Neuropharmacological Comparison of Subcortical Actions of Anticholinergic Compounds

14

Neuropharmacological Comparison of Subcortical Actions of Anticholinergic Compounds R.P. WHITE A N D A. S. R U D O L P H Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units,Memphis, Tennessee (U.S.A.)

Numerous reports attest to a direct action of the belladonna alkaloids on the cerebral cortex. They preferentially accumulate in cortical tissue6l and produce a greater percentage loss of acetylcholine in the cerebral cortex than in subcortical areaslg920. The local effects of cholinergic drugs topically applied to the cortex are antagonized by the belladonna alkaloidsQ~37~41 and atropine administered by close arterial injection will unquestionably produce unilaterally cortical EEG synchrony41. Systemically administered, these alkaloids block the EEG activation pattern, but not the behavioral flight reaction derived from hypothalamic stimulation in rabbits3’; an obvious ‘dissociation’ between electrocorticogram and behavior. However, it should be emphasized that these alkaloids abolish conditioned behavior in rabbits32935 and induce abnormal behavior in dogs5~59963.Indeed, many behavioral effects produced by these alkaloids in dogs (e.g., blindness, intermittent sleep, absence of placing reflexes) resemble those produced by decortication5~59.Since the normal electrocorticogram is absent in decorticate dogs, it is not too surprising that some drugs might cause an EEG ‘sleep’ pattern without inducing sleep behaviorally. The experiments of Loeb et ~ 1 . 3 0show the difficulty of demonstrating, at least electrographically, a subcortical site of action for these compounds. Single shock responses recorded from the midbrain reticulum are not reduced by atropine. Moreover, electrostimulation of the reticular formation will still inhibit thalamocortical recruiting indicating that ieticulo-thalamic functions are not impaired by atropine. These investigators confirmed that the EEG activation pattern obtained from highfrequency stimulation of subcortical structures is abolished by atropine, but emphasize this effect might be produced by atropine at the cortical level. On the other hand, there is much data indicating the belladonna alkaloids exert important subcortical effects. Di-isopropylfluorophosphate (DFP) given unilaterally into the nucleus caudatus produces specific contraversive body movements which are antagonized by atropine58. Rhythmic leg movements induced by electrostimulation of the subthalamus are inhibited by the belladonna alkaloidszg. The EEG synchrony produced by atropine given at the cortical level does not prevent the EEG activation induced by electrostimulation of the reticular formation, but atropine administered intravenously abolishes this reticular effect on the cortex, indicating a subcortical

SUBCORTICAL ACTIONS OF ANTlCHOLINERGICS

15

action of atropine is necessary to obtain complete blockade of the EEG activating mechanism41. Physostigmine given intravenously can abolish single shock responses recorded from the midbrain reticulum, an effect which is completely antagonized by the belladonna alkaloids61. However, in all studies with intact animals, including responses from single neurons7, such results may depend on important reverberating or corticofugal connections with various subcortical areas, thus making it difficult to assess which sites are primarily affected by these drugs. Also, injections of drugs directly into the brain substance may excite receptors not normally reached by the vascular route nor normally excited by adjacent neurons. It is significant, therefore, to find reports indicating that the belladonna alkaloids exert actions in animals in which the telencephalon and/or most of the prosencephalon have been extirpated and that cholinergic mechanisms are operant in the brainstem. Teuchmann47 found that scopolamine reduced spinal flexor reflexes in thalamic (decorticate) cats, but not in decerebrate (midcollicular transected) or spinal cats. These results clearly indicate a diencephalic site of action of scopolamine. DeMaarlO confirmed this finding with both scopolamine and atropine, further showing by removing most of the prosencephalon that the ventro-caudal portion of the diencephalon (roughly the area of the subthalamus) was the site for this inhibitory action. Further evidence that there are cholinergic receptors in this general area was afforded by Desmedt and Schlagll who showed that eserine greatly increased the discharge of single midbrain neurons in cats with transections through the posterior aspects of the diencephalon. The administration of acetylcholine directly into the lateral reticular formation causes EEG activation and behavioral alertness2], providing further evidence that EEG arousal may result from excitation of subcortical cholinergic me~hanisms33~42~5~. Also, some unilateral body movements produced by the injection of DFP into one carotid artery persist after complete removal of the diencephalon57, indicating again that cholinergic receptors are present below the thalamus. Most reports suggest that the newer anticholinergic psychotomimetics, the piperidyl benzilates, have similar central actions to the belladonna alkaloids. Both groups of drugs, for example, block the EEG activation caused by physostigmine53,55,61 and reduce brain acetylcholine content20 in laboratory animals to a degree that appears related to their reported psychotogenicity in humans. The piperidyl benzilates appear to differ in effect only quantitatively from the belladonna alkaloids, i.e., in their affinity for different cholinergic receptors53. They have similar behavioral effects in dogs17159 and man26. Both groups of compounds also block the electrographic effects of physostigmine recorded from the midbrain reticulum in intact rabbits61. However, since physostigmine clearly produces electrographic signs of excitation above the midbrain in rabbits45, it is possible that the midbrain electrographic changes reported were secondary to drug effects on structures more anterior in the neuraxis. The work described here was performed, therefore, to ascertain whether anticholinergic agents could antagonize the electrographic effects induced by physostigmine on ‘spontaneous’ brain waves and on single shock responses recorded from the midbrain reticulum of rabbits in which all activity of the prosencephalon was eliminated. References p . 24-26

