 4 of 7 animals given I mg/kg of scopolamine and 2-3 mg/kg of methamphetamine in that order. Methamphetamine had negligible EEG effects but abolished evoked potentials suggesting scopolamine has an EEG blocking action cephalad to the midbrain. Similar results were obtained in 1 of 3 animals given imipramine instead of scopolamine. We were unable to confirm the report of Long~~ and Silvestrini (1958) that scopolamine prevents depression of single shock responses caused normally by high doses of pentobarbital (15 mg/kg i.v., 25-40 mg/kg i.p.). However, higher doses of pentobarbital are apparently required to suppress the evoked response since in 3 of 5 animals 13-18 mg/kg i.v. were necessary to diminish this response which is tdgher than expected from control data (Table 1), although 20-30 mg/kg always depressed or abolished the evoked response. Moreover, we initially administered 1 mg/kg of scopolamine compared to their maximum dose of 0.2 mg/kg and all our drugs were given intravenously.
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prevented this action. Catechol likewise abolished (4 rabbits) or markedly reduced (2 rabbits) the cortical evoked response and neither phenoxybenzamine or imipramine prevented this effect. In low doses (0.5-1 mg/kg) d, amphetamine and methamphetamine failed to alter the cortical potentialin 4 animals, but in I methamphetamine (2 mg/kg) flattened this response for approximately 12 min. Methylphenidate (10 mg/kg) reduced evoked cortical potentials in 1 of 3 animals whereas pentetrazol diminished this response in 2 rabbits only after seizures. In 2 separate animals, paraldehyde and pentobarbital in lower doses failed to appreciably alter the cortical potential but in anesthetic doses suppressed this activity. Scopolamine, imipramine, chlordiazepoxide, phenoxybenzamine or thioridazine either had no effect or slightly increased the single shock cortical response when administered to 3 or more animals, nor did JB-329 or atropine exert appreciable action here in 2 animals. DISCUSSION
These studies clearly show that a drug-induced EEG synchronization or activation may be accompanied by an abolition, decrease, increase or no change in single shock responses recorded from midbrain reticular substance. While studying the anesthesia state, Longo and Silvestrini (1958) first noted such disparities between EEG patterns and evoked reticular potentials. However, high doses of pentobarbitai and all clinically used general anesthetics produce EEG synchronization and markedly depress or abolish midbrain evoked responses (cf Nagai 1963) and it is not surprising that paraldehyde also exerts these effects. That CNS stimulants such as phys.. ostigmine and methamphetamine can also abolish single shock responses while (unlike anesthetics) producing EEG arousal is of theoretical interest. This effect on the midbrain evoked response seems analogous to the attenuation or occlusion caused by repetitive physiologic or dectrophysiologic excitation of afferent nerves (French et a/. 1952; Hern~ndez-Pe6n and Effects on cortical evoked responses Physostigmine flattened this response in 3 Hagbarth 1955). It is likely that at least some stimcontrol animals and in 5 rabbits previously given ulants produce sufficient neuronal excitation to phenoxybenzamine, thioridazine or pentobar- mimic the central effects of high frequency stimbital (6 mg/kg); scopolamine (! and 10 mg/kg) ulation. Moreover, no response to a single in.. Electroeneeph. elin. Neurophysiol., 1965, i9:16-24
22
R.P. WHITEet ai.
coming signal would be expected if stimulant drugs caused all neurons capable of reacting to discharge maximally. Recordings from single reticular units indicated repetitive stimuli may activate some neurons and depress others (Hern/mdez-Pe6n and Hagbarth 1955) but activation of such units is commonly associated with stimulation (Machne et al. 1955; Desmedt and Schlag 1957; Schlag and Brand 1958), whereas ether (Schlag and Brand 1958) and thiopental (Yamamoto and Schaeppi 1961) in high doses predominantly decrease unitary discharge. Regardless of the mechanism, evidently some stimulants readily mimic electrographic features of CNS excitation caused by marked physiological or electrophysiological stimulation. Serotonin and LSD-25 will also reduce potentials produced in midbrain reticulum by single shock stimuli (Hart et al. 1962) and amphetamine and LSD-25 have been reported (Hance 1959) to reduce such potentials in the central gray, an area considered by Brodal (1957) closely related functionally with the reticular formation. Such reports, coupled with those herein, demonstrate that the midbrain enhanced evoked responses recorded after Metrazol, strychnine (Arduini and Arduini 1954) or caffeine in cats with diencephalic lesions (Jouvet et al. 1957) are by no means a universal characteristic of CNS stimulants. Why some stimulants are more effective than others in abolishing reticular evoked responses, however, remains undetermined. That different mechanisms o1' stimulation are involved is indicated by the specificity with which some agents can block excitant effects of certain drugs (Fig. 2). it is also evident that in lower doses, or during recovery periods, stimulants can cause EEG arousal patter~Js ':,ithout abolishing midbrain evoked potentials and that such a dissociation is accentuated by appropriate CNS depressants (Fig. 2). This finding would suggest that the "spontaneous" EEG mechanism can be a more sensitive index to drug action than the "superimposed" single shock reaction. Our findings concur with previous reports that atropine (2-4 mg/kg) in cats (Loeb et al. 1960), scopolamine (0.25--I mg/kg) in rabbits (Longo and Silvestrini 1958), chlorpromazine (5-10 mg/kg) in cats (DeMaar et ai. 1958) and sedative
