Nabtic Trammi I¢,Tt I T the Lateral G.Tnicvdate / ')Ldcus of the ( At

Psychopharmacology. Pharmacologic effects on behavior. Ed.by: Pennes, H.H. Hoeber-Harper, New York 1958, p. ]_73-2o2.. LSD 633/BOL

CHAPTER Vlll

Effect,s _f a Series tf lndolcs on ,S)'nabtic Trammi_i¢,tt i_t the Lateral G.tnicvdate /_')ldcus of the (_at

EDWARD V. EVARTS

SINCE tloffman's origillal observation (20) on the psychologic effects of d-lysergie acid diethylamide (LSD), its mechanism of action has been the subject of considerable investigation. The observations of Woolh_v a_d Shaw (21) and of Gaddum (11) on the antagonism of LSD to the effects of 5-hydroxytryptamine on smooth muscle have suggested the possibility that the psychologic effects of LSD in ma_ may be the result of an interaction between LSD and 5-hydroxytryptamine in the central nervous system. The observations by Pletscher et al. (16) on the effects of reserpine on 5-hydroxytryptamine levels in the brain have suggested that the neuropsyehologic effects of reserpine may also be related to an interaction with the latter. Re- cently, Gaddum (]2) and Rothlin (19) have criticized the theory that mltagonism of LSD to an action of 5-hydroxytrypta- 173

I r! 174 PsYt'HOPtiAI_MACOLOGY mine is involved in the effects of LSD on the brain; both in- vestigators point out that a number of 5-hydroxytryptamine antagonists, the in vitro potency of which (as tested on a smooth muscle preparation) is equivalent to that of LSD, do not produce psychologic effects of the type produced by LSD. It would appear, then, that the degree to which the effects of LSD on cerebral function are related to 5-hydroxytryptamine is still a matter of speculation. The experiments to be described were carried out to obtain further data concerning certain electro- physiologic effects of 5-hydroxytryptamine, LSD, and of a series of tryptamine and LSD derivatives. It seemed that such data might be relevant to the question of interaction between LSD and 5-hydroxytryptamine in the central nervous system. One of the eleetrophysiologic effects of LSD is depression of synaptie transmission in the lateral genieulate nucleus (9). The present study reports observations on the effects of a number of indole derivatives on lateral genieulate transmission. These indoles mav be divided into the two general categories of tryptamine derivatives and of LSD derivatives. The effects of these agents alone and the interaction between these agents and LSD were observed. There were several reasons for selecting the lat_.ral genieulate nucleus as a site at which to test the effects ,_f and interactions between the various indole derivatives First, the f¢mn of file genieulate response to optic nerve shock is separal_le into easily measured presynaptie and postsynaptie conq_

quaternary ammonium ion to cross the blood-brain barrier. These factors must necessarily limit the interpretations which may be based on the data to be presented.

MATERIALS AND METHODS

Cats weighing 2.5 to 4.5 Kg. were anesthetized with 30 mg./Kg, sodium pentobarbital (Nembutal), intraperitoneally. The optic nerve was.dissected free; stimulating electrodes were placed on the cut edge of the sclera (+) and on the optic nerve (-). Stimuli to the optic nerve were nearly square pulses of approximately 30 _tsee. duration. The intensity of optic nerve stimulus required to produce a near maximal geniculate response varied from preparation to preparation, over a range of 1,5 to approximately 5 volts. The geniculate response was recorded with a stereotaxically placed steel needle insulated to the tip; the tip diameter was approximately 100 F" The cortical

CH3 H H il HO CH2CH2NHz _---_FCH2CH2N_CH3 • I VI _N- _ /CH3CH3+ _ H H

III HO CH?.CH2N,.cH3 HO CH2CHtN,,c41.19

H H

IV {_---_-CH2CH2N_ CH3 CH_lO_"_---Tr- CH2CH2N_CH3 VIII _,_,,,N _ CH3 CH30_ CH3 H H

Fzo. 16. Structures of tryptamine derivatives whose effects on genieulate transmission were studied. Compounds I, III, and IV caused depression of geniculate • transmission; the remainder were without effect on the lateral gerticaalate body or the cortical responses to optic nerve shock. EFFECTS OF INI)OLES ON SYNAPTIC TI_.NSNIISSION 177 response to stinmlation of the optic nerve was recorded from the lateral gyrus with a pore electrode filled with Tyrode's solution. The brain was covered with a pool of continuously flowing Tyrode's solution which was maintained between 36 and 38 °. The cat's body temperature was also maintained at 36 to 38 °. - Drugs were injected intravenously or via the carotid artery on the side from which geniculate recordings were obtained. Eight tryptamine derivatives (Fig. 16) and four LSD derivatives (Fig. 17) were used. Dimethyltryptamine, _ bufotenine, b and 5-hydroxytryptamine _ were obtained as the free base and dissolved as the creatinine sulfate complex. Tryptamine, c 5-hydroxy- .3-(fl- dibutylaminoethyl )indole, d 5,6- dimethoxy- 3- (fl- dim ethylaminoethyl )indole, _ and g- ( fl- (2-methylpiperidyl ) ethyl)indole h were obtained in the form of the hydroehloride. Bufotenidine" was obtained as the iodide. LSD t and 2-bromo LSI) t were obtained as the tartrate. 2-Oxy LSD, _ a metabolic product of LSD, was formed from LSD by in vitro incubation of LSD ill a microsomal enzyme preparation (1). Gramine" was obtained as the free base, and dissolved as the hydrochlo- ride. 2,2'-Bis LSD disulfide _ was obtain6d as the free base. All of these drugs were obtained in solid form and dissolved in Tyrode's solution shortly before administration. In the case of the last two, a small amount of dilute HC1 was required to pro- mote solubility. The volume injected over approximately l0 sec. was 0.5 to 1.0 ml. regardless of the amount of drug administered. The drags were injected through a polyethylene canula placed in a small muscular branch of the common carotid artery, and ad- vanced to a point at which the tip of the canula was within the

