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Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain
Brain Research, 94 (1975) 57-66
57
© ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands
INTERACTIONS BETWEEN LYSERGIC AClD DIETHYLAMIDE AND DOPAMINE-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN RAT BRAIN
KERN VON H U N G E N , SIDNEY ROBERTS AND DIANE F. HILL
Department of Biological Chemistry, School q[ Medicine and the Brain Research Institute, University qf California Center for the Health Sciences, Los Angeles, Calif. 90024 (U.S.A.) (Accepted March 13th, 1975)
SUMMARY
Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-bromo-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation ofadenylate cyclase activity by equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10#M concentration, D-LSD not only completely eradicated the response to 10 E~M dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80/~M) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1/~M D-LSD. Activation of striatal adenylate cyclase by either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the fl-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and, in relatively high concentration (1130 ~M), partially blocked the activation by l0/~M dopamine, but was without effect on the stimulation by 10/~MD-LSD. The present results indicate that serotonin antagonists, in general, are potent inhibitors of
58 catecholamine-induced stimulation of adenylate cyclase systems in brain celt-lrce preparations. In addition, these results, coupled with earlier findings on the capacity of D-LSD to interact with serotonin-sensitive adenylate cyclase systems from rat brain'S3,24 and other neural systemsTM, strongly suggest that this hallucinogenic agent is capable of acting as an agonist at central dopamine and serotonin receptors, as well as functioning as an antagonist at dopamine, norepinephrine, and serotonin receptors in the brain. I NTRODUCTION
Considerable evidence supports the concept that the psychotomimetic drug, Dlysergic acid diethylamide (D-LSD), may exert certain of its effects on behavior by interactions with serotonin receptors in the central nervous system1,5,1s,ag. This hypothesis is based principally on the chemical similarity between D-LSD and serotonin and the effects of this hallucinogenic agent on central2,4,~° and peripheral 9,2s serotonergic mechanisms. Recent investigations suggest that adenylate cyclase systems may be involved in these central actions of D-LSD. Thus, o-LSD has been shown to stimulate adenylate cyclase activity, as well as antagonize the activation of this enzyme system by serotonin, in cell-free preparations from rat brain colliculi23,z4 and cockroach ganglion ~6. Although adenylate cyclase activity in collicutar preparations from newborn rats was highly sensitive to serotonin, stimulation by low concentrations of D-LSD was relatively small and the serotonin-btocking action of this drug was incomplete ~4. These observations suggested that the hallucinogenic effects of D-LSD might also involve interactions with central receptors for neurotransmitters other than serotonin. The present investigations provide support for this concept by revealing that D-LSD and other serotonin antagonists have profound effects on catecholamine-sensitive adenylate cyclase systems in rat brain. A preliminary report of some of this work has appeared 26. MATERIALS AND METHODS
The methods employed for preparation of brain subcellular fractions and measurement of adenylate cyctase activity have been described in detail elsewhere22,2.L Brains were obtained from young adult male rats of an inbred Sprague-Dawley strain. These rats were approximately 6 weeks old and weighed about 200 g. Most of the experiments were carried out on particulate fractions from various brain regions prepared by centrifugation of the homogenized tissue at 10,0C0 × g for 20 min. Adenylate cyclase activity was determined from the conversion of [14C]ATP to cyclic AMP by a procedure 22 which was based on the method of Krishna et al, 13. The incubation medium (0.1 ml) contained the following components in the final concentrations indicated: 3.6 mM (0.5 /tCi) [8-14C]ATP, 5.0 mM MgC12, 1.0 mM cyclic AMP, 40 mM Tris-HC1 (pH 7.3 at 37 °C), 10 mM phosphoenolpyruvate, 4 /~g pyruvate kinase, 15 mM (NH4)eSO4 (added with pyruvate kinase), 20 mM caffeine, 0.1 mg bovine serum albumin, 0.2 mM EGTA, 5 #g phosphatidylserine and 50-1130