doses of pentobarbital (5-10 mg]kg) in rabbits (Longo and Silvestrini 1958)need not depress and may actually augment reticular evoked responses while producing EEG synchronization. Morphine (Chin and Domino 1961), meprobamate (Kletzkin and Swan 1959) and lower doses of chloralosane (Arduini and Arduini 1954) or thiopental (King 1956) also clearly possess these characteristics. In addition, chlordiazepoxide, phenoxybenzamine, thioridazine, imipramine, JB-329, lower doses of paraldehyde and even enormous doses of atropine or scopolamine were fc,und to exert these actions. Excepting chloralosane, paraldehyde or the barbiturates (in high doses), none of these drugs are classic anesthetics and certainly their actions on midbrain evoked potentials contrast with the anesthetic state where evoked [potentials are unequivocally depressed or abolished. The inference that atropine (Loeb et al. 1960) and meprobamate (Kletzkin and Swan 1959) do not depress excitability of the ascending activating system because these drugs fail to depress midbrain evoked response (in contrast to anesthetic doses of barbiturates) seems untenable in view of the large number of drugs which share this property, it is possible, rather, that EEG synchronizing drugs reduce the multifarious stimuli which impinge on the reticular formation, thereby reducing tonic occlusive phenomena and "spontaneous" neuronal activity. !t such a reduction in activity were sufficiently greater than that expected from the elimination of proprioceptive activity by curarization, ar~ enhanced single shock evoked response might be anticipated in the absence of surgical anesthesia. Similar hypotheses have been advanced to explain enhanced reticular potentials following chlorpromazine (DeMaar et al. 1958) and morphine (Chin and Domino 1961). The fact that thiopental (Yamamoto and Schaeppi 1961) and chlorpromazine (Bradley 1957) can greatly reduce "spontaneous" firing of individual reticular units without "blocking" evoked discharges, indicates that large populations of neurons may react in a similar manner. Moreover, physiologic EEG slowing is associated with enhanced cortical responses (cf. Desmedt and La Grutta (1957) and sedative drugs also facilitate hippocampal evoked potentials (Costa et al. Electroenceph. clin. NeurophysioL, 1965, 19:16--24
23
DRUG-INDUCED DISSOCIATION
1961). If depression of evoked potentials were the only index of drug-induced depression, atropine and many CNS depressants would have no significant actions on the neuraxis (cf. White 1965). Certainly enhanced evoked responses are commonly associated with drugs which induce EEG synchronization and apparently the failure of a drug to depress midbrain evoked potentials in animals immobilized by curariform agents or brain lesions does not prove an absence of an effect. Whether the drugs studied affect directly or indirectly the midbrain reticulum remains problematic. No similar electrographic analysis, even intrareticular stimulus-response data, will resolve this point in intact animals. If they act extrareticularly, evidence of pharmacologically distinct neurons (cholinergic, etc.) discharging into the reticular formation is provided by the relatively specific blockade exerted by some drugs against several CNS stimulants. Our findings with intrareticular responses imply a direct effect. Moreover, micro-electrode studies suggest a direct action for physostigmine (Desmedt and Schlag 1957), epinephrine and acetylcholine (Bradley 1957). in any case, all evidence supports the suggestion of Bradley (1957) "that it is unlikely that this complex system is pharmacologically homogeneous". SIJMMARY
I. Sixteen drugs were given i.v. to curarized rabbits to clarify neuropharmacologic relationships between single shock responses in midbrain reticulum to single sciatic shocks and EEt'; patterns. The results revealed that drugs can produce marked dissociation between the two phenomena. 2. Physostigmine, catechol and relatively high doses of methamphetamine or d-amphetamine, in that order of effectiveness, indued EEG activation and consistently abolished the reticular evoked response. Lower doses of the amphetamines and various doses of methylphenidate, caffeine and, especially, pentetrazol often produced EEG activation without impairing, and may even enhance, the evoked response. 3. High doses of paraldehyde and high doses of pentobarbital abolished or markedly diminished the reticular single shock responses while
producing EEG synchronization. Similar tracings followed convulsant doses of pentetrazol. 4. Lower doses of pentobarbital or paraldehyde and wide dose ranges of chlorpromazine, thioridazine, phenoxybenzamine, chlordiazepoxide, imipramine, atropine, scopolamine and JB-329 also produced EEG synchronization but failed to impair, and often augmented, the evoked reticular potential. 5. Using drug combinations: (a) some drugs blocked all the electrographic actions of others (e.g., scopolamine antagonized physostigmine), whereas others completely failed to exert any blocking effects (e.g., phenoxybenzamine failed to alter the actions of physostigmine) and (b) some agents blocked only partially the bioelectric effects of others. 6. These results emphasize that different neuropharmacological mechanisms act upon the midbrain reticular formation, whether directly or indirectly, and indicate this reticulum is not pharmacologically homogeneous or isolated. The authors gratefully acknowledge the technical help of Miss Mary Martha Whalen and Mr. Joseph J. Bclluomini. REFERENCES ARDUINi, A. and ARDUI~I, M. G. Effects of drugs and
metabolic alterations on brain stem arousal mechanism. J. Pharmacoi. exp. 7her., 1954, 110: 76-85. BRADLEY, P. B. Mic,'oek ~.rode approach to the neuropharmacology
of the reticular formation. In S.
GARATTINIand V. GHETTI(Eds.), Psychotropic drugs. Elsevier, Amsterdam, 1957: 207-216. BRODAL,A. The reticular formation of the brain stem: anatomical aspects and functional correlations. 01 iver and Boyd, London, 1957, 87 p. CHIN, J. H. and DOMINO, E. F. Effects of morphine on brain potentials evoked by stimulation of the tooth pulp of the dog. J. Pharmacol. exp. Ther., 1961, 132: 74-86. COSTA, E., MORPURGO,C. and REVZIN, A. M. Theoretical implications of the chemotherapy of depressions. Recent Advanc. Biol. Psychiat., 1961, 3: 122-139. DEMAA.n_, E. W. J., MARTIN, W. R. and UNNA, K. R. Chlorpromaz|ne ll: the effects of chlorpromazine on evoked potentials in the midbrain reticular formation. J. PharmacoL exp. ?'her., 1958, 124: 77-85. DESMEDT,J. E. and LA GRUTTA, G. The effect of selective inhibition of pseudocholinesterase on the spontaneous and evoked activity of the cat's cerebral cortex. J. Physiol. (Paris), 1957, 136: 20--40. DESMEDT,J. E. et SCHLAG,J. Mise en 6vidence d'616ments
cholin©rgiques dans ia formation r6ticui6e m6senc6phalique. J. PhysioL (Paris), 1957, 49: 136-138.
Electroeneeph. clin. Neurophysiol., 1965, 19:16--24
24.