a Supplied by Upiohn Comp, ny. bSupplied by Dr. Evan Homing, National Heart Institute, National Institutes of tlealth, Bethesda, Md. c Supplied by the Eastman Kodak Company. a Supplied by Dr. Gordon Walker, National Heart Institute, National Institutes of Health. e Supplied by Dr. Melvin Fish, National Heart Institute, National Institutes of Ilealth. t Supplied by Sandoz Company. a Supplied by Dr. Julius Axelrod, Naional Institute of Mental Ilealth, National Institutes of Ileahh. h Supplied by Eli Lilly Company. i Supplied by Dr. Bernard Witkop, National Institute of Arthritis and Metabolic Diseases, National Insti- tutes of Health. 17_ PSYCHOPHARMACOLOCY

/C2H5 IC2H5

H H H

L SD 2-OXY LSD 2-BROMO L.,qD

c4N_CZH5 C2H5",.u I_C C2H5 C2 H5"o_pC

H H

2,2'-BIS LSD DISULFIDE GRAMINE

Flc. 17. Structures of four LSD derivatives and granfine. Of the former, only LSD caused depression of geniculate transmission; the other three were without effect on the geniculate or cortical responses to optic nerve shock. Gramine did not depress geniculate transmission; LSD administered after gramine did not block geniculate transmission. common carotid. Femoral blood pressure was recorded with a mercury manometer.

RESULTS Effects on Geniculate Transmission

The effects of the indole derivatives on geniculate transmission are summarized in Table 88. Of the 18 agents used, 4 depressed geniculate transmission; the remaining 9 agents had no observable effects on the geniculate response to optic nerve t" FFECTS OF' INDOLES ON SYNAPTIC TRANSN'IISSION J.7,(}

Table 38. Effects of a Series of Indoles on Genieulate Transmission ...... Decrease _ in Dose, postsynaptic respon*e Drug a mg./Kg. 1 mi,. after i,qe,'tion, f'_

! 0.2 70 (5O-}O0) II 0.2 III 0.2 60 (50-90) fV 0.4 9O (S0- _00) V 4.0 VI 0.2 VI I 1.(} VIii ,3.0 LSD frO3 80 (80-/00) 2-Oxv I.SI) 2-Br_ml_ 1.SI) 0.3 2.2'.BJs LSD disulfide 1.5 Gramim' 15.0

" For the stn_chlres, s_',. _',e,,_rcs 16 and 17. Based on c,h;mge in a.mpiitude of postsynaptie response to a single, near maximal optic norve shock; figures in parentheses indicate observed range in the efl','cts _,f the acti',t" _'_nnpounds.

stimulation. The three active tryptamine derivatives were tryptamine (1), bufotenine (llI), and dimethyltryptamine (IV). Although the percentage decrease of the postsynaptie response at the peak effect of tryptamine was of the same order as that caused bv bufotcuine and dimethyltryptamine, the duration of effect in the case of tryptamine was considerably less than with the other two agents (:omplete recovery of genieulate transmission occurred within :3 to 6 rain. after tryptanfine administration (0.12 mg./Kg.), whereas recovery following comparah]e doses of bufotenine and di,,wthyltryptamine (fig mg,/Kg.) required 80 to 60 rain. (Figs. 1,5-19). l/eeruitment of the type occurring during depression of geniculate transmission by tryptamine and dimethyltryptamine is also caused by the two other active indoles. The increase in latency of the cortical response is associated with the effects of tryptamine on the geniculate postsynaptic response (Fig. 18); during the period of geniculate 1 _1 ) PSYCHOPItARNIACOLOGY depression the c_Jrtical response corresponds to the second, or at times to the third, _q_tic nerve stimulus. Whereas tryptamine, dimethyltryptamine, and bufotenine caused depression of geniculate transmission, 5-hydroxytryptamine had no such effect (Fig. 20). In this experiment, the optic n_,rv_, was stmmlated every 2 see. with alternate maximal and submaximal stimuli; a total close of 0.1 nag. 5-hydroxytryptamine was injected via the carotid artery. Following the injection, there was no significant change in the amplitude of the postsynaptie response to either maximal or submaximal optic nerve stimuli. A similar lack of effect has been noted following both larger and smaller doses of the drug.

Effects on Cortical Response to Geniculate Radiation Shock In a previous study (9) it was observed that much greater doses of LSD and bufotenine than those which blocked geniculate transmission did not decrease the cortical response to stimulation of geniculate radiation fibers. In the present experiments it has been found that dimethyltryptamine and tryptamine, which depress the geniculate response to optic nerve shock, do not reduce the cortical response to stimulation of geniculate radiations (Fig. 21). Effects of Asphyxia The effects of LSD and bufotenine on geniculate transmission are transiently reversed by asphyxia (Fig. 22). A similar effect of asphyxia occurs in the case of dimethyltryptamine and tryp- famine. The reversal of the LSD effects occurs within less than 30 sec. of asphyxia. A comparable period of asphyxia in a preparation which has received no drugs (except Nembutal) does not depress geniculate transmission, or may cause a slight increase in the amplitude of the postsynaptic response (2) (Fig. 23). A change in the configuration of the geniculate response occurs approximately 2 rain. after occlusion of the trachea. When depression of the geniculate responses occurs as the result of asphyxia, the pattern of depression differs from that produced by LSD and the 8 active tryptamine derivatives. During CONTROL 4'