59 /zg brain protein. Incubation was conducted in air for 5 rain at 37 °C. Protein was measured by the method of Lowry et al. 14. Biogenic amines, psychotropic drugs and all related compounds used in these investigations were of the highest purity obtainable. Sigma supplied L-norepinephrine hydrochloride, dopamine hydrochloride, serotonin creatinine sulfate, and propranolol hydrochloride. The following compounds were obtained from Sandoz: D-LSD, L-lysergic acid diethylamide (L-LSD), 2-bromo-D-lysergic acid diethylamide bitartrate (BOL), 1-methyl-D-lysergic acid diethylamide (MLD), 1-methyl-D-lysergic acid butanolamide (UML), thioridazine hydrochloride and N,N-dimethyl-4-hydroxytryptamine (psilocin). California Corp. for Biochemical Research was the source of N,Ndimethyltryptamine monooxalate and N,N-dimethyl-5-hydroxytryptamine monooxalate hydrate (bufotenine). Mescaline monooxalate was obtained from Aldrich Chemical Company, cyproheptadine hydrochloride from Merck, Sharp and Dohme, chlorpromazine and trifluoperazine hydrochlorides from Smith, Kline and French, haloperidol from McNeil Labs and promethazine hydrochloride from Wyeth Labs. RESULTS
Previous investigations from this laboratory revealed that adenylate cyclase systems in cell-free preparations from cerebral cortex of the adult rat were highly responsive to norepinephrine and dopamine, but were quite insensitive to serotonin 22, 2.5. Nevertheless, the serotonin-blocking agents, D-LSD and BOL, in 10/JM concentration, completely abolished the stimulation of cerebral adenylate cyclase activity by 10/zM norepinephrine plus I 0/zM dopamine 26. Comparable results were obtained in the present studies when serotonin antagonists were tested against equimolar amounts of either norepinephrine or dopamine at concentrations of these amines which produced maximal stimulation of adenylate cyclase activity in particulate preparations from adult rat cerebral cortex (see ref. 22). Norepinephrine (1130/zM) and dopamine (IC0/~M) resulted in increases in cerebral adenylate cyclase activity of approximately 60% and 50 ~o, respectively (Table I). The addition of D-LSD, BOL, or UML, each in 100/~M concentration, obliterated or markedly reduced these responses. Catecholamine-sensitive adenylate cyclase systems in particulate preparations from the hippocampus of adult rats were also strongly inhibited by D-LSD. The cerebral and hippocampal adenylate cyclase systems closely resembled one another in their responses to the catecholamines. Parallel studies from this laboratory demonstrated that D-I_SD was capable of producing a small stimulation of adenylate cyclase activity, as well as partially inhibiting the response to serotonin, in particulate preparations from newborn rat colliculi23, 24. These collicular preparations appeared to be unusually rich in serotonin receptors and poor in catecholamine receptors for adenylate cyclase24. However, o-LSD evoked a considerably larger increase in adenylate cyclase activity in particulate preparations from corpus striatum of adult rats 26. These striatal preparations contain adenylate cyclase systems which respond to norepinephrine or dopamine with large increases in activity but are relatively unresponsive to serotonin (see below). Adenylate
60 TABLE 1 INFLUENCE OF LYSERG1C ACID DERIVATIVES ON ACTIVATION OF ADENYLATE CYCLASE B' t CATECHOLAMINFS IN PARTICULATE PREPARATIONS FROM CEREBRAL CORTEX AND HIPPOCAMPUS OF A D U L T RATS
']7he particulate preparation from the cerebral cortex was a crude mitochondrial fraction obtained by centrifuging the 1000 i< g supernatant fraction from the cerebral homogenate at 10,000 ;;i g for 20 min 22. The preparation from the hippocampus was obtained by centrifuging directly the hippocampal homogenate at 10,000 ;~: g for 20 min. Values for cyclic AMP formed are means J_ S.E. for triplicate samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the lysergic acid derivative. P values were calculated for the difference between the increase in cyclic AMP formed with only the catecholamine added and the increase with both the catecholamine and the lysergic acid derivative added. The catecholamines and lysergic acid derivatives were tested at 100 ltM concentration.