R.P. WHITE et aL
I~SSA~tD, A. E. Neurophysiological frontie~ in brain research. In J. D. FRENCH (Ed.), Frontiers in brain research. Columbia University Press, New York, 1962: 70-96. FIFKOV~, E. and MAR~ALE,J. Stereotaxic atlases for the cat, rabbit and rat. In J. BURE~et al. (Eds.), Electrophysiological methods in biological research. Academic Press, New York, 1962 (Appendix I): 426-467. FRENCH, J. E., VON AMERONGEN, F. K. and MAGOUN, H. W. An activating system in brain stem of monkey. Arch. NeuraL P~ychiat. (Chic.), 1952, 68: 577-590. HANCE, A. J. The effects of chlorpromazine, thiopentone, amphetamine, and d-lysergic acid diethylamide on conduction within an extra-lemniscai system in the brain stem of the cat. J. Physiol. (Land.), 1959, 145: 41-42. HART, E. R., RODRIGUEZ, J. M. and MARRAZZl, A. S. Reticular synaptic inhibition by serotonin and lysergic acid diethylamide (LSD-25). Pharmacologist, 1962, 4: 175. HERN~NDEZ-PEON,R. and HAGBARTH,K. E. Interaction between afferent and cortically induced reticular responses. J. Neurophysiol., 1955, 18: 44-55. JOUVET, M., BENOIT, O., MARSALLON,A. et COURJON,J. Action de la caf~ine sur l'activit(~ ~lectrique c~r~,brale. C. R. Sac. Biol. (Paris), 1957, 151: 1542-1545. KINO, E. E. Differential action of anesthetics and interneuron depressants upon EEG arousal and recruitment responses. J. Pharmacol. exp. ?'her., 1956, 116: 404-417. KLETZKIN, M. and SWAN, K. The effects of meprobamate and pentobarbital upon cortical and subcortical responses to auditory stimulation. J. Pharmacoi. exp. ?'her., 1959, 125: 35-39. LOEe, C., MAON,, F. and Rossl, G. F. Electrophysiological analysis of the action of atropine on the central nervous system. Arch. itai. Biol., 1960, 98: 293-307.
LoNoo, V. C. et SILVESnUNI,B. Contribution ~ l'6tude des rapports entre le potentiel r6ticulaire 6voqu6, l'~tat d'anestl~sie et l'activit~ 61ectrique c~r6brale. Electroenceph, clin. NeurophysioL, 1958, 10:111-120. MACHNE,X., CALMA,I. and MAOOUN,H. W. Unit activity of central cephalic brain stem in EEG arousal. J. Neurophysiol., 1955, 18: 547-558. NAOAI, S. H. General anesthetics: effects upon physiological systems. In W. S. ROOT and F. G. HOrMANN (Eds.), Physiological pharmacology. Academic Press, New York, 1963, 1: 43-98. RANDY, C. T., COLLINS, W. F., DAVIS, H. S. and Dn.LON, W. H. Differential susceptibility of afferent pathways to anesthetic agents in the cat. Amer. J. Physiol, 1958, 192: 305-310. SCHLAG,J. and BRAND,H. An analysis of electrophysiological events in cerebral structures during ether anesthesia. Electroenceph. clin. NeurophysioL, 1958, 10: 305-324. WHIne, R. P. Relationship between cholinergic drugs and EEG activation. Arch. int. Pharmacodyn., 1963, 145: 1-17. WHITE, R. P. Some motor and electrical signs of drug action. In W. A. H|MWlCH and J. P. SCHAD~reds.), Progress in brain research. Elsevier, Amsterdam, !965, 16: 169-183. WHITE, R. P. and CARLTON, R. A. Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia (Bed.), 1963, 4: 459-47 I. WHI~, R. P. and DAIONEAULT,E. A. The antagonism of atropine to the EEG effects of adrenergic drugs. J. Phar,nacol. exp. ?'her., 1959, 12.~: 339-346. Y/.MAMOTO, S. and SCHAEPPI,U. Effects of pentothal on neural activity in somatosensory cortex and brain stem in cat. Electroenceph. clin. Neurophysiol., 1961, 13: 248-256.
Reference: WHITE,R. P., SEWELLJR., H. H. and RUDOLPH,A. S. Drug-induced dissociation between evoked reticular potentials and the EEG. Electroenceph. ciin. Neurophysi~l., 1965~ !9: 16-24.
ELECTROENCEPHALOGRAPHY AND CLINICALNEUROPHYSIOLOGY DRUG,INDUCED
BETWEEN
EVOKED
RETICULAR
DISSOCIATION POTENTIALS
AND
THE
EEG 1
RICHARD P. WHITE, HOBARTH. SEWELLJR. AND ALLANS. RUDOLPH Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tenn. (U.S.A.)
(Accepted for publication:November23, 1964) INTRODUCTION Numerous studies indicate that drugs can produce EEG or behavioral effects wilich appear unrelated to their actions on reticular potentials evoked by peripheral stimulation (see Discussion). Since many conflicting inferences have resulted from comparing the effects of a few drugs, it seemed pertinent to study a wide variety of compounds, and their antagonists, at various dose levels to ascertain whether drugs may be grouped systematically according to their actions on the reticular evoked responses and their effects on the EEG. In addition, such a study seems important because any demonstrable effect of a drug on the reticular formation should help reveal its participation in central pharmacodynamic mechanisms. To this end 16 drugs were studied in 147 animals. METHODS Adult albino rabbits weighing between 2.3 and 3.1 kg were used and all surgery was performed under local anesthesia (1% procaine). The animals were immobilized with decamethonium or d-tubocurarine (1 mg/kg) given slowly i.v. and placed under artificial respiration with tidal volumes and rate set to maintain control heart rate, blood pressure and EEG tracings. Body temperature was maintained with heating pads. EEGs were obtained from steel electrodes inserted into the cranium over the right motor and left limbic areas of the cortex (White and Daigneault 1959) and recorded with a Grass t This investigation was supported by U.S. Public Health Servicegrants K3-NB-15279and NB-04305 from the National Institute of Neurological Diseases and Blindaess. Portions of this article have been published in abstract form (Fed. Prec., 1964,23: 560; Pharmacologist, 1964,6" 170).