_"_|

NEMBUTALIZED CAT G-7-SG

2 MSEC 200JL,/-V CORTEX 200/d.V GENICULATE

FIG. 18. Effects of tryptamine on geniculate transmission. Upper tracings, cortical, and lower tracings, lateral geniculate, response to a train of 5 near- maximal optic nerve shocks. Times indicate period after injection. Tryptamine (0.12 mg./Kg.> caused almost complete block of geniculate transmission with rapid recovery; 2 min. after the injection recovery was clearly _resent, with recruitment of the geniculate postsynaptic response, and was complete within 6 min. Note that the presynaptic optic tract spike remains unaltered during depression of geniculate postsynaptic response. T, presynaptic (tract) spike; S, postsynaptic (soma) spike. The upward deflection is the positive. CONTROL 5'

5" 21'

15"

[i r'ZE

NEMBUTALIZEO CAT 10-19-54

200 _V 2 MSEC EFFEUI_ OF INDOLES ON SYNAPTIC TKANSMISSION 183 acute asphyxia there is no recruitment; the response to each of the series of optic nerve shocks is depressed. In some cases, the response to the third or fourth stimulus of the series is depressed to a greater degree than is the response to the first shock. Interactions between LSD and Other Indoles in Effects on Geaiculate Tmmmissiou ,_-

The inactive indoles (Table 38) were tested as possible antagonists to LSD. Experiments were carried out to determine tkv degree to which a given compound would prevent (if administered before LSD ) or reverse (ff administered after LSD) the LSD-indueed depression of the lateral genieulate postsynaptie response. 5-Hydroxytryptamine, administered before or after / LSD, caused no consistent change in the LSD effects. Gramine, however, did cause a significant decrease in the effects of sub- sequently administered LSD. In initial experiments, gramine was injected into the carotid in quantities up to 80 mg./Kg.; no change in geniculate or cortical responses to optic nerve shock was observed, nor was there a change in the amplitude or form of the spontaneous cortical activity. The geniculate excitability cycle to paired optic nerve shocks was equally unaf- fected by gramine. LSD in doses up to 0.06 mg./Kg., administered after gramine, did not cause any change in the genieulate response. It seemed possible that the absence of effect by LSD following gramine might have been the result of a gramine- induced constriction of the carotid artery. Such constriction, if present, might have decreased the blood flow via the carotid artery into which injections were made, and might therefore have lessened the apparent effect of LSD. To avoid this possibility, both LSD and gramine were administered via the femoral vein; the LSD was washed in with sufficient fluid to insure its entry into the systemic circulation. Gramine, 15 mg./Kg., was administered to 6 cats at 0 and 3 nan., followed by 0.08 mg./Kg.

19. Effects of dimethyltryptam!ne on geniculate transmission; response to a tnfln of 5 near-maximal optic nerve shocks. Dlmethyltryptamine (0.3 mg./Kg.) caused marked depression of postsynaptic rmpome; despite almost complete after first shock of train, recruitment occurred, and at. 15 see. and 1 rain. ths postsynaptic response to fourth shock of train was larger than in control period. 184 pSYCHO_COLOC_

; 13"

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u • r- 7" • eoeee • z 6- • 01_- • ee • • • • mt.- 5" • • • • • • • 0 • • a. • 4. " 0 ' .. 3' o -i J IL

io /o s'o 6o io _Ubi SEROTONM LI_ 8[CONOI

F*o. 90. EEects o£ 5..hydro_ne on S_deadats tnmmission in m_ lmtalJzedm,,,.i,,ud(z)cat. _Amplitudesnerveshocksof lXlUbymldiCdm_ andrespafter(xlmtntmcto submaxim_Ud tn|ectlo,al liCe.)com.- pound. No dgfiillcant change in occurred postsynapttc responses to maxmaxt or , _uhnauamadl md_

LSD at 6 and 9 min. (total dose of LSD, 0.10 mg,/Kg.); 7 cats received 0.08 mg./Kg. LSD at 0 and 8 _. (total dose, 0.11t mgJKg. ) without previous gramine (Table 89). In the gramine- treated animals LSD caused no reduction in Postsynaptic spike 1 amplitude; in those which had not received gramine LSD caused a mean decrease of 54 per cent in the spike amplitude. The difference in the effects of LSD under the two conditions is statistically significant (p > 0.01). Gramine also prevented the effects of LSD on spontaneous cortical activity (Fig. 24). _In animals not receiving gramine, LSD caused a marked decrease in the amplitude of spontaneous cortical aeti_ty; following gramine, LSD did not have this effect (Fig. 25). EFFECTS OF INDOLES ON SYNAPTIC TRANS_flSSION 185

Table 39. Antagonism of Gramine to Effects of LSD on Geniculate Transmission

Gramine, 15 mg./Kg, at 0 and 3 rain. + .... LSD, 0.08 mg./Kg, at 0 and 3 Min. LSD, 0.08 mg./Kg, at 6 and 9 rain. Soma spike Soma spike Experiment decrease Experiment decrease No. at 5'45"-6', % No. at 11'45"-12", %

3-4 75 4-1 -13 3-8 10 4-4 0 3-9 82 4-6 2 3-10 52 4-7 --13 3-11 21 4-11 5 3-16 80 4-12 5 3-17 59 Mean decrease a 54 -2

a The difference between the two groups is significant (P < 0.01), allowing 7 degrees of freedom to take into account the unequal variance in the two groups.