16

R. P. WHITE A N D A. S. R U D O L P H METHODS

Data were obtained from thirty-one adult albino rabbits. All drugs were injected through a cannulated saphenous vein with dosages computed from the active base. Surgery was performed under local anesthesia (1 procaine) to avoid the effects that residual amounts of general anesthetics may exert on the drugs studied61. Great care was exercised so that no alarm reactions (body movements, blood pressure changes) were observed during the surgical procedures. In one group of rabbits some peripheral signs of action of the anticholinergics were studied; namely, effects on pupil size, light reflex and bradycardia induced by excitation of the peripheral end of the transected right vagus nerve (Table 1). TABLE I COMPARISON OF ANTICHOLINERGIC BLOCKADE AT PERIPHERAL AND CENTRAL SITES IN THE RABBIT. NUMBERS IN PARENTHESES ILLUSTRATE MG/KG DOSES GIVEN YIELDING THE RESULTS INDICATED. PHYSOSTIGMINE GIVEN AFTER THE ANTICHOLINERGIC AGENT

I.V.

--

-~

Evoked pot. of midbrain animal

EEG from intact rabbit

Vagal blockade

Light reflex

Mydriasis

Atropine Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes 0.5) Yes ( 0.1)

Present ( 5.0) Sleep ( 5.0)* Present [ 0.2) Sleep ( 0.1)

Scopolamine Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes ( 0.5) Yes ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( O S ) * Sleep ( 0.3)

JB-329 Physostigmine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.2)

Yes ( 0.5) Yes ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( OS)* Sleep ( 0.4)

JB-318 Physostigrnine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) Yes ( 0.1)

Yes ( 0.5) Less ( 0.1)

Present ( 2.0) Present ( 0.2)

Sleep ( 0.5)* Sleep ( 0.1)

JB-340 Physostigrnine

Yes ( 0.5) Yes ( 0.1)

None ( 0.5) None ( 0.1)

Yes (0.5 ) Yes ( 0.1)

Present (20.0) Reduced ( 0.2)

Sleep (50.0) Alert ( 0.1)

JB-305 Physostigmine

No (12.0)

Yes

(15.0)

Mild (15.0) N o ( 0.1)

Present (15.0) Reduced ( 0.2)

Sleep (15.0) Alert ( 0.1)

Drug

* Dosages known to consistently

__

block EEG effects of at least 0.1 mg/kg of physostigmine55.

In the second group, the midbrain was isolated functionally from the prosencephalon. First, the common carotid arteries were tied ;this procedure did not cause unconsciousness. The cerebrum was carefully ablated with a sharp scalpel and the diencephalon was transected from the midbrain by electrocautery and in most cases completely removed (Fig. 1). The animal was then curarized (1 mg/kg d-tubocurarine), put under artificial respiration, and placed in a stereotaxic instrument. Three electrodes were inserted into the midbrain with the help of the stereotaxic atlas of FiflcovS and MarSala14. The exposed surface of the superior colliculus, however, was the starting point for placing most of the electrodes (e.g., 3P and 2L from rostra1 and medial aspects, respectively). The depth of the electrodes was usually 7 mm from the surface

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

17

of the superior colliculus, although the morphology of the response rather than a predetermined site was selected for the recordings. Electrode position was verified by sectioning the fixed brain (Fig. I). The EEG and evoked potentials were obtained from these electrodes. The evoked potentials were produced by single shocks applied to one sciatic nerve (0.1 msec duration with supramaximal voltage) and/or to the pontine region of the tegmentum. These were recorded on the EEG tracings or by means of a cathode ray oscilloscope, the latter recorded photographically (Fig. 2). In some experiments one additional electrode was placed in the fluid rostra1 to the midbrain

Fig. 1. Gross appearance of the brain and the histological position of the electrodes in the nucleus reticularis tegrnenti of the midbrain from which recordings were obtained.

but no biological activity was recordable. The exposed surfaces of brain were covered with a thin layer of warm mineral oil. Further details on methods used in this study for obtaining single shock responses may be found elsewhereGI. The anticholinergic drugs studied were : scopolamine hydrobromide, atropine sulphate, JB-329 (Ditran), JB-318, JB-340, and JB-305. The J B compounds were References p . 24-26

18

R. P. W H I T E A N D A. S. R U D O L P H

”’!!!t

EVOKED RESPONSE

EEG

CONTROL

CONTROL

-*a C

.

.

CONTROL

CONTROL

H.F.S. SCIATIC N.