Polygraph. Blood pressure was recorded from one femoral artery by a Statham P23 transduc~:r connected to the polygraph. In most animals the contralateral sciatic nerve was stimulated with single shocks of near maximal strengths from 3 to 7 V with 0.1 msec duration squ~.re waves. In eleven experiments midbrain evoked responses were elicited by excitation of the bulbo-pontine reticular substance (Loeb et al. 1960). Initially the responses were picked up by bipolar concentric steel electrodes, insulated except at the tips, with the tips separated by 0.5 ram, but later unipolar electrodes were employed with the referential one located in the nasal bone. Comparable recordings of drug induced changes were obtained by either technique. The midbrain reticular electrode was positioned stereotaxically and located according to the atlas of Fitkovfi and Margale (1962). Infiltration anesthesia was performed at pressure points with the stereotaxic apparatus. The electrode position was verified by sectioning the fixed brain. In 109 animals the midbrain electrodes were placed either at AP 9, V - 2 to - 4 , L 1.5 to 3, or more com,aonly at AP 10.5, V - 1 to -3.5, L 1.5 to 3 with 97% of all electrodes being located in the nucleus reticularis tegmenti in an area adjacent to, and parallel with, the periaqueductal gray. These locations correspond to sites described by Randt et al. (1958)in cats and by Chin and Domino (1961) in dogs where reticular evoked potentials were commonly recorded. Action potentials were also recorded from the sciatic nerve central to the point of stimulation in 16 animals and from the medial lemniscus (AP 9, V --6, L 1) in 9 to ascertain whether changes in nerve excitability of conduction might account for some of the findings. In most experiments the morphology of the response rather than strictly topographic criteria Electroenceph. clin. NeurophysioL, 1965, 19:16-24
DRUG-INDUCEDDISSOCIATION was used to select the recording point. At least 20 single responses were studied during control periods and again after giving the experimental drug. The single shocks were applied at random intervals, usually with at least 20 sec separating each response. Responses produced by the single shocks were amplified (Grass preamplifier) and displayed on a Dumont dual beam oscilloscope. In approximately 25% of the animals cortical evoked responses were also recorded simultaneously. The drugs studied were: pentobarbital, parab dehyde, chiorpromazine (Thorazine), thioridazine (Mellaril), chlordiazepoxide (Librium), phenoxybenzamine (Dibenzyline), imipramine (Tofranil), scopolamine, atropine and JB-329 (l-ethyl-2-pyrrolidyl-methyl-phenyl-cyclopentyl. glycolate hyd rochloride, Ditran, Lakeside Laboratories). The last compound is a hallucinogen which exerts central atropine-like actions in animals (White and Carlton 1963). The degree of EEG depression induced by these agents was tested physiologically by noise and touch stimuli and further by high frequency (e.g., 60 c/sec) sciatic stimulation with varying voltage, t Also studied were: physostigmine, catechol, methamphetamine, d-amphetamine, methylphenidate (Ritalin), caffeine and pentetrazol (Metrazol). In addition, neostigmine was administered to ascertain whether its peripheral actions could reproduce any central effects of physostigmine (White 1963). All drugs were injected through a cannulated saphenous vein and dosage was computed from the active base. Fifty-seven animals were given a single drug in graded doses and the The authors and four volunteers assessed the sensations derived from supramaximal single shock and repetitive (60 c/see) stimuli by applying skin electrodes treated with cardiopaste and firmly pressed against the ulnar nerve. The latter stimuli produced an unpleasant, but not painful, paresthesia. Submaximal stimuli as used in these animal experiments produced a "tingle" sensation. Nevertheless, in the animals care was taken to apply stimuli only long enough to obtain an effect, to avoid any unnecessary discomfort. Single shock stimuli applied to somatic nerves seem to be ignored by unanesthetized free moving cats (Fessard 1962). To compare this neuropharmacologic with physiologic studies (e.g., French et aL 1952), where similar stimuli were used, general anesthetics were not employed. That residual amounts may significantly affect the actions of the drugs studied herein is indicated in many of our findings.
|7
effects of low and high doses were compared in the same animal. Fifteen to 20 min separated each injection. Results obtained with the highest dosage were verified in at least 2 separate animals by giving the drug in a single injection. A total of 59 animals received the drugs as a single injection. In 74 of these 116 animals the drug was permitted to wear off, in the remaining a cross-over design was employed where a second drug was administered 15-20 min later to ascertain whether it could alter the effects of the first. In addition, 31 animals were given drug combinations where the time interval between injections was less than 10 min. Each drug was administered at a rate which produced minimal effects on blood pressure. Experiments in which the blood pressure decreased more than 25 % of control were arbitrarily discarded. RESULTS Effects of EEG "'activators" The results are shown in Table I and Fig. 1. Physostigmine and catechol abolished midbrain reticular evoked responses and induced EEG activation. This effect on the reticular evoked potential lasted 4-16 min and recovery was slow (3-9 min), the EEG effect outlasting the abolition of the reticular response. In some animals an ephemeral enhanced evoked potential occurred during the administration of these drugs. Neostigmine or saline injections had none of these effects. Methamphetamine would also produce abolition of the single shock responses but only at higher doses than that required to induce EEG activation. Otherwise, this drug closely resembled physostigmine. Conduction was not impaired by any of these drugs. Residual effects of either physostigmi~,e or methamphetamine were still demonstrable after recovery of the midbrain evoked response
were
administered
to 2
separate animals in which the midbrain eveked response was elicited by intrareticular pontine excitation with identical results, i.e., EEG activation and abolition of the single shock evoked potential. Significantly, repetitive stimulation of Electroenceph. clin. Neurophysiol., 1965, 19:16-24
18
R.P. WroTEet aL TABLE I Drug effects on reticular evoked responses and on the EEG
(Plus refers to augmentation, zero to no change, minus to decrease and an x to abolition of evoked responses. Ratio represents number per total of rabbits receiving the drug indicated*) Drug
A. EEG deactivators Pentobarbital
Paraldehyde Chlorpromazine Thioridazine Phenoxybenzamine Chlordiazepoxide Imipramine Atropine Scopolamine JB-329 B. EEG activators Physostigmine Catechol Methamphetamine
Amphetamine Methylphenidate Caffeine Pentetrazol
Dose mg/kg
EEG effects
3-6 8-10 15-30 40- 100 !20-200 I-I0 0.5-5 I-5 1-20 I-5 0.5-20 0.2-20 0.5-2
synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony synchrony
0. I 6--I 2 0.5-I 2 3-5 0.5-I 2-3
activation activation activation activation activation activation activation activation
I-I0 5 10=15 2.5-5 5- I0 5-15
Effects on evoked responses +
0
6/17 1/4
10/17 2/4
2/6
3/6
8/10 4/9 6/9 9/14 4/11 3/16 4/23 2/5
2/10 5/9 3/9 5/14 7/11 12/16 19/23 3/5
~ 1/17 I/4 3/8 1/6 4/7
Main effect on amplitude of evoked responses ×
5/8 3/7
1/16
3/12
7/12
1/12 I/3
3/14
2/14 3/7
2/9
9/14 I/7 6/9
activation
I/2
I/2
activation activation activation seizure
I/6 4/5 I/4
2/6 I/5 I/4
27/27 16/I 6 1/12 2/3 6/6 3/7
I/9 I/6
2/6
2/4 6/6
no change to greater variable decreased to abolished variable decreased to abolished increased no change to greater increased increased no change to greater no change to greater no change to greater no change to greater abolished abolished variable decreased to abolished abolished no change decreased to abolished no change no change variable increased variable abolished
~"Data obtained from 116 animals with 59 receiving by single injection the drug indicated and 57 given the drug in graded doses. Data were grouped when effects on evoked potentials were similar regardless of dose and when a single injection of a high dose verified the results obtained with graded doses. The number of animals given physostigmine or catechol appears excessive because they represent 5 control experiments plus others where prior administration of CNS depressants failed to block their actions.