Additional experiments wexe carried out to determine the duration of LSD antagonism after gramine administration, and to discover whether or not gramine would reverse the effects of previously administered LSD. It was found that the period following gramine administration during which the effects of LSD are blocked is short; when administered 15 rain. following gramine, there was a clear decrease in postsynaptic spike amplitude. Gramine administered following onset of the LSD effect did not cause restoration of normal geniculate transmission. Discussion

Of the 8 tryptamine derivatives administered in the doses in- dicated (Table 38), 3 cause a decrease in the amplitude of the geniculate postsynaptic response; 2 of the 3 active agents, bufotenine and dimethyltryptamine, have approximately equal activity, both in terms of peak effect and of duration of action. Tryptamine produces a peak effect comparable to that of bufotenine and dimethyltryptamine; the duration of this effect, how- ,,; ]86 PSYCIIOPHARMACOLOGY

CONTROL I'

NEMBUTALIZlrD CAT 6-7"56

200 ]./-V 2 MSEC

Ftc. 21. Effects of tryptamine on cortical response to stimulation of genieulato radiations. Cortical response to a single shock was not affected by intracam_ administration of 1.0 mg./Kg,, a dose approximately eighffold that whidg in this particular cat, caused complete block of postsynaptie geniculate response to • single shock applied to optic nerve.

ever, is quite brief. The relatively brief effect of tryptamine (as compared to dimethyltryptamine and bufotenine) may be the result of rapid destruction in vivo. It might be expected that the N-dimethvl substitution on dimethyltryptamine and bufotenine would decrease the rate of oxidation by amine oxidase (4), and lead to a more prolonged pharmacologic effect. The "-" absence of any effects on geniculate transmission following intracarotid administration of 5-hydroxytryptamine, and the ab- EFFE(YFS OF INDOLES ON SYNAI'TIC TRAN_,\_ISSI ()_, 1_7 sence of any consistent interaction between it and LSI), n_ay be the resuit of rapid in vivo destruction of 5-hydrox)tryptamine or of its failure to cross the blood-brain barrier in suf-

l,J_ 80"

NEMBUTAL|ZED CAT 8-19-54,

'_ MSEC

200/.L_

FIc. 22. Antagonism of asphyxia to effects of LSD on geniculate trazlsmissiozl; gemculate response to a train of 6 near-maximal optic nerve shocks. Upper left, depression of genieulate transmission caused by intracarotid administration of 0.05 mg./Kg. LSD. The times in seconds indicate the elapsed time after tracheal occlusion. Note return of geniculate depression, but absence of r_cr,dtment, 80 see. after traeheal occlusion. fieient quantity; since there are no adequate data on either of these points, the absence of effects following 5-hydroxytryptamine and the lack of a demonstrable interaction between it and LSD cannot be taken as clear evidence against the theory tl_at the effects of LSD on the central nervous s)stem are the result of block of some physiologic action of 5-hydrox)'tr)'ptanfine, 188 PSYCHOPHARMACOLOGY

Large doses of the remaining 4 tryptamine derivatives produced no change in the geniculate response. The absence of effect fol-

_t CONTROL

NEMBUTALIZED CAT 6-5"56

2 MSEC 200 /.LV GENICULATE

TRACHEA OCCLUDED AT O" 200 /J,.V CORTEX

FIc. 23. Depression of geniculate transmission by asphyxia. Upper tracing, the geniculate, and lower tracing, the cortical response to a train of 6 near-maximal optic nerve shocks before and at various intervals following tracheal occlusion. At ,3 min., amplitude of postsynaptic geniculate spike is clearly depressed; no recruitment occurs during asphyxia-produced depression. Cortical response --.to optic nerve shock is markedly depressed at 2 rain. after tracheal occlusion, when depression of geniculatc postsynaptic response has not yet occurred. EFFECTS OF IN'DOLES ON S"i_APTIC TRANSMISSION 189

I I' ,i,!I , i'1 ,!, t¢ GRAMINE

I zoop.v

6 SECONDS

.j , _)

2OO _v " I GRAMtNE "_

LSD

Fro. 24. Antagonism of gramine to effects of LSD fn nemb_ltMized cats. Trac- ings of spontaneous cortical activity show absence of effect of gramine alone, marked reduction in barbiturate spindles caused by LSD alone, and prevention of LSD effects on spontaneous cortical activity when gramine has been administered before LSD.

lowing bufotenidine (VI) may be the result of the failure of this quaternary ammonium ion to penetrate the blood-brain barrier. The inactivity of 5-hydroxy-g-(B-dibutylaminoethyl) indole (VII), in which butyl groups are substituted for the methyl groups of bufotenine, indieates that extension of the alkyl ,i side chains attached to the amino gro_p decreases the activity of this series of agents; unfortunately, the diethvl and dipropyl analogues were not available for testing. The structures of the 190 PSYCHOPHAR_I A(_OLOCY

CONTROL CONTROL

GRAMINE + LSD LSD .

I2oo_v (c.l I2OOFLV {c.) FIc. 25. Antagonism of gramine to effects of LSD in nembutalized cats. Upper tracin , the geniculate, and lower tracing, the cortical response to single optic nerve ts_lock. Note absence of effects of LSD administered after granune (left) and depression caused by LSD alone (tight).

inactive corrq)ounds V and VIII (Fig. 16) indicate other structural alterations which decrease the genieulate-blocking effects of these agents. Of the various LSD derivatives tested, only EFFECTS OF INrDOLES ON SYNAPT1C TRANSMISSION ]()1