0.2 MG

PHYSOSTIGMINE

CRo

0 . 2 MG. PHYSOSTIGMINE

RECOVERY

t

50 MSEC.

2 MG. SCOPOLAMINE

44

0.2 MG. PHYSOSTIGMINE

+‘“J--y’w.’r

9

2 HG.

JE-310

4

0.2 MG.

0 . 2 MG.

PHYSCSTIGMINE

PHVSOSTICMINE

Fig. 2. EEG pattern and the single shock responses obtained from the midbrain of four rabbits in which the diencephalon and cerebrum were removed (see Fig. 1). Tracings at A show control (extreme left) recordings of the EEG, which is low in amplitude, and the single shock responses superimposed (abovedots)upontheEEG,andthesingle shockresponseasit appears on the cathode ray oscilloscope. Subsequently seen (from left to right) is the abolition of the single shock responsa resulting from high frequency (30 c/s) stimulation of one sciatic nerve without a change in the EEG; recovery; and lastly that 0.2 mg/kg of physostigmine also abolishes the evoked potentials. Tracings at B show (left to right) control recording; attenuation of the evoked responses by physostigrnine; restoration of the shock responses by scopolamine; and lastly that after scopolamine, physostigmine no longer affects theevokedresponses. Tracingsat Cshowcontrol responses; that JB-318 didnotchange these responses but blocked the usual effect of physostigrnine. Tracings at D show that, in contrast to JB-318 or scopolamine, the anticholinergic agent JB-340 fails to antagonize the usual effect of physostigmine.

piperidyl benzilates obtained from the Lakeside Laboratories, Milwaukee, Wisconsin ; the first two are psychotomimetics whereas the last two are not2. Their structural formulae are given elsewherez955. All these drugs in adequate dosage produce EEG synchrony in intact rabbit@. The doses used in these experiments were comparatively high to assure a reasonable effect in the ablated animals since a dose-response study was not feasible because of the high mortality of the midbrain preparation. The agonist employed was physostigmine sulfate because it is a cholinergic substance with known CNS stimulant properties53. However, prostigmine bromide was also administered to some animals to ascertain the degree to which cholinergic stimulation peripherally might contribute to the central phenomena studied. Mean blood pressure was recorded on a Grass Polygraph from one femoral artery. The blood pressure rose abruptly 20 to 70 mm Hg immediately following the brain transection by electrocautery and gradually retcrned to control value or to below normal. In the latter case, saline was administered to restore the blood pressure to control value. Normal body temperature was maintained by use of a heating pad.

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

19

RESULTS

The single shock responses recorded from the reticular formation of the midbrain animal may be easily seen superimposed on the EEG pattern and readily recorded on the cathode ray oscilloscope (Fig. 2). These evoked potentials are remarkably stable in amplitude and shape in most of the ablated animals. However, as the frequency of electrical stimulation is increased to about 6 c/s or more they are reduced in amplitude (attenuated) and at higher frequencies (20-60 c/s) are abolished (Fig. 2). Virtually the same effects were obtained whether the stimuli were applied to the sciatic nerve or in the brainstem, caudal to the recording electrodes. Evoked responses did not always appear in every midbrain lead, but those obtained from different leads resemble each other in response to high frequency stimulation and to the drugs administered. An EEG was recorded from all leads placed in the midbrain. Physostigmine (0.2 mg/kg) produced changes in the single shock responses similar to those obtained with high frequency stimulation ; namely, attenuation, to less than 50 % of control, or less often, abolition of the evoked potential (Fig. 2). Scopolamine, atropine, JB-329 and JB-318 clearly antagonized this action of physostigmine (Fig. 2, Table I) whether given before or after physostigmine. All of these anticholinergic agents restored the evoked response, in the latter case before one-half of the dose was administered, indicating the doses given (Table I) were higher than necessary to reverse the physostigmine effect. Furthermore, these compounds completely antagonized the effects of an additional dose of physostigmine (Fig. 2). In contrast, JB-305 and JB-340 failed to block the attenuation of the evoked response produced by physo,tigmine (Fig. 2, Table 1). Neostigmine failed to mimic the effect of physostigmine, suggesting that the latter’s action is central in origin. This suggestion is strengthened by the finding that JB-340, which possesses strong anticholinergic properties peripherally (Table I), failed to alter this central manifestation of physostigmine (Fig. 2). Other differences between central and peripheral actions among the anticholinergic compounds are shown in Table I. The very low amplitude (4 to 33 p V ) EEG tracing obtained from the midbrain of these animals was not significantly changed by any of the anticholinergic drugs or by electrostimulation (Fig. 2). Even anesthetic doses of pentobarbital (25 mg/kg) when administered slowly did not increase the amplitude. These findings are in contrast to those obtained in intact rabbits where a midbrain EEG pattern is seen which closely resembles the electrocorticogram in magnitude and in response to these drugs and to electrical stimulation53. The low amplitude may reflect a reduction in blood flow in these ablated animals. On the other hand, a continuum of frequencies that shift spontaneously from about 3.5 to 8 c/s was usually evident with the most common frequency range being 4 to 7. Also, a typical high amplitude (180 pVor more) seizure pattern was obtained by injecting Metrazol (5-15 mg/kg) and, in some animals, occurred spontaneously. These last two findings, coupled with the fact that the single shock response was prominent in these ablated animals, suggest that the low amplitude electroreticulogram may be caused by a reduction in the number of midbrain neurons References p . 24-26