the sciatic nerve also abolished the intrareticular single shock response in control experiments, thus mimicking this drug action. Caffeine, methylphenidate and especially damphetamine induced electrographic changes similar to methamphetamine but in our experience manifested greater dose-~esponse variation (Table l). Indeed, such drags (in contrast to physostigmine) we originally believed were unable to abolish midbrain evoked responses. Pentetrazol in subconvulsive doses usually increased the amplitude of the reticular evoked re-
sponses but during seizures produced by this drug, evoked potentials were unobtainable. Abolition of the evoked response was strikingly evident during the post-ictal period (Fig. 1) where apparently brain depression is marked since the EEG is synchronous and high frequency excitation with supramaximal (e.g., 9 V, 60/see) stimuli failed to causc EEG activation. Conduction was not impaired in these animals. Return of the EEG alerting response and of the single shock response occurred about 20 rain after all traces of seizure activity subsided. Electroe:)ceph. clin. NeurophysioL, 1965, 19:16--24
DRUG-INDUCED DISSOCIATION
EE~I ~p.V A
EVO.EO i,oc~
RESPONSE,,.l.- - 50 NSEC
2 .~EC
CONTROL
/
I MG METHAMPHETAMINE /
/
I
CONTROL
19
l
10MIN LATER
O.I MG PHYSOSTIGMINE
CONTROL
T I
0o...o
I ,..o
2 MG M[THAMPHETAMINE
G PI,IHE[NOXYBENZAMINE T l
I
EVOI
EVOKED RECOVERY
,0 MG CATECHOL
t
Fig. I Dissociation between the EEG (right motor) and single shock reticular responses induced by drugs. Control records for each experimental animal are shown on the left. A (from left to right): methamphetamine (I mg/kg) induces EEG activation but evoked response remains unaltered; additionally 2 mg/kg abolishes the reticular evoked potential; recovery of the evoked response which precedes return of the control EEG sleep pattern. B: physostigmine causes EEG activation and abolishes evoked response for protracted period; recovery of evoked potential. C: phenoxybcnzamine induces EEG synchrony and slightly enhances evoked response; catechol exerts an activating effect similar to physostigmine and methamphetamine (third excerpts above) despite presence of phenoxybenzamin©; recovery. D: pentobarbital (18 mg/kg) produces EEG synchrony and abolishes reticular evoked potential (contrast with phenoxybenzamine in above tracing); physostigmin¢ significantly alters EEG but not the evoked record; recovery. E: pentetrazol (5 mg/kg) causes only EEG activation (similar to I mg/kg of methamphetamine, A); EEG synchrony and abolished evoked potential 9 rain after a seizure episode; recovery. Shock artifacts m~de visible by Zip-a-tone line (exception: stimulus artifact in tracings D corresponds to left edge of panel).
Effects of EEG "synchronizing" agents These drugs could be grouped into two categories. One, consisting of pentobarbital and paraldehyde, produced EEG synchrony in lower doses and often enhanced evoked responses. In higher doses these drugs further depressed the EEG pattern and depressed or abolished the evoked potential (Table I, Fig. 2). The second category, consisting of chior-
promazine, thioridazine, phenoxybenzamine, chlordiazepoxide, imipramine, JB-329, atropine and scopolamine all produced a similar degree of EEG synchrony within a wide dose range, often augmented the evoked response and rarely depressed this response even in high doses (Table 1), Chlordiazepoxide (2 mg/kg), phenoxybenzamine (2 mg/kg) and scopolamine (1-2 mg/kg) produced identical effects when the midbrain E/ectroenceph. clin. Neurophysiol., 1965, 19:16-24
R.P. WHITE et
20
EEG17~V
EVOKEOL~.~V
RESPONSE 5OMSEC
CONTROL
T
CONTROL
D
al.
l
IOMG CHLOROIAZEPOXIOE 'T
5 MG
O.IMG PHYSOSTIGMINE
T
0.2 MG SCOPOLAMINE
0.1MG PHYSOS'~IGM~NE
T
20 MG SCOPOLAMINE
I MG METHAMPHETAMINE l
25 MG PENTOSARBITAL
i CONTRC'..
I
CONTROL
I
/'
5 MG PHENOXYBENZAMINE
2 MG METh'AMPHETAMINE
T
0,1MG PHYSOSTIGMINE
0.1MG PHYSOST;GMINL"
I MG SCOPOLAMINE
I
0
E
"
Fig. 2 Illustrates antagonism and fundamental differences among drugs on the EEG (right motor) and reticular evoked responds. Control tracirlgs for each experimental animal are shown on the left. A (from left to right): chlordiazepoxide (10 mg/kg) produces EEG synchrony and enhances evoked potential; physostigmine actions are not blocked by chlordiazepoxide; low dose of scopolamine reverses physostigmine effects. B: thioridai, ine has effects like chlordiazepoxide (above A); physostigmine actions are not blocked by thiorida;,ine; scopolamine (20 mg/kg) reverses the physostigmine effects but even this enormous dose fails t~ impair the evoked response. C: pentobarbital (5 mg/kg) produces efl'ects similar to above ataraxics; methamphetamine (I mg/kg) antagonizes EEG effects of pcntobarbital but evoked response persist,J; anesthetic dose of pentobarbital (25 mg/kg) produces changes not seen with either of the previous drug injections. D: phenoxybcnzamine (5 mg/kg) blocks actions of methamphetamine (2 mg/kg) but not those of physostigmine. E: evoked responses obtained by intrareticular stimulation. Scopolamine (I mg/kg) reverses the effects of physostigmine (0. I mg/kg) and blocks a 0.2 mg/kg dose, Shock artifacts made visible by Zip-a-tone lines.
evoked response was elicited by intrareticular stimulation. Furthermore, only scopolamine blocked the usual actions of physostigmine in these animals (Fig. 2).