Lysergic acid derivative
Cyclic AMP Jbrmed (nm:~le~/mgprotein/h) Basal
bwrease with norepinephrine
Stimulation ( %)
Increase w i t h dopamine
Stimulation (%)
5.64 1.48 0.49 2.47
63 16 6 29
4.21 0.78 0.11 0.93
47 9** 1"* 11'*
49 1"**
2.85 ± 0.73 0.08 ~ 0.53
Cortex None D-LSD BOL UML
8.91 9.13 8.02 8.66
± ± ± ±
0.04 0.29 0.29 0.10
± 0.12 ± 0.30* :~::0.43* ± 0.11'
{_ 0.45 ± 0.44 ± 0.34 A: 0.40
Hippocampus None D-LSD
7.43 ± 0.67 8.53 ± 0.52
3.64 :L 0.69 0.08 ~_ 0.58
38 l***
* P < 0.001. ** P < 0.01. *** P << 0.02.
cyclase activity in particulate preparations from corpus striatum of the adult rat was quite sensitive to the stimulatory action of D-LSD, but was unaffected by even high concentrations of L-LSD (Table I I, exp. 1). Significant activation of the striatal enzyme system was obtained with 0.1 # M D-LSD (Fig. 1). Half maximal activation was obtained with about 0.5 # M D-LSD and maximal activation with 5-10 # M o-LSD. The maximal degree of stimulation of striatal adenylate cyclase activity was about 30~. The sensitivity of adenylate cyclase to dopamine in cell-free fractions from the rat corpus striatum has been well documented12,15, 25. In the present investigations, adenylate cyclase activity in particulate preparations from striatal tissue of the adult rat was stimulated about 5 0 ~ by l0 # M dopamine (Table II). Equimolar concentrations of the dopamine-blocking agents, haloperidol (exp. 2) and chlorpromazine (exp. 3) eradicated this response. Other dopamine-blocking agents (e.g., trifluoperazine or thioridazine) had similar actions (see also, ref. 7). However, the response of adenylate cyclase to l0 # M dopamine was also eliminated or markedly reduced by equimolar concentrations of D-LSD and several other serotonin antagonists, e.g., BOL, MLD and cyproheptadine (exp. 4). The dopamine-blocking potency of these serotonin antagonists was similar to that of the phenothiazine antagonists.
61
~= o
6
(aE •-= -~
3
v
2
I O.I
I 0.3
I I
I 2 LSD
I 5
I I0
I I 2030
I 50
I IOO
(/~M)
Fig. 1. Dose-response curve for adenylate cyclase activity in the 10,000 × g particulate preparation from corpus striatum of adult rats in the presence of D-LSD. Results are expressed as increase in cyclic AMP formation in response to various concentrations of D-LSD. Each value represents the mean :~ S.E. for triplicate samples. Basal adenylate cyclase activity was 24.73 ~ 0.43 nmoles/mg protein/h. Compounds other than D-LSD which blocked the response of adenylate cyclase to dopamine in particulate preparations of striatal tissue from adult rats also inhibited the response to D-LSD (Table II). These substances included the antipsychotic dopamine-blocking agents, haloperidol, chlorpromazine, thioridazine and trifluoperazine, as well as the serotonin antagonists, cyproheptadine, BOL and MLD. The fl-adrenergic blocking agent, propranolol, even when present in 10-fold greater concentration than dopamine or D-LSD, had no effect on the activation of striatal adenylate cyclase by these compounds. Promethazine and L-LSD, which have no antipsychotic action, also failed to block activation of this enzyme system by dopamine and D-LSD. No blocking agent other than D-LSD was in itself capable of stimulating striatal adenylate cyclase. Serotonin, i n concentrations of 10 or 100 # M , prod uced about l 0-15 % increase i n the activity of striatal adenylate cyclase and partially inhibited stimulation of this enzyme system by 10/~M dopamine but had no effect on the activation by 10 # M D-LSD (Table Ill, exp. 1). A very high concentration of serotonin (10C0 # M ) was required to produce a strong inhibition of the dopamine stimulation (exp. 2). Several other drugs related to serotonin which have hallucinogenic properties were also tested in this system. Mescaline, psilocin, dimethyltryptamine and bufotenine were all without effect on basal adenylate cyclase activity in particulate preparations from the corpus striatum of adult rats. However, like serotonin per se, the indoleamines, dimethyltryptamine and bufotenine, appeared to inhibit partially the response to dopamine in these preparations.