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R. P. W H I T E A N D A. S. R U D O L P H

being discharged “spontaneously” as a result of the destruction of reverberating circuits normally active in the intact brain. DISCUSSION

The four anticholinergic compounds (atropine, scopolamine, JB-329, and JB-3 18) which blocked the attenuating effects of physostigmine on the evoked potentials are well known psychotomimetics. These findings agree with previous studies indicating that drugs which block the EEG activation caused by physostigmine are either psychotomimetics or antiparkinson agents in man53J4*55>62.Moreover, the literature clearly indicates that such compounds exert both actions clinically53.Parsidol (ethopropazine), for example, is a phenothiazine used in the treatment of Parkinson’s disease which, in contrast to the ataractic phenothiazines, will block the EEG activation induced by physostigmine62, will arrest the pseudoparkinsonism produced by chlorpromazine29, and in adequate dosage will induce hallucinations39. Similarly, benactyzine readily blocks the EEG effects of physostigmine54; may be used to treat parkinsonism24; and is capable of producing psychotic episodes51. Lastly, the psychotomimetic JB-3 18 has an antitremor action in patients’. The present study affords new evidence that anticholinergic compounds can antagonize an effect of a cholinergic drug at the midbrain level. Although the changes induced by physostigmine in the midbrain reticulum may be indirect, e.g., secondary to excitation of other brainstem structures, the results clearly show that these changes take place in the absence of the prosencephalon and indicate they are central in origin. Therefore, the antagonism of atropine, scopolamine, JB-329 (Ditran) and JB-3 18 to this effect ofphysostigmine can occur centrally at sites below the diencephalon. This is not true of JB-340, which exerts its strong anticholinergic actions only peripherally. These results, coupled with the finding that JB-318 is less active on the iris than JB-340, but more active centrally in antagonizing physostigmine, emphasize that central and peripheral cholinolytic properties of a drug may be of a different order of magnitude and that inferences concerning the central actions of these drugs should not be based upon results obtained from peripheral tissue53. In high doses, for example, Darstine (mepiperphenidol) mimics many of the peripheral effects of atropine in humans but is far less active centrally60; whereas, the newer anticholinergic psychotomimetics (piperidyl benzilates) are evidently superior tools for neuropsychiatric research because they produce more gradual EEG and psychological changes as the dose is increased, induce richer hallucinogenic episodes, and have less autonomic side effects than the belladonna alkaloidss3. There is a growing body of pharmacological evidence indicating that a family of related cholinergic receptors are involved in synaptic transmission. There is, for example, pharmacological evidence indicating both muscarinic and nicotinic receptors are capable of independently causing EEG activationz5150but that nicotinic receptors may not be present in the cerebral cortex37. Atropine readily blocks the EEG arousal caused by acetylcholine42 but does not block the effects of this substance on the Renshaw ce1112. Conversely, dihydro-/3-erythroidine inhibits the Renshaw neuron

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

21

but fails to block EEG activation. At least two distinct cholinergic receptors are involved in sympathetic ganglionic transmissionl3. Also, atropine and scopolamine inhibit spinal flexor reflexes only at subthalamic sites; whereas, caramiphen produces a similar inhibition at areas below this level of the neuraxislo. The intracerebral injection of cholinergic substances will produce a wide variety of behavioral effects (rage, sleep, arousal, catatonia, etc.) depending on the area injected 21.It is not surprising, therefore, that differences have been reported among the “anticholinergic psychotomimetics” including the belladonna alkaloids. A survey of the literature, however, indicates these differences are quantitative in nature rather than qualitative53. These compounds apparently also have a dual action in impairing central cholinergic mechanisms: they block the usual EEG effects of cholinergic agents55 and they decrease brain acetylcholine content20. These two actions appear to be related; the most potent blockers of cholinergic drugs seem to be most active in decreasing brain acetylcholine. Moreover, the effects on the EEG53,55*59, behavior55959 and on brain acetylcholine levels20 caused by the anticholinergic psychotomimetics reach a maximum with comparatively low doses. Since scopolamine36.59 and atropine59 d o not produce notable sedation in intact rabbits even in enormous doses59 but do produce a “Iissive” effect in decorticate rabbits36959, it is apparent that the subcortical and cortical actions of the drugs differ in this species. The subcortical effects of these drugs may be related to their antiparkinson actions in humans. At least, our findings lend support to the hypothesis that antiparkinson agents counteract a subcortical hyperactive cholinergic mechanism24,28,49,62. The early work of Veit and Vogt48 may help explain why low doses of these belladonna alkaloids produce sedation in dogs and in higher doses disorientation or deliriumlike behavior59. They found the concentration of scopolamine in the cerebral cortex and midbrain to be comparable after low doses of scopolamine (2 mg/kg), but after high doses (10 mg/kg) the concentration in the cortex was about 2.5 times greater than in the midbrain. Therefore, in low doses normal function may be inhibited throughout the neuraxis producing effects on the EEG and midbrain reticular evoked responses similar to those produced by other sedatives in animals53-61. In low doses both are also sedatives in man32138~60.In higher doses, only the functions of the cerebrum may be further impaired significantly, producing in dogs many of the characteristics of decortication5.59 without producing classical signs of anesthesia, either behaviorally, electroencephalographically, or on midbrain evoked responses34.53. Indeed, the behavioral changes induced in dogs (blindness, slow compulsive gait, etc.) resemble those obtained with LSD256. Moreover, some motor effects produced by amphetamine are enhanced8359 suggesting that adrenergic or non-cholinergic mechanisms may be “released” during atropine or scopolamine toxicity. However, the so-called stimulation produced by these drugs does not mimic that induced by amphetamine in dogs59 and has no reliable analeptic value clinically. Indeed the “stimulation” seen with these alkaloids is interrupted by periods of sleep in dogs, monkeys and man53. Moreover, the EEG activation caused by adrenergic drugs is blocked by anticholinergic agents56 suggesting some central “adrenergic” systems depend ultimately on cholinergic References p . 24-26