LSD itself was active; the inactivity of 2-bromo LSD and 2- oxy LSD in our experiments parallels their failure to have psychologic effects in man (1, 5). In view of tile high doses of LSD required to affect geniculate transmission, it is extremely unlikely that alteration of geniculate activity of the type described here is causally related to the" " disturbances of visual perception which LSD causes in man. LSD intravenously in doses of 0.16 mg./Kg, caused a decrease of approximately 50 per cent in the amplitude of the postsynaptic response to a single optic nerve shock in the nembutalized preparation; the dose of LSD required to produce a comparable effect in the conscious preparation is approximately fivefold (8). In the monkey, transient blindness followed doses of LSD of the order of 1.0 mg./Kg. (7). While failing to indicate a mechanism of LSD action in man, the effects of LSD and the other indoles on geniculate transmission may possibly serve to predict the occurrence of psychotomimetic effects following administration of smaller doses of these drugs to man. Fabing and Hawkins (10) have shown that bufotenine causes hallucinations in man. Isbell (14) has also found that bufotenine causes hallucinations and other perceptual disorders in man. On the basis of our experiments, it might be predicted that dimethvl- tryptamine would have psychologic effects similar to thoseof bufotenine; tryptamine, because of its very brief duration of action, to be less active; and the remaining tryptatmine derivatives to be relatively inactive as psyehotomimetie agents. To date, dimethyltryptamine has not been administered to man; the results of studies of its effects in human subjects will help to clarify the extent to which the effect of these indoles on geniculate transmission mav be used to predict their actions in man. Gramine [8-(N-dimethylaminomethvl )indole] is an alkaloid originally isolated from chlorophyll-deficient barley mutants, which has since been synthesized. There is relativeiy little information on its pharmacologic properties. Powell and (;hen (17) have reported that gramine hvdrochlolide intravenously, in quantities of 80 to 40 rag./Kg., causes a tral_sient lowering 192 PSYCHOI'HAR3,_ACOLOGY followed by a secondary rise in blood pressure of anesthetized cats. The results of our experiments indicate that, in nembutalized cats, gramine is antagonistic to the effects of subse- quently administered LSD. This antagonism is seen in the fact that gramine blocks the effects of LSD both on spontaneous cortical activity and on transmission in the lateral geniculate nucleus. The mechanism of this antagonism is, of course, a matter of speculation. However, the work of Erspamer (6) and of Gaddum and tlameed (1,3), showing that gramine is a serotonin antagonist, may indicate an explanation of LSD-gramine interaction. Gramine and LSD possess the common property of being serotonin antagonists; moreover, gramine is structually similar to both bufotenine and dimethyltryptamine, both of which have LSD-like effects on genicnlate transmission. These facts are compatible with the notion that gramine and LSD may com- pete for the same receptor site, and that the antagonism seen in the present experiments may be a function of this competition. The failure of gramine to reverse the effects of previously administered LSD may result from the fact that LSD enters into a relatively irreversible combination with the receptor site. We do not, as yet, have an explanation for the short duration of the period following administration of gramine during which the action of LSD is blocked. Possibly, gramine is rapidly metab- olized, and this rapid degradation of gramine is the cause for the short duration of its action. Unfortunately, there are at present no data on the metabolic fate of gramine. It has been shown that gramine blocks the effects of subse- quently adnmdstered LSD in nembutalized cats. Since.the nembutalized preparation is considerably more sensitive to LSD than is the _manesthetized preparation, it is possible that the demonstrated interaction between LSD and gramine may be a function of the Nembutal anesthesia. Experiments on interaction between ,Zramine and LSD in unanesthetized preparations have not vet been carried out. It is therefore emphasized that the reported interaction between gramine and LSD may be peculiar to the particular experimental conditions under which •it was observed. EFFECTS OF 1NDOLES ON SYNAPTIC 'I'RANSMISSION 1,_)3

It may be pointed out that 2 of the agents which failed to alter geniculate transmission are 5-hydroxytryptamine antagonists; these agents, 2-bromo LSD and gramine, did not have effects of the type which follow LSD. The results add to the " growing evidence that serotonin antagonism itself is not sufficient to explain the effects of LSD on the central nervous system.

REFERENCES

1. Axe[rod, J., Brady, R. O., Witkop, B., and Evarts, E. E. The dis- tribution and metabolism of lysergic acid diethylamide. Ann. New York Acad. Sc. 66: 485, 1957. 2. Bishop, P. O., and McLeod, J. G. Nature of potentials associated with synaptic transmission in lateral genieulate of cat. I. Neuro- physiol. 17: 387, 1954. 3. Bishop, G. H., and O'Leary, J. L. Electrical activity of the lateral geniculate of cats following optic nerve stimuli. I. Neurophysiol. 3: 308, 1940. 4. Blaschko, H. Enzymic oxidation of amines. Brit. M. Bull. 9: 146, 1953. 5. Cerletti, A., and Rothlin, E. Role of 5-hydroxytryptamine in mental diseases and its antagonism to lysergic acid derivatives. Nature, London 176: 785, 1955. 6. Erspamer, V. Gramine derivatives antagonistic to 5-hydro×ytrvp- tamine (enteramine). Science 121: 869, 19_5. 7. Evarts, E. V. Some effects of bufotenine and lysergic acid diethylamide on the monkey. A.M.A. Arch Nct, rol & Psychiat. 75: 49, 1956. 8. Evarts, E. V. A review of the neurophysiological effects of lysergic acid diethylamide (LSD) and other psychotomimetic agents. Ann. New York Acad. Sc. 66: 479, 1957. 9. Evarts, E. V., Landau, W., Freygang, W. H., Jr., and Marshall, W. H. Some effects of lysergic acid diethylamide and bufotenine on electrical activity in the cat's visual system. Am. I. Physiol. 182: 594, 1955. 10. Fabing, H. D., and ttawkins, J. R. Intravenous bufotenine injcc- _ tion in the human being. Science 12.'3: 886, 1956. 194 }3S YCHOPHA1ANIACOLOGY