62 TABLE lI INFLUENCE OF BLOCKING AGENTS ON ACTIVATION OF ADENYLATE CYCLASE BY DOPAMINE AND
D-LSD
IN PARTICULATE PREPARATIONS FROM CORPUS STRIATUM OF A D U L T RATS
The particulate fractions were prepared by centrifuging striatal homogenates at 10,000 > g for 20 rain. Values for cyclic A M P formed are means i S.E. for 3-5 samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the blocking agent. P values were calculated for the difference between the increase in cyclic A M P formed with only 10 ~tM dopamine or 10/~M D-LSD and the increase with the blocking agent added as well.
Agent
Cyclic' AMP Jbrmed Basal Cnmoles/mg protein~h)
Increase with dopamine
Increase with D-LSD
nmoles/mg protein/h
%
nmoles/mg protein/h %
25.19 4- 0.52 31.08 A: 0.73 24.53 4- 0.40
12.41 4- 0.69 1.59 4- 0.95* 12.61 4- 0.71
49 5 51
5.89 4- 0.90
23
6.45 4- 0.68
26
24,06 30.35 24.59 23.95 24.20 25.64
~: 0.37 4- 0.33 + 0.90 4- 0.91 4- 0.53 4- 0.63
10.66 2.26 4.63 0.05 12.75 10.58
~ 1.12 4- 0.42** ± 1.02"** ± 1.00"* 4- 0.74 4- 0.75
44 7 19 2 53 41
6.29 £ 0.49
26
5.30 2.05 7.98 5.10
22 9 33 20
18.93 23.16 24.43 18.23 17.78 19.54 18.95 20.09 20.33 21.37 19.12
:t: 0.23 4- 0.30 4- 0.31 ± 0.24 4- 0.27 4- 0.50 4- 0.34 i 0.63 4- 0.56 i 0.47 4- 0.39
9.62 4.22 0.72 5.95 1.45 3.60 1.59 1.87 -- 0.81 8.06 8.75
i 0.28 ± 0.86** i 0.76* :t: 0.42** 4- 1.00"* 4- 0.62* 4- 0.42* 4- 0.67* ± 0.61" i 0.89 4- 0.50
51 18 3 33 ;3 I8 8 9 -- 4 38 46
23.87 29.09 23.35 22;3t 22.58 21.06 23.88 23.09
i 0.46 ± 0.27 4- 0.36 + 0.55 4- 0.32 4- 0.29 :t: 0.23 4- 0.16
11.89 1.93 0.51 0.48 1.28 1.46 1.14 1.20
4- 0.65 4- 0.40* 4- 0.51" 4- 1.07"* 4- 0.71" 4- 0.45* _4- 0.23* 4- 0.25*
50 7 2 2 6 7 5 5
Experiment 1 None o-LSD, 10 # M L-LSD, 80/~M
Experiment 2 None D-LSD, 10 ArM Haloperidol, 1 # M Haloperidol, 10 # M Propranolol, 10/~M Propranolol, 100/~M
± 4± 4-
0.97 1.18~ 0.90 0.86
Experiment 3 None D-LSD, 1/~M D-LSD, 10 # M Chlorpromazine, 1/~M Chlorpromazine, 10 p M Thioridazine, I / ~ M Thioridazine, 10 p M Trifluoperazine, 1 / z M Trifluoperazine, 1 0 / t M Promethazine, 1 p M Promethazine, 1 0 / t M
5.50 =3:0.39
29
5.49 2.42 5.38 1.42 3.84 1.08 4.61 6.61
30 I4 28 7 19 5 22 35
4- 0.57 4- 0.31"* 4- 0.68 ± 0.70** ± 0.67 4- 0.57** 4- 0.71 _% 0.57
Experiment 4 None D-LSD, 10 # M BOL, 10 p M BOL, 100 p M Cyproheptadine, 10 p M Cyproheptadine, 100/zM MLD, 10 p M MLD, 100 p M *
P < 0.001.