Effects of drug combination Scopolamine (0.2-2 mg/kg), atropine (2 mg/kg) or JB-329 (0.5-2 mg/kg) blocked completely and consistently both of the electrophysiological actions of O.1 mg/kg of physostigmine.
In some experiments when the antagonistic dose was minimal (e.g., 0.5 mg/kg atropine) a physostigmine-induced EEG activation occurred without a concomitant suppression of the evoked potential--another example of drug-induced dissociation. Imipramine in 3-5 mg/kg doses markedly shortened in 3 animals and abolished the actions of physostigmine (0.1 mg/kg) in another. In contrast, pentobarbital (5-10 mg/kg), Electroenceph. clin. Neurophysiol., 1965, 19:16-24
DRUG-INDUCED DISSOCIATION
chlorpromazine (1-10 mg/kg), thioridazine (1-5 mg/kg), phenoxybenzamine (2-4 mg/kg) and chlordiazepoxide (1-20 mg/kg) failed to block the usual actions of 0.1 mg/kg of physostigmine (Fig. 2), but any of these CNS depressants elevated the dose of methamphetamine necessary to produce EEG activation and to abolish the evoked potential. Lower doses of pentobarbital (2 mg/kg) or chlordiazepoxide (2 mg/kg) promptly restored single shock responses following high doses (3 mg/kg) of methamphetamine in 7 of 8 animals. The usual actions of catechol (6-12 mg/kg) were not blocked by 5 mg/kg of phenoxybenzamine (Fig. 1), 5-10 mg/kg of pentobarbital, or 1 mg/kg doses of scopolamine. Also, there was no obvious synergism between the EEG activators in abolishing the evoked potential (e.g., 5 mg/kg of caffeine followed by 0.5 mg/kg of methamphetamine) but too few experiments were performed to make definite inferences. Another case of dissociation occurred in 4 of 7 animals given I mg/kg of scopolamine and 2-3 mg/kg of methamphetamine in that order. Methamphetamine had negligible EEG effects but abolished evoked potentials suggesting scopolamine has an EEG blocking action cephalad to the midbrain. Similar results were obtained in 1 of 3 animals given imipramine instead of scopolamine. We were unable to confirm the report of Long~~ and Silvestrini (1958) that scopolamine prevents depression of single shock responses caused normally by high doses of pentobarbital (15 mg/kg i.v., 25-40 mg/kg i.p.). However, higher doses of pentobarbital are apparently required to suppress the evoked response since in 3 of 5 animals 13-18 mg/kg i.v. were necessary to diminish this response which is tdgher than expected from control data (Table 1), although 20-30 mg/kg always depressed or abolished the evoked response. Moreover, we initially administered 1 mg/kg of scopolamine compared to their maximum dose of 0.2 mg/kg and all our drugs were given intravenously.
21
prevented this action. Catechol likewise abolished (4 rabbits) or markedly reduced (2 rabbits) the cortical evoked response and neither phenoxybenzamine or imipramine prevented this effect. In low doses (0.5-1 mg/kg) d, amphetamine and methamphetamine failed to alter the cortical potentialin 4 animals, but in I methamphetamine (2 mg/kg) flattened this response for approximately 12 min. Methylphenidate (10 mg/kg) reduced evoked cortical potentials in 1 of 3 animals whereas pentetrazol diminished this response in 2 rabbits only after seizures. In 2 separate animals, paraldehyde and pentobarbital in lower doses failed to appreciably alter the cortical potential but in anesthetic doses suppressed this activity. Scopolamine, imipramine, chlordiazepoxide, phenoxybenzamine or thioridazine either had no effect or slightly increased the single shock cortical response when administered to 3 or more animals, nor did JB-329 or atropine exert appreciable action here in 2 animals. DISCUSSION
These studies clearly show that a drug-induced EEG synchronization or activation may be accompanied by an abolition, decrease, increase or no change in single shock responses recorded from midbrain reticular substance. While studying the anesthesia state, Longo and Silvestrini (1958) first noted such disparities between EEG patterns and evoked reticular potentials. However, high doses of pentobarbitai and all clinically used general anesthetics produce EEG synchronization and markedly depress or abolish midbrain evoked responses (cf Nagai 1963) and it is not surprising that paraldehyde also exerts these effects. That CNS stimulants such as phys.. ostigmine and methamphetamine can also abolish single shock responses while (unlike anesthetics) producing EEG arousal is of theoretical interest. This effect on the midbrain evoked response seems analogous to the attenuation or occlusion caused by repetitive physiologic or dectrophysiologic excitation of afferent nerves (French et a/. 1952; Hern~ndez-Pe6n and Effects on cortical evoked responses Physostigmine flattened this response in 3 Hagbarth 1955). It is likely that at least some stimcontrol animals and in 5 rabbits previously given ulants produce sufficient neuronal excitation to phenoxybenzamine, thioridazine or pentobar- mimic the central effects of high frequency stimbital (6 mg/kg); scopolamine (! and 10 mg/kg) ulation. Moreover, no response to a single in.. Electroeneeph. elin. Neurophysiol., 1965, i9:16-24
22
R.P. WHITEet ai.