11. Gaddum, J. H. Antagonism between lysergie acid diethylamide and 5-hydroxytryptamine. ]. Physiol. 121: 15P, 195,3. 12. Gaddum, J, H. Serotonin-LSD interactions. Ann. New York Acad. Sc. 66: 643, 1957. 13. Gaddum, J. H., and Hameed, K. A. Drugs which antagomze 5-hydroxytryptamine. Brit. 1. Pharmaeol. 9: 240, 1954. 14. Isbell, H. Personal communication. 15. Marrazzi, A. S., and Hart, E. R. Relationship of hallucinogens to adrenergic cerebral neurohumors. Science 121: 365, 1956. 16. Pletscher, A., Shore, P. A., and Brodie, B. B. Serotonin release as a possible mechanism of reserpine action. Science 122: 374, 1955. 17. Powell, C. E., and Chen, K. K. Tile action of gramine. Proc. Soc. Exper. Biol. & Med. 5_: 1, 1945. 18. Purpura, D. P. Electropbysiological analysis of psychotogenie drug action. A.M.A. Arch. Neurol. & Psychiat. 75: 122, 132, 1955. 19. Rothlin, E. Lysergic acid diethylamide and related substances. Ann. New York Acad. Sc. 66: 668, 1957. 20. Stoll, W. A. Lysergsimre-di_ithylamid, ein Phantasticum aus der Mutterkorngruppe. Schweiz. Arch. NeuroI. u. Psychiat. 60: 279, 1947. 21. Woolley, D. W., and Shaw, E. A biochemical and pharmaco- logical suggestion about certain mental disorders. Proc. Nat Acad So. 40: 228, 1954.

DIYK:USSION

Dr. Marrazzi: Dr. Evarts has made an imposing beginning in correlating structure to fimction at a particular synapse, but it is characterized by the weakness of any insufficiently long series. Although this is very well known in the endless correlations that have been made, we all keep on making them nevertheless. The emphasis placed on synaptic effects is somewhat different and agrees more with Dr. Pribram's statement. In other words, we are more impressed with the similarities of synaptic effects than with the differences. Although there are differences, we tend to attribute them to differences in threshold rather than to differences in kind of action. This is useful for comparing a series of compounds from which, on the basis of past experience, not an aU-or-none phenomenon but graded effects are ex- EFFECTS OF INDOLES ON SYNAPTIC TI4ANSNIISS1ON 1_5

petted. But in that case, the technic ought to be so adapted as to get graded effects. The excitability-cycle technic described by Dr. Evarts does do that, but it seems to me that much of his work-and he will correct me if I am wrong-is based on super- maximal stimuli, and these are not conducive to bringing out. changes in degree. In other words, we tend to consider all the synapses of the optic-geniculate-striate system as having similarities, so that the differences would be those of degree. It is based, of course, on our work, already quoted by Dr. Evarts, showing that LSD in 8 mg. doses produces inhibition in the optic cortex, via the transcallosal system, whereas serotonin can produce inhibition in 1 mg. doses, and dimethylserotonin (bufotenine) is twice as potent as that. We also found strong evidence that epinephrine had inhibitory effects on synapses in the optic-geniculate-striate system, although we did not localize it further at that time. Maybe I can induce Dr. Pennes, who did some work in our laboratory, to give you some idea of his preliminary data on the various sites of action in that system. In any case, we would be inclined to look for a way of showing an action which we think is exercised at the geniculate synapses. Even though these synapses may differ anatomically, predominantly, they do have some axodendritic synapses of the same type as are thought to be susceptible in the cortex; we therefore are inclined to attribute the differences obtained so far to variations in technic. Why is this important? Well, for instance, the presence or absence of the 5-hs'drox 5" group ha._ been emphasized. If serotonin does not act at the genieulate synapses, then the role of the 5-hydroxy group as a critical, discriminating part of the molecule loses its significance, and this is true even if its action at this site is only minimal. An- other fact is that both bufotenine and the dehvdroxvlated bufotenine have an effect, although the latter has the hvdroxv group and the former does not. In other words, the hydroxy group is important but not absolutely essential. Actually, the fact that dimethoxybufotenine, which involves a characteristic muzzling of the 5 position, fails to act, would indicate that the 5-hydroxv 196 PSYCHOPIIAItMACOLOGY