** P < 0.01. *** P < 0.02. P < 0.05.
5.22 ~ 0.53 3.19 0.94 1.85 1.72 4.31 1.54
=~: 0,41 ± 0.92"* :L 0.38** 4- 0.40** ± 0.38 4- 0.50~
22 !4 .... 4 8 8 8 7
63 TABLE III INFLUENCE OF SEROTONINON ACTIVATIONOF ADENYLATE CYCLASEBY DOPAMINEIN PARTICULATE PREPARATIONS FROM CORPUS STRIATUMOF ADULT RATS
The particulate fractions were prepared by centrifuging striatal homogenates at I0,000 x g for 20 rain. Values for cyclic AMP formed are means ± S.E. for 3 4 samples in a representative experiment. Where indicated, basal values shown were obtained in the presence of se, otonin. P values were calculated for the difference between the increase in cyclic AMP formed with only 10 ttM dopamine or D-LSD added and the increase with both 10 I~M dopamine or D-LSD and serotonin added.
Serotonin (/~M)
Basal (nmoles/mg protein/h)
Cyclic AMP formed Increase with D-LSD
Increase with dopamine nmoles/mg protein/h
%
nmoles/mg protein/h
%
23.87 ~ 0.46 27.59 ± 0.83 27.23 -E 0.52
11.89 ::E 0.65 10.18 ± 0.88 8.67 ± 0.61"
50 37 32
5.22 -3- 0.53 4.37 ±. 0.97 6.31 ± 0.85
22 16 23
23.84 26.33 25.82 25.79
14.60 12.52 11.83 3.31
61 48 46 13
Experiment 1 0 10 100
Experiment 2 0 10 100 1000
~ 0.48 ± 0.29 ± 0.49 :k: 0.22
:~: 0.68 ± 0.67 ± 0.57** ± 0.26***
* P < 0.05. ** P <~ 0.01. *** P <~ 0.001.
DISCUSSION
Earlier investigations from this laboratory revealed that D-LSD and several other serotonin antagonists significantly but incompletely blocked stimulation of adenylate cyclase activity by serotonin in cell-free preparations from the colliculus of newborn rats 23,24. The dopamine-blocking agent, chlorpromazine, had a similar effect in this serotonin-sensitive system. The present results indicate that serotonin antagonists block even more strongly the stimulation of adenylate cyclase activity by norepinephrine or dopamine in cell-free preparations from areas of the adult rat brain which are sensitive to these catecholamines; e.g., cerebral cortex, hippocampus and corpus striatum. Palmer and Burks 17 previously reported that D-LSD and BOL were potent blocking agents for the increase in cyclic AMP induced in rat brain slices by norepinephrine. The physiological significance of the blocking action of serotonin antagonists on catecholamine receptors for adenylate cyclase in the central nervous system is supported by the results of electrophysiological studies which have indicated that the effects of norepinephrine on central neural pathways can be inhibited by D-LSD 3. In our earlier investigations, several of the compounds which produced partial blockade of serotonin-induced stimulation of adenylate cyclase activity in particulate
64 fractions from rat colliculus were themselves capable of stimulating adenylate cyclase activity in these preparations z4. These compounds included chlorpromazine as well as the serotonin antagonists, D-LSD and cyproheptadine. In contrast, only D-LSD stimulated adenylate cyclase activity in catecholamine-sensitive areas of the rat brain in the present studies (see also ref. 26). The greatest stimulation was observed with cellfree preparations from the corpus striatum. This area of rat brain is replete with dopaminergic neurons 11 and contains adenylate cyclase systems which are markedly stimulated by dopamine 12,t~,'~5. These considerations and the fact that activation of adenylate cyclase by D-LSD in striatal preparations could be blocked by the dopamineblocking agents, chlorpromazine and haloperidol, but not by the norepinephrineblocking agent, propranolol, suggest that this stimulation was specifically mediated through dopamine receptors. This conclusion was reenforced by the finding that serotonin itself did not appear to interact strongly with catecholamine receptors tbr adenylate cyclase in rat brain. A high concentration of this indoleamine (1 mM) was required to produce a strong inhibition of the stimulation of striatal adenylate cyclase activity by dopamine. Weiss and Costa 27 earlier reported that this concentration of serotonin