coming signal would be expected if stimulant drugs caused all neurons capable of reacting to discharge maximally. Recordings from single reticular units indicated repetitive stimuli may activate some neurons and depress others (Hern/mdez-Pe6n and Hagbarth 1955) but activation of such units is commonly associated with stimulation (Machne et al. 1955; Desmedt and Schlag 1957; Schlag and Brand 1958), whereas ether (Schlag and Brand 1958) and thiopental (Yamamoto and Schaeppi 1961) in high doses predominantly decrease unitary discharge. Regardless of the mechanism, evidently some stimulants readily mimic electrographic features of CNS excitation caused by marked physiological or electrophysiological stimulation. Serotonin and LSD-25 will also reduce potentials produced in midbrain reticulum by single shock stimuli (Hart et al. 1962) and amphetamine and LSD-25 have been reported (Hance 1959) to reduce such potentials in the central gray, an area considered by Brodal (1957) closely related functionally with the reticular formation. Such reports, coupled with those herein, demonstrate that the midbrain enhanced evoked responses recorded after Metrazol, strychnine (Arduini and Arduini 1954) or caffeine in cats with diencephalic lesions (Jouvet et al. 1957) are by no means a universal characteristic of CNS stimulants. Why some stimulants are more effective than others in abolishing reticular evoked responses, however, remains undetermined. That different mechanisms o1' stimulation are involved is indicated by the specificity with which some agents can block excitant effects of certain drugs (Fig. 2). it is also evident that in lower doses, or during recovery periods, stimulants can cause EEG arousal patter~Js ':,ithout abolishing midbrain evoked potentials and that such a dissociation is accentuated by appropriate CNS depressants (Fig. 2). This finding would suggest that the "spontaneous" EEG mechanism can be a more sensitive index to drug action than the "superimposed" single shock reaction. Our findings concur with previous reports that atropine (2-4 mg/kg) in cats (Loeb et al. 1960), scopolamine (0.25--I mg/kg) in rabbits (Longo and Silvestrini 1958), chlorpromazine (5-10 mg/kg) in cats (DeMaar et ai. 1958) and sedative
doses of pentobarbital (5-10 mg]kg) in rabbits (Longo and Silvestrini 1958)need not depress and may actually augment reticular evoked responses while producing EEG synchronization. Morphine (Chin and Domino 1961), meprobamate (Kletzkin and Swan 1959) and lower doses of chloralosane (Arduini and Arduini 1954) or thiopental (King 1956) also clearly possess these characteristics. In addition, chlordiazepoxide, phenoxybenzamine, thioridazine, imipramine, JB-329, lower doses of paraldehyde and even enormous doses of atropine or scopolamine were fc,und to exert these actions. Excepting chloralosane, paraldehyde or the barbiturates (in high doses), none of these drugs are classic anesthetics and certainly their actions on midbrain evoked potentials contrast with the anesthetic state where evoked [potentials are unequivocally depressed or abolished. The inference that atropine (Loeb et al. 1960) and meprobamate (Kletzkin and Swan 1959) do not depress excitability of the ascending activating system because these drugs fail to depress midbrain evoked response (in contrast to anesthetic doses of barbiturates) seems untenable in view of the large number of drugs which share this property, it is possible, rather, that EEG synchronizing drugs reduce the multifarious stimuli which impinge on the reticular formation, thereby reducing tonic occlusive phenomena and "spontaneous" neuronal activity. !t such a reduction in activity were sufficiently greater than that expected from the elimination of proprioceptive activity by curarization, ar~ enhanced single shock evoked response might be anticipated in the absence of surgical anesthesia. Similar hypotheses have been advanced to explain enhanced reticular potentials following chlorpromazine (DeMaar et al. 1958) and morphine (Chin and Domino 1961). The fact that thiopental (Yamamoto and Schaeppi 1961) and chlorpromazine (Bradley 1957) can greatly reduce "spontaneous" firing of individual reticular units without "blocking" evoked discharges, indicates that large populations of neurons may react in a similar manner. Moreover, physiologic EEG slowing is associated with enhanced cortical responses (cf. Desmedt and La Grutta (1957) and sedative drugs also facilitate hippocampal evoked potentials (Costa et al. Electroenceph. clin. NeurophysioL, 1965, 19:16--24
23
DRUG-INDUCED DISSOCIATION
1961). If depression of evoked potentials were the only index of drug-induced depression, atropine and many CNS depressants would have no significant actions on the neuraxis (cf. White 1965). Certainly enhanced evoked responses are commonly associated with drugs which induce EEG synchronization and apparently the failure of a drug to depress midbrain evoked potentials in animals immobilized by curariform agents or brain lesions does not prove an absence of an effect. Whether the drugs studied affect directly or indirectly the midbrain reticulum remains problematic. No similar electrographic analysis, even intrareticular stimulus-response data, will resolve this point in intact animals. If they act extrareticularly, evidence of pharmacologically distinct neurons (cholinergic, etc.) discharging into the reticular formation is provided by the relatively specific blockade exerted by some drugs against several CNS stimulants. Our findings with intrareticular responses imply a direct effect. Moreover, micro-electrode studies suggest a direct action for physostigmine (Desmedt and Schlag 1957), epinephrine and acetylcholine (Bradley 1957). in any case, all evidence supports the suggestion of Bradley (1957) "that it is unlikely that this complex system is pharmacologically homogeneous". SIJMMARY
I. Sixteen drugs were given i.v. to curarized rabbits to clarify neuropharmacologic relationships between single shock responses in midbrain reticulum to single sciatic shocks and EEt'; patterns. The results revealed that drugs can produce marked dissociation between the two phenomena. 2. Physostigmine, catechol and relatively high doses of methamphetamine or d-amphetamine, in that order of effectiveness, indued EEG activation and consistently abolished the reticular evoked response. Lower doses of the amphetamines and various doses of methylphenidate, caffeine and, especially, pentetrazol often produced EEG activation without impairing, and may even enhance, the evoked response. 3. High doses of paraldehyde and high doses of pentobarbital abolished or markedly diminished the reticular single shock responses while
producing EEG synchronization. Similar tracings followed convulsant doses of pentetrazol. 4. Lower doses of pentobarbital or paraldehyde and wide dose ranges of chlorpromazine, thioridazine, phenoxybenzamine, chlordiazepoxide, imipramine, atropine, scopolamine and JB-329 also produced EEG synchronization but failed to impair, and often augmented, the evoked reticular potential. 5. Using drug combinations: (a) some drugs blocked all the electrographic actions of others (e.g., scopolamine antagonized physostigmine), whereas others completely failed to exert any blocking effects (e.g., phenoxybenzamine failed to alter the actions of physostigmine) and (b) some agents blocked only partially the bioelectric effects of others. 6. These results emphasize that different neuropharmacological mechanisms act upon the midbrain reticular formation, whether directly or indirectly, and indicate this reticulum is not pharmacologically homogeneous or isolated. The authors gratefully acknowledge the technical help of Miss Mary Martha Whalen and Mr. Joseph J. Bclluomini. REFERENCES ARDUINi, A. and ARDUI~I, M. G. Effects of drugs and
metabolic alterations on brain stem arousal mechanism. J. Pharmacoi. exp. 7her., 1954, 110: 76-85. BRADLEY, P. B. Mic,'oek ~.rode approach to the neuropharmacology
of the reticular formation. In S.