group is of somc importance; in addition, there is the fact that it acts prophylactically against other active members of the series. We believe this to be an indication that, with careful adapta- tion of the technic, true inhibition might be demonstrated. We have an illustration of this in our own work with adrenochrome, which theoreticallv should work, and actually does, but with 2,000 rag., instead of 20 mg. doses as is the case with epinephrine, or 1 rag. doses as is the case with serotonin. This is of no great significance so far as chemical structure is related to function. Some aspects of the technic are not adapted to bringing out the particular kind of graded reaction which I believe is neces- sary in this type of evaluation. Nevertheless, Dr. Evarts has made a very fine beginning. Dr. Purpura: I am happy to have the opportunity to discuss Dr. Evarts' paper since I have followed his work with considerable interest. The analvsis he has just presented emphasizes one particular generalization discussed previously, that indoles in general, and LSD in particular, appear to be effective inhibitory, agents at certain synapses. In our own experience, axodendritie synapses seemed to show a greater sensitivity to LSD than axosomatie synapses. The same has been found for serotonin, which also inhibits axodendritic synapses. In unanesthetized, paralyzed (by succinylcholine) cats, the dendritic postsynaptic potential evoked by a direct cortical shock was found to be reduced following 50 btg. LSD per kilogram. I think this has also been confirmed by Dr. Grenell, who administered the LSD intra-arteriallv. Subsequent injection of 10 [xg. serotonin per kilogram further reduced this dendritic --.potential in a synergistic manner. It is also interesting to note that 10 rag. Nembutal per kilogram completely abolished both effects, although the mechanism is not as yet clear. However, it is important to keep in mind that the presence or absence of Nembutal may lead to different effects when studying synap- tically active agents. There is little doubt that at least low doses EFFECTS OF INDOLES ON SYNAPTIC TRANSMISSION 197 of Nembutal exert an anti-inhibitory action on certain dendritic synapses. .-- Similarly, it can be shown that the long latency of relayed reticular response to sciatic stimulation picked up at a lower mesencephalic level is also reduced by LSD and serotonin. Thus, in our experience, serotonin has not shown antagonism to LSD but rather synergism. Returning now ,to another point, we recently have been in- vestigating the mechanism of LSD action at axodendritic synapses and have been led to conclude that LSD and perhaps serotonin may activate inhibitory postsynaptie receptor sites. For this reason, I think Dr. Evarts' work may be actually telling us something about the possible nature of an inhibitory transmitter. That these exist seems certain from the results of some of the work on cross-perfusion experiments, as well as some work Dr. Grundfest and I have been doing on the nature of dendritic potentials. Note that all these agents appear to be inhibitory. I would like to call your attention to the fact that strychnine, one of the most powerful convulsants known, also has an indole grouping and appears to exert its convulsant action by blocking inhibitory synapses. It is quite likely that the kind of analysis which Dr. Evarts has instituted, when carried out on other systems, may give us some ideas about the nature of an inhibitory transmitter. Dr. Rapport: The attempt to correlate chemical structure with biologic function, which Dr. Evarts' study represents, is enticing. Although an apparent relation may look simple and correspondingly attractive, in essence it is really exceptionally complex. Now it is not especially constructive to call attention to complexity, but it may be worth recalling that the relatio_s of chemical structure to biologic activity have been studied i_k the epinephrine series for many years withont yielding any broad generalizations. We must be satisfied with the unsophis- ticated conclusion that certain structural features exert a larK,', influence whereas others are less important. No penetratin_ in- sight into the mechanism of action has vet been gained. With the class of compounds under consideration here, the indoles, 198 I'SYf l 1( _PltARXIACOLOGY the chemist is still quite helpless when it comes to explaining some of tile most elementary phenomena. For example, in the series going from tryptamine to N-methyltryptamine to N,N- dimethyltryptamine, there is a remarkable change toward in- creased solubility in ether, which might well be related to the penetration of these compounds into the brain. We do not yet clearly understand why the change in solubility is so marked when the change in structure is so slight. Similarly, if tile simple compounds studied by' Dr. Evarts do not present a consistent picture, it is not necessarily true that the exceptions are sufficient to disprove a given hypothesis. At this stage it is preferable to emphasize tile positive. The study of Dr. Brodie and his group showing that reserpine affects the release of serotonin from its binding sites is certainly fascinating. It pinpoints at least one action of reserpine, now amply confirmed, which without doubt contributes to the pharmacologic effects of the drug. The effect of reserpine on binding sites is not the same for all ceils. The release of serotonin from platelets and from brain is more complete and more sus- tained than its release from the gastrointestinal tract. Even were we to grant that reserpine action is in large part mediated through serotonin function, it would not help us very much in understanding the basic mechanism. It would be only one small step, but an important one. The fact that chlorpromazine has many pharmacologw actions in common with reserpine, without sharing its capacity to interfere with serotonin binding, would appear to detract seriously from the hypothesis of serotonin mediation. We are still in our biochemical infancy with respect to explaining such complex effects, but a substantial contribu- tion has been made by studies with 5-hydroxytryptophan, the biochemical precursor of serotonin. 5-Hydroxytryptophan, an amino acid, is not a compound which would be expected to ex- hibit pharmacologic activity. But Udenfriend et al. (3) have reported that it produces pharmacologic effects in dogs resem- bling those observed after LSD administration. What is even more provocative, the in vivo effects are apparently antagonized by ehlorpromazine. We are thus able to reconsider both chlor- EFFECTS OF INDOLES ON SYNAPTIC "IRAN_;.klISSION ]t_,) promazine and reserpine actions in connection with serotonin function, although clearly the processes in which each drug acts are distinct. It is encouraging to see how biochemical information on the separate steps involved in a complex biologic mechanism can serve to reconcile inconsistencies which appear only as the result of our own efforts toward simplification. Chairman Pennes: The hallucinogen, mescaline, administered intravenously or into the carotid artery, depresses the primary evoked response of optic cortex to photic or electric stimulation in the anesthetized cat. Although LSD inhibition occurs at the geniculate level, inhibition after mescaline is exerted at the cortical level because it is obtained in response to radiation stimulation. However, with optic nerve stimulation, the radiation spike remains intact when the remainder of the response is profoundly depressed by the drug. If Dr. Bishop, whom I see in the audience, still clings to his analysis that the first spike in the primary evoked response represents postgeniculate input to the cortex, this would also indicate that the locus of action of mescaline is mainly cortical. On the other hand, topically applied mescaline sulfate (0.1-0.5 per cent) in buffered solution produced no consistent change in the primary evoked response but did cause spontaneous cortical discharge and also facilitated repetitive after-discharge. Rovetta (2) has reported qualitatively similar results with mescaline in anesthetized eats. His work was done exclusively with photic stimulation. Intra- venously, the drug produced a 15 to 20 per cent diminution in both positive and total amplitude of cortical evoked responses. I observed this degree of inhibition with intravenous doses of 5 to 10 rag. per kilogram and with intracarotid doses of 05 to 1 mg. per kilogram (1). Higher intracarotid doses ('2_ mg./Kg.) usually produced an almost complete inhil_ition of the response, with the exception of the radiation spike. Rovetta likewise found a different effect with topical mescaline (.5-,'33per cent). These very high concentra';ons enornqouslv enhanced a "later" com- ponent of the evoked response which was otherwise not iden- tified; the effects in his figures bear a close resemblance to those noted by myself in which repetitive positive after-discharge _(X) PS YCIIOI'ItA R_ IA ( ;OLOG'_" tended to encroach upon the second phase of the evoked response. I/ovetta found no consistent effect on the "first" phase of the evoked response which usually doubled in the first stage of mescalinization and later might even be decreased in amplitude. Dr. Purpura: We have confirmed Dr. Bishop's story on the nature of the striate cortex response by using intravenously administered d-tubocurarine. Complete synaptie blockade produced by 8 mg. per kilogram of this agent abolishes all but the first brief spike record from the optic cortex when the radiations are stimulated. All subsequent spikes are therefore referable to intracortical synaptic activity. Chairman Pennes: In Dr. Evarts' nembntalized cats, I believe that even very large doses of I,SD either had no effect or occa- sionally facilitated the cortical response to optic radiation stimulation. This occurred in the presence of oMiteration of the spontaneous eleetrocortieal activity by the drug. Is it possible that the observed facilitation was an mmmsking of primary evoked cortical response in association with the depression of the spontaneous background activity? I believe Bremer has con- sidered this a mechanism in the auditor_ system. Dr. Evarts: Extremely large doses of"L,_D did not depress the cortical response to geniculate radiation stimulation. In some animals, there was an increase in this response following intracarotid administration of LSD in doses of 1 mg. per kilogram. At the same time, in nembutalized cats, the spontaneous cortical activity was obliterated by doses of LSD much smaller than those which failed to depress the cortieal response to genieulate radiation stimnlation. The complete obliteration of the spontaneous cortical activity which resulted from administration of large doses of LSD in nembutalized animals may have been in some measure related to tae augmentation of the cortical response which sometimes occurred following even larger (1 mg./Kg. ) doses of LSD. Chairman Pennes: So far as the observed genieulate depression by I,SD is concerned, would not an alteration in spontaneous electric aetivity by the drug be a factor to consider EFFECTS OF' INDOLES ON SYNAPTIC TRANSMISSION ;20] insofar as the locus of action of LSD is involved? Would it be conceivable that variation in tonic inflow to the lateral geniculate body accompanying the over-all change in the spontaneous EEG might depress geniculate excitability so that postsynaptie response would be suppressed without direct action of LSD at -. the geniculate body? Dr. Evarts: I don't think we have completely ruled out this possibility. There are several points against this hypothesis, however. Our studies in unanesthetized cats with chronicallv implanted electrodes have shown that LSD reduces the genicu- " late postsynaptic response without simultaneously obliterating spontaneous cortical activity. In these unanesthetized ,prepara- t_ions, even large doses of LSD (1 mg./Kg. ) failed to cause any consistent augmentation of the cortical response to geniculate radiation stimulation. The dosage of LSD required to cause a given reduction in the geniculate postsynaptic response is much greater in the conscious unanesthetized cat than in the nembutalized preparation. Chairman Pennes: You have observed depression of the geniculate postsynaptic response without change in the EEG, I take it. Dr. Evarts: In the conscious preparation, LSD (1 mg./Kg.) depresses the postsynaptie geniculate response without causing obliteration of spontaneous cortical activity. Following such doses of LSD, the record of spontaneous cortical activity gen- erally shows 8 to 5 per second slow activity, which is, howew,r, responsive to auditory stimulus. The slow waves may be transiently obliterated by a sudden auditory stimulus, with restoration of what appears to be a fairly normal pattern of spontaneous cortical activity. The degree of genieulate depression does not vary with these variations in the spontaneous cortical activity. Dr. Marrazzi: Bearing on the same poiut, I don't hear Dr. Grenel] speaking up but he has gotten LSD inhibition on the undercut cortex. Dr. Grenell: We have every reason to believe that in the locally perfused cortex, in whieil the Nembutal has been washed out with the control solution, and which is then perfused with 20 ° I)SYCHOPHARNIA(;OLOGY