completely inhibited the stimulation of adenylate cyclase by norepinephrine in rat pineal homogenates. The present observations strongly suggest that central catecholamine receptors do not have the specificity formerly attributed to them. It is not possible from these results to decide whether more than one site of interaction exists on these receptors. However, the fact that stimulation of adenylate cyclase by D-LSD in dopaminesensitive areas of the brain was eradicated by dopamine-blocking agents, as well as by serotonin-blocking agents, suggests that dopamine and D-LSD interact with the same receptor at the same site. The capacity of D-LSD to stimulate adenylate cyclase activity in brain was first suggested by the findings of Uzunov and Weiss 21. These investigators reported that D-LSD increased cyclic AMP levels in rat brain in vivo. The possibility that the stimulatory action of D-LSD was mediated by catecholamine receptors was supported by the observation that this action was prevented by administration of the phenothiazine tranquillizer, trifluoperazine21. Trifluoperazine was also capable of inhibiting the norepinephrine-induced accumulation of cyclic AMP in rat brain slices and pineal homogenates~°, as well as the stimulation of adenylate cyclase activity by dopamine in homogenates of rat corpus striatum 7,15 (this paper). The present investigations provide direct evidence that D-LSD can stimulate dopamine receptors and may thereby activate dopaminergic mechanisms in the brain. This conclusion is consistent with the observation of Fogs that D-LSD produced stereotypy in rats, a condition associated with dopaminergic stimulation6. In addition, our results indicate that D-LSD is capable of blocking the interaction of dopamine and norepinephrine with their specific receptors. These investigations, along with earlier findings from this laboratory 24,28, suggest that the psychotomimetic actions of D-LSD may be related to complex interactions of this drug with central receptors for serotonin, dopamine and norepinephrine, rather than a single action on one neurotransmitter system.
65 ACKNOWLEDGEMENTS This research was supported Foundation
by research grants from the National
(GB-30300) and the National
Institute of Neurological
Science
Diseases and
Stroke, U.S. Public Health Service (NS-07869).
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66 20 UZUNOV, P., AND WEISS, B., Effects of phenothiazine tranquillizers on the cyclic 3',5"-adenosine monophosphate system of rat brain, Neuropkarmacology, 10 (1971) 697-708. 21 UzuNov, P., AND WEISS, B., Psychopharmacological agents and the cyclic AMP system of r~tt brain. In P. GREEN6ARD, R. PAOLETTIAND G. A. RoBIsON (Eds.), Advanc. Cyclic Nucleotide Re.~., Vol. 1, Raven Press, New York, 1972, pp. 435-453. 22 VON HUNGEN, K., AND ROBERTS,S., Adenylate-cyclase receptors for adrenergic neurotransmitters in rat cerebral cortex, Europ. J. Biockem., 36 (1973) 391-401. 23 VON HUNGEN,K., ROBERTS,S., AND HILL, D. F., Serotonin and catecholamine activation of brain adenylate cyclase: effects of psychoactive drugs, Trans. Amer. Soc. Neurockem., 5 (1974) 150. 24 VON HUNGEN, K., ROBERTS, S., AND HILL, D. F., Serotonin-sensitive adenylate cyclase activity in immature rat brain, Brain Researck, 84 (1975) 257-267. 25 VON HUNGEN, K., ROBERTS, S., AND HILL, D. F., Developmental and regional variations in neurotransmitter-sensitive adenylate cyclase systems in cell-free preparations from rat brain, J. Neuroekem., 22 (1974) 811-819. 26 VON HUNGEN, K., ROBERTS, S., AND HILL, D. F., LSD as an agonist and antagonist at central dopamine receptors, Nature (Lond.), 252 (1974) 588-589. 27 WEISS, B., AND COSTA, E., Selective stimulation of adenyl cyclase of rat pineal gland by pharmacologically active catecholamines, J. Pkarmacol. exp. Tker., 161 (1968)310-319. 28 WOOLLEV,D. W., AND SHAW, E., A biochemical and pharmacological suggestion about certain mental disorders, Proc. nat. Acad. Sci. (Wask.), 40 (1954) 228-231.