22

R. P. W H I T E A N D

A. S. R U D O L P H

processes. Also, the belladonna alkaloids greatly potentiate the depressant actions of ether (personal observations) and pentobarbital69 in dogs. They also potentiate the action of barbiturates in rats19 and monkeys5Q.Such synergism is a common characteristic of CNS depressants. Hence the “excitation” or disorientation induced by toxic doses of the belladonna alkaloids may be considered “pseudostimulation” caused by an inhibition of cholinergic mechanisms and an imperfect “release” of non-cholinergic mechanisms. Most reports dealing with the mechanism by which anticholinergics produce central actions are consonant with the hypothesis that they inhibit cholinergic systems and that their behavioral effects may result from a functional “imbalance”4,53. The tricyclic antidepressants (e.g., desipramine) evidently possess mild anticholinergic actions centrally, reducing the effects of cholinergic agents3.40, while simultaneously may enhancing the actions of adrenergic agents40943, so that a dual a~tion4~~43~46~53 account for their clinical efficacy. In this regard, it would be of interest to administer to endogenously depressed patients small doses of both atropine and amphetamine to ascertain whether this combination is also beneficial to such patients. In humans, of atropine toxicity the EEG synchrony64 and hallucinogenic manife~tationsl~9~~964 revert to normal after the intramuscular administration of 4 mg of physostigmine, presumably by restoring cholinergic functions centrally. Similarly, the belladonna alkaloids and physostigmine are antagonists in their effects on conditioned behavior of laboratory animals22135. Another cholinergic drug, tetrahydroaminoacrin (THA), in 60 mg doses i.v. will antagonize the hallucinations, stupor and other effects of 10 mg of Ditran (JB-329) in humanslB.Moreover, the psychotomimetic effects of Ditran are changed to a coma-like condition with small doses of chlorpr0mazine2~, and perhaps because the sedative properties of chlorpromazine are intensified or “released”, adrenergic phenomena are antagonized. The independent nature of the EEG and the evoked responses seen in this study indicate that different processes or neurons are involved in each phenomenon. The evoked phenomenon is also more specific, being obtained only in certain leads; whereas, an EEG was obtained from all locations of the midbrain. Independent variations between the EEG and evoked responses are also evident in intact a n i m a W . It is possible that the neuroglia contribute significantly to the EEG pattern16, and evoked potentials are specific signals so that, at least under the influence of drugs, they may vary independently. Since the anticholinergic agents failed to alter the EEG pattern from these “midbrain animals”, but do change this pattern in intact rabbits61, it is possible that their main site of action is on cholinergic links above the midbrain23v40.52.On the other hand, such drugs may not be able to affect these abnormal waves because impulses responsible for normal EEG patterns were destroyed, thereby preventing any cholinolytic action at the midbrain level. In this regard, atropine fails to change the electrocorticogram obtained from the acute “isolated hemisphere” preparation42 so that the drug apparently does not affect the electroencephalogram in such abnormal preparations. From more physiological experiments, however, Rinaldi41 concluded that atropine must have actions both at cortical and midbrain sites,