LSD directly, wc get aI_ inhi})itory response in the area where the anesthetic has been washed out. If you undercut the cortex in the nembutalized animal you still get the marked inhibition of the response to systemically injected LSD. Dr. Evarts: I would like to make one final point concerning Dr. Marrazzi's comment on the degree to which our observations on some of the actions of several indoles may be viewed as a study of the relation of structure to activity. I can only say that I completely agree with Dr. Marrazzi's conclusion that our observations cannot be said to constitute a structure-activity study. Indeed, our study was not intended to be such. We did feel, however, that information concerning the action of some structurally related substances would be of value in getting at some notion of the patterns of activity which are relevant to the mechanism of action of agents such as LSD and bufotenine.

REFERENCES

1. Pennes, H. H. The effects of mescaline on evoked cortical optic responses in the anesthetized eat, in Proceedings of the tttrund Table on Lysergic Acid Diethylamixte and Mescali,u" i71Experi- mental Psychiatry. New York, Grune & Stratton, 1955, pp. 61-62. 2. Rovetta, P. Effect of mescaline and L.S.D. on evoked ,'_ponses, especially of the optic s'cstt.lrn Of the cat. Electroc*u'_7,qmlog. & Clin Ncurol_hysqo[. 8: 15. 1956. 3. I'denfriend, S., \Vcis,b_ i, H, and Bogdanski, D. F. Increase in tissuv ,,.rotonin following administration of its precursor 5-hy- dlox)'trvt)tophan. ] Biol. Cbe_. 224: 803, 1957.