GARATTINIand V. GHETTI(Eds.), Psychotropic drugs. Elsevier, Amsterdam, 1957: 207-216. BRODAL,A. The reticular formation of the brain stem: anatomical aspects and functional correlations. 01 iver and Boyd, London, 1957, 87 p. CHIN, J. H. and DOMINO, E. F. Effects of morphine on brain potentials evoked by stimulation of the tooth pulp of the dog. J. Pharmacol. exp. Ther., 1961, 132: 74-86. COSTA, E., MORPURGO,C. and REVZIN, A. M. Theoretical implications of the chemotherapy of depressions. Recent Advanc. Biol. Psychiat., 1961, 3: 122-139. DEMAA.n_, E. W. J., MARTIN, W. R. and UNNA, K. R. Chlorpromaz|ne ll: the effects of chlorpromazine on evoked potentials in the midbrain reticular formation. J. PharmacoL exp. ?'her., 1958, 124: 77-85. DESMEDT,J. E. and LA GRUTTA, G. The effect of selective inhibition of pseudocholinesterase on the spontaneous and evoked activity of the cat's cerebral cortex. J. Physiol. (Paris), 1957, 136: 20--40. DESMEDT,J. E. et SCHLAG,J. Mise en 6vidence d'616ments
cholin©rgiques dans ia formation r6ticui6e m6senc6phalique. J. PhysioL (Paris), 1957, 49: 136-138.
Electroeneeph. clin. Neurophysiol., 1965, 19:16--24
24.
R.P. WHITE et aL
I~SSA~tD, A. E. Neurophysiological frontie~ in brain research. In J. D. FRENCH (Ed.), Frontiers in brain research. Columbia University Press, New York, 1962: 70-96. FIFKOV~, E. and MAR~ALE,J. Stereotaxic atlases for the cat, rabbit and rat. In J. BURE~et al. (Eds.), Electrophysiological methods in biological research. Academic Press, New York, 1962 (Appendix I): 426-467. FRENCH, J. E., VON AMERONGEN, F. K. and MAGOUN, H. W. An activating system in brain stem of monkey. Arch. NeuraL P~ychiat. (Chic.), 1952, 68: 577-590. HANCE, A. J. The effects of chlorpromazine, thiopentone, amphetamine, and d-lysergic acid diethylamide on conduction within an extra-lemniscai system in the brain stem of the cat. J. Physiol. (Land.), 1959, 145: 41-42. HART, E. R., RODRIGUEZ, J. M. and MARRAZZl, A. S. Reticular synaptic inhibition by serotonin and lysergic acid diethylamide (LSD-25). Pharmacologist, 1962, 4: 175. HERN~NDEZ-PEON,R. and HAGBARTH,K. E. Interaction between afferent and cortically induced reticular responses. J. Neurophysiol., 1955, 18: 44-55. JOUVET, M., BENOIT, O., MARSALLON,A. et COURJON,J. Action de la caf~ine sur l'activit(~ ~lectrique c~r~,brale. C. R. Sac. Biol. (Paris), 1957, 151: 1542-1545. KINO, E. E. Differential action of anesthetics and interneuron depressants upon EEG arousal and recruitment responses. J. Pharmacol. exp. ?'her., 1956, 116: 404-417. KLETZKIN, M. and SWAN, K. The effects of meprobamate and pentobarbital upon cortical and subcortical responses to auditory stimulation. J. Pharmacoi. exp. ?'her., 1959, 125: 35-39. LOEe, C., MAON,, F. and Rossl, G. F. Electrophysiological analysis of the action of atropine on the central nervous system. Arch. itai. Biol., 1960, 98: 293-307.
LoNoo, V. C. et SILVESnUNI,B. Contribution ~ l'6tude des rapports entre le potentiel r6ticulaire 6voqu6, l'~tat d'anestl~sie et l'activit~ 61ectrique c~r6brale. Electroenceph, clin. NeurophysioL, 1958, 10:111-120. MACHNE,X., CALMA,I. and MAOOUN,H. W. Unit activity of central cephalic brain stem in EEG arousal. J. Neurophysiol., 1955, 18: 547-558. NAOAI, S. H. General anesthetics: effects upon physiological systems. In W. S. ROOT and F. G. HOrMANN (Eds.), Physiological pharmacology. Academic Press, New York, 1963, 1: 43-98. RANDY, C. T., COLLINS, W. F., DAVIS, H. S. and Dn.LON, W. H. Differential susceptibility of afferent pathways to anesthetic agents in the cat. Amer. J. Physiol, 1958, 192: 305-310. SCHLAG,J. and BRAND,H. An analysis of electrophysiological events in cerebral structures during ether anesthesia. Electroenceph. clin. NeurophysioL, 1958, 10: 305-324. WHIne, R. P. Relationship between cholinergic drugs and EEG activation. Arch. int. Pharmacodyn., 1963, 145: 1-17. WHITE, R. P. Some motor and electrical signs of drug action. In W. A. H|MWlCH and J. P. SCHAD~reds.), Progress in brain research. Elsevier, Amsterdam, !965, 16: 169-183. WHITE, R. P. and CARLTON, R. A. Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia (Bed.), 1963, 4: 459-47 I. WHI~, R. P. and DAIONEAULT,E. A. The antagonism of atropine to the EEG effects of adrenergic drugs. J. Phar,nacol. exp. ?'her., 1959, 12.~: 339-346. Y/.MAMOTO, S. and SCHAEPPI,U. Effects of pentothal on neural activity in somatosensory cortex and brain stem in cat. Electroenceph. clin. Neurophysiol., 1961, 13: 248-256.
Reference: WHITE,R. P., SEWELLJR., H. H. and RUDOLPH,A. S. Drug-induced dissociation between evoked reticular potentials and the EEG. Electroenceph. ciin. Neurophysi~l., 1965~ !9: 16-24.