SUBCORTICAL ACTIONS OF ANTJCHOLINERGICS

23

Although it is questionable whether the results described here are specifically related to the many diverse behavioral effects produced by anticholinergic compounds, they do provide evidence that the midbrain reticulum is affected - probably directly by cholinergic and anticholinergic agents. They further show that pharmacological changes may be induced in the midbrain reticular formation that are not dependent on higher centers and demonstrate the importance of testing anticholinergic compounds upon a background of cholinergic stimulation. Alone, none of the anticholinergic drugs given systemically depress midbrain evoked responses, but neither do ataractics or sedatives61. However, the latter two groups of drugs fail to block the effect ofphysostigmine on single shock responses recorded from the midbrain of intact rabbits; whereas, the piperidyl benzilates and belladonna alkaloids are antagonistic to physostigmine53~61.Similarly, atropine alone will not inhibit synaptic activity of the cerebral cortex, but will block the effects of acetylcholine given by close arterial injection44. Lastly, our positive findings question the implication of Giarman and Pepeu20 that scopolamine has no important action on the “rostral” midbrain because in rats it failed to reduce significantly acetylcholine levels in this area. The fact that these investigators found a reduction of 14% in “rostral” midbrain agrees with the report of Veit and Vogt48 that scopolamine does enter the midbrain. Also, with higher doses Veit and Vogt found about 2.5 times more scopolamine in the cortex than in the midbrain. Giarman and Pepeu showed, similarly, that scopolamine reduced acetylcholine levels of the cerebrum 2.5 times greater than the 14% in the midbrain. Our findings indicate that at least some anticholinergic agents enter regions of the brainstem to antagonize the actions of cholinergic agents and support the inferences of 0 t h e r s 7 J 5 ~ ~that 8 ~ ~chclinoceptive ~ neurons are present in the brain below the diencephalon which may be pharmacologically altered by anticholinergic compounds. SUMMARY

Electrographic recordings were obtained from the midbrain reticular formation of rabbits in which the prosencephalon (cerebrum and diencephalon) was extirpated. These recordings consisted of “spontaneous” brain waves (EEG) and evoked potentials produced by applying single shock stimuli to one sciatic nerve or to the pontine region of the reticular formation. Physostigmine (0.2 mg/kg) significantly reduced or abolished the single shock responses. In contrast, atropine, scopolamine, and four piperidyl benzilates (JB-329, JB-3 18, JB-340, JB-305) did not reduce the amplitude of the evoked potentials. However, atropine, scopolamine, JB-3 18 and JB-329 completely blocked the effect of physostigmine on the single shock responses; whereas, JB-340 and JB-305 had no such effect. Possible relationships between the ability of anticholinergic compounds to produce psychotic episodes or ameliorate Parkinson’s disease and their ability to block the electrographic effects of physostigmine in experimental animals were discussed. It was concluded that the reduction of the single shock response caused by physostigmine was central in origin because (1) this same effect was obtained by high freReferences p . 24-26

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quency stimulation of the pontine reticulum, (2) it was not produced by neostigmine, and (3) JB-340, a compound which exerts strong anticholinergic actions peripherally, was unable to block this action of physostigmine. The “spontaneous” electrical activity recorded from the midbrain reticulum in these experiments was low in amplitude and was not significantly changed by the above drugs. Pentobarbital also failed to change these patterns but a Metrazol seizure pattern could be induced. Important differences between recording antagonistic actions of drugs on single shock responses and on “spontaneous” electrical activity was therefore emphasized. It was also stressed that the use of a suitable agonist (e.g., physostigmine) may be necessary to reveal significant effects of, and differences among, many centrally acting drugs. REFERENCES 1 AeooD, L. G. (1957) Some relations between chemicalstructure andphysiologicalaction of mescaline and related compounds, in Neuropharmacology. Josiah Macy, Jr. Foundation, New York, pp. 229-234. 2 ABOOD,L. G., OSTFELD, A. AND BIEL,J. H. (1959) Structure-activity relationship of 3-piperidyl benzilates with psychotogenic properties. Arch. int. Pharmacodyn., 120, 186-200. 3 BENESOVA, O., BOHDANECK~, Z. AND GROFOVA, I. (1964) Electrophysiological analysis of the neuroleptic and antidepressant actions of psychotropic drugs in rabbits. Znt. J. Neuropharmacol., 3,479-488. 4 BIEL,J. H., NUHFER, P. A., HOYA,W. K., LEISTER, H. A. AND ABOOD,L. G. (1962) Cholinergic blockade as an approach to the development of new psychotropic agents. Ann. N. Y.Acad. Sci., 96, 251-262. 5 BIJLSMA, U. G. AND BROUWER, J. E. (1928) Die Wirkung des Skopolamins in Kombination mit Cyanid, Kohlenoxyd und Luftverdunnung, Arch. exp. Path. Pharmakol., 138, 190-207. 6 BOGDANSKI, D. F., WEISSBACH, H. AND UDENFRIEND, S. (1958) Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharrnacol., 122, 182-194. 7 BRADLEY, P. B. (1957) Microelectrode approach to the neurnpharmacology of the reticularformatiorr. Psychotropic Drugs. Eds. S. Garattini, V. Ghetti. Elsevier, Amsterdam, 207-216. 8 CARLTON, P. L. AND DIDAMO, P. (1961) Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmacol., 132, 91-96. 9 CHATFIELD, P. 0. AND PURPURA, D. P. (1954) Augmentation of evoked cortical potentials by topical application of prostigmine and acetylcholine after atropinization of cortex. EEG Clin. Neurophysiol., 6, 287-298. 10 DEMAAR, E. W. J. (1956) Site and mode of action in the central nervous system of some drugs used in the treatment of Parkinsonism. Arch. int. Pharmacodyn., 105, 349-365. 11 DESMEDT, J. E. AND SCHLAG, J. (1957) Mise en evidence d’elements cholinergiques dans la formation reticulee mesendphalique. J . Physic/. (Paris), 49, 136-1 38. 12 ECCLES, J. C., ECCLES, R. M. AND FATT,P. (1956) Pharmacological investigations on a central synapse operated by acetylcholine. J. Physiol. (Lond.), 131, 154-169. 13 ECCLES, R. M. AND LIBET,B. (1961) Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol., 157, 484-503. E. AND MARSALA, J. (1962) Sereotaxic atlases for the cat, rabbit and rat. In J. BureS 14 FIFKOVA, et al., Electrophysiological Methods in Biological Research. Academic Press, New York, Appendix I: 426-467. 15 FORRER, G. R. (1958) Atropine coma therapy: Report of a death. J. Michigan State Med. Soc., 57, 996-998. 16 GALAMBOS, R. (1961) A glia-neural theory of brain function. Proc. Nut. Acad. Sci.,47, 129-136. 17 GERSHON, S. AND BELL,C. (1963) A study of the antagonism of some indole alkaloids to the behavioural effects of “Ditran”. M e d exp., 8, 15-27. 18 GERSHON, S. AND OLARIU,J. (1960) JB-329 - A new psychotomimetic. Its antagonism by tetrahydroaminacrin and its comparison with LSD, mescaline and sernyl. J. Neuropsychiat., 1,283-292.

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19 GIARMAN, N. J. AND PEPEU,G. (1962) Drug-induced changes in brain acetylcholine. Brit. J. Pharmacol., 19, 226-234. 20 GIARMAN, N. J. AND PEPEU,G. (1964) The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol., 23, 123-1 30. 21 HERNANDEZ-PEON, R., CHAVEZ-IBARRA, G., MORGANE, P. J. AND TIMO-IARIA, C. (1963) Limbic cholinergic pathways involved in sleep and emotional behavior. Exper. Neurul., 8, 93-1 11. 22 HERZ,A. (1967) Some actions of cholinergic and anticholinergic drugs on behaviour. This volume. 23 HIMWICH, H. E. AND CUCULIC, Z. (1967) An examination of a possible cholinergic link in the EEG arousal reaction. This volume. 24 HIMWICH,H. E. AND RINALDI,F. (1957) The antiparkinson activity of benactyzine. Arch. int. Pharmacodyn., 110, 119-127. 25 ILYUTCHENOK, R. J. (1963) Problems of chemical perceptibility of the bruin stem reticular formation. Psychopharmacological Methods, Eds: Z. Votava, M. Horvath, 0. Vinaf. Pergamon Press, Oxford, England, pp. 115-122. 26 ISBELL, H., ROSENBERG, D. E., MINER,E. J. AND LOGAN, C. R. (1964) Tolerance and cross tolerance to scopolamine, n-ethyl-3-piperidyl benzylate (JB-318) and LSD-25. Neuropsychopharmacology, 3,440-446. 27 ITIL,T. M. (1966) Quantitative EEG changes induced by anticholinergic drugs and their behavioral cxrelates in man. Rec. Adv. B i d . Psychiat., 8, 151-173. 28 JENKNER, F. L. AND WARD,JR., A. (1953) Bulbar reticular formation and tremor. Arch. Neirrol. Psychiat. (Chicago), 70,489-502. 29 KRUSE,W. (1960) Treatment of drug-induced extrapyramidal symptoms. Dis. New. Syst., 21, 79-8 1, 30 LOEB,C., MAGNI,F. AND ROW,G. F. (1960) Electrophysiological analysis of the action of atropine on the central nervous system. Arch. ital. Biol., 98, 293-307. 31 LONGO,V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 32 LONGO, V. G. (1966) Mechanisms of the behavioral and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18, 965-996. 33 LONGO,V. G. AND SILVESTRIM, B. (1957) Effects of adrenerkic and cholinergic drugs injected by the intra-carotid route on electrical activity of brain. Proc. Soc. exp. Biol., 95, 43-47. 34 LONGO,V. G. AND SILVESTRINI, B. (1958) Contribution a l’etude des rapports entre le potentiel reticulaire Bvoque, l’etat d’anesthtsie et l’activite electrique cerebrale. EEG Clin. Neurophysiol., 10,111-120. 35 MCGAUGH, J. L., DEBARAN, L. AND LONGO,V. G. (1963) Electroencephalographic and behavioral analysis of drug effects on an instrumental reward discrimination in rabbits. Psychophurmacofogia, 4, 126-1 38. 36 MEHES,J. (1929) Studien iiber den Skopolaminschlaf und seine Verstarkung durch Morphium. Arch. exp. Path. Pharmakol., 142, 309-322. 37 NICKANDER, R. C. AND YIM,G. K. W. (1964) Effects of tremorine and cholinergic drugs on the isolated cerebral cortex. Int. J. Neuropharmacol., 3, 571-578. 38 OSTFELD, A. M. AND ARUGUETE, A. (1962) Central nervous system effects of hyoscine in man. J. Pharmacol., 137, 133-139. 39 PFEIFFER, C. C., (1959) Parasymphathetic neurohumors; possible precursors and effect on behavior. Int. Rev. Neurobiol., 1, 195-244. 40 RATHBUN, R. C. AND SLATER, I. H. (1963) Amitriptyline and nortriptyline as antagonists of central and peripheral cholinergic action. Psychopharmacologiu, 4, 114-125. 41 RINALDI,F. (1956) Direct action of atropine on the cerebral cortex of the rabbit. Progr. Brain Res., 16, 229-244. 42 RINALDI,F. AND HIMWICH, H. E. (1955) Cholinergic mechanisms involved in function of mesodiencephalic activating system. Arch. Nrurol. Psychiat., 73, 396-402. 43 SIGG,E. 3.(1962) The pharmacodynamics of imipramine. The first Hahnemann Symposium on Psychosomatic Medicine. Lea and Febiger, Pub., 671-678. 44 SIGG,E. B., DRAKONTIDES, A. B. A N D DAY,C. (1965) Muscarinic inhibition of dendritic postsynaptic potentials in cat cortex. Int. J. Neurupharmucol., 4,281-289. 45 STEINER, W. G. AND HIMWICH, H. E. (1962) Central cholinolytic action of chlorpromazine. Science, 136, 873-874. 46 SULSER, F., BICKEL, M. H. AND BRODIE, B. B. (1964)The action of desmethylimipramine in counteracting sedation and cholinergic effects of reserpine-like drugs. J. Pharmacol., 141, 321-330.

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47 TEUCHMANN, J. (1949) The action of diallilbarbituric acid and of scopolamine on the spinal reflexes of the decapitated, the decerebrated and the decorticated cat. Arch. in/. Pharmacodyn., 79, 257-262. 48 VEIT,F. AND VOGT,M. (1935) Verteilung von Arzneistoffen auf verschiedene Regionen des Zentralnervensystems, zugleich ein Beitrag zu ihrer quantitativen Mikrobestimmung im Gewebe. Arch. f: exper. Path. u. Pharmakol., 178, 534-559. 49 VERNIER, V. G. AND UNNA,K. R. (1956) Theexperimental evaluationofantiparkinsoncompounds. Ann. N . Y.Acad. Sci., 64,690-704. 50 VILLARREAL, J. E. AND DOMINO,E. F. (1964) Evidence for two types of cholinergic receptors involved in EEG desynchronization. The Pharmacologist, 6, 192. 51 VOJTECHOVSKY, M. (1967) Experimental psychosis induced by benactyzine. This volume. 52 WHITE,R. P. (1963) Relationship between cholinergic drugs and EEG activation. Arch. in/. Pharmacodyn., 145, 1-17. 53 WHITE,R. P. (1966) Electrographic and behavioral signs of anticholinergic activity. Rec. Adv. Biol. Psychiat., 8, 127-139. 54 WHITE,R. P. AND BOYAJY, J. D. (1960) Neuropharmacological comparison of atropine, scopolamine, benactyzine, diphenhydramine and hydroxyzine, Arch. in/. Pharmacodyn., 127, 260-273. 55 WHITE,R. P. AND CARLTON, R. A. (1963) Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia, 4, 459-47 I . 56 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effects of adrenergic drugs. J. Pharmacol., 125, 339-346. 57 WHITE,R. P. AND HIMWICH,H. E. (1957) Analysis of forced circling induced by DFP and ablation of cerebral structures. Am. J. Physiol., 189, 513-516. 58 WHITE,R. P. AND HIMWICH, H. E. (1957) Circus movements and excitation of striatal and mesodiencephalic centers in rabbits. J . Neurophysiol., 20, 81-90. 59 WHITE,R. P., NASH,c. B., WESTERBEKE, E. J. AND POSSANZA, G . 3. (1961) Phylogenetic comparison of central actions produced by different doses of atropine and hyoscine. Arch. int. Pharmacodyn., 132, 349-363, 60 WHITE,R. P., RINALDI, F. AND HIMWICH, H. E. (1956) Central and peripheral nervous effects of atropine sulfate and mepiperphenidol bromide (Darstine) on human subjects. J. Appl. Physiol., 8,635-642. 61 WHITE,R. P., SEWELL, H. H., JR. AND RUDOLPH, A. S. (1965) Drug-induced dissociation between evoked reticular potentials and the EEG. EEG Clin. Neurophysiol., 19, 16-24. 62 WHITE,R. P. AND WESTERBEKE, E. J. (1961) Differences in central anticholinergic actions of phenothiazine derivatives. Exp. Neurol., 4, 317-329. 63 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: Morphine, N-allylnormorphine and atropine. Proc. Soc. exp. Biol., N . Y., 79, 261-264. 64 WILSCN, W. P. (1961) Observations on the effect of toxic doses of atropine on the electroencephalogram of man. J. Neuropsychiat., 2, 186-190.