57
© ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands
INTERACTIONS BETWEEN LYSERGIC AClD DIETHYLAMIDE AND DOPAMINE-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN RAT BRAIN
KERN VON H U N G E N , SIDNEY ROBERTS AND DIANE F. HILL
Department of Biological Chemistry, School q[ Medicine and the Brain Research Institute, University qf California Center for the Health Sciences, Los Angeles, Calif. 90024 (U.S.A.) (Accepted March 13th, 1975)
SUMMARY
Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-bromo-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation ofadenylate cyclase activity by equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10#M concentration, D-LSD not only completely eradicated the response to 10 E~M dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80/~M) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1/~M D-LSD. Activation of striatal adenylate cyclase by either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the fl-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and, in relatively high concentration (1130 ~M), partially blocked the activation by l0/~M dopamine, but was without effect on the stimulation by 10/~MD-LSD. The present results indicate that serotonin antagonists, in general, are potent inhibitors of
58 catecholamine-induced stimulation of adenylate cyclase systems in brain celt-lrce preparations. In addition, these results, coupled with earlier findings on the capacity of D-LSD to interact with serotonin-sensitive adenylate cyclase systems from rat brain'S3,24 and other neural systemsTM, strongly suggest that this hallucinogenic agent is capable of acting as an agonist at central dopamine and serotonin receptors, as well as functioning as an antagonist at dopamine, norepinephrine, and serotonin receptors in the brain. I NTRODUCTION
Considerable evidence supports the concept that the psychotomimetic drug, Dlysergic acid diethylamide (D-LSD), may exert certain of its effects on behavior by interactions with serotonin receptors in the central nervous system1,5,1s,ag. This hypothesis is based principally on the chemical similarity between D-LSD and serotonin and the effects of this hallucinogenic agent on central2,4,~° and peripheral 9,2s serotonergic mechanisms. Recent investigations suggest that adenylate cyclase systems may be involved in these central actions of D-LSD. Thus, o-LSD has been shown to stimulate adenylate cyclase activity, as well as antagonize the activation of this enzyme system by serotonin, in cell-free preparations from rat brain colliculi23,z4 and cockroach ganglion ~6. Although adenylate cyclase activity in collicutar preparations from newborn rats was highly sensitive to serotonin, stimulation by low concentrations of D-LSD was relatively small and the serotonin-btocking action of this drug was incomplete ~4. These observations suggested that the hallucinogenic effects of D-LSD might also involve interactions with central receptors for neurotransmitters other than serotonin. The present investigations provide support for this concept by revealing that D-LSD and other serotonin antagonists have profound effects on catecholamine-sensitive adenylate cyclase systems in rat brain. A preliminary report of some of this work has appeared 26. MATERIALS AND METHODS
The methods employed for preparation of brain subcellular fractions and measurement of adenylate cyctase activity have been described in detail elsewhere22,2.L Brains were obtained from young adult male rats of an inbred Sprague-Dawley strain. These rats were approximately 6 weeks old and weighed about 200 g. Most of the experiments were carried out on particulate fractions from various brain regions prepared by centrifugation of the homogenized tissue at 10,0C0 × g for 20 min. Adenylate cyclase activity was determined from the conversion of [14C]ATP to cyclic AMP by a procedure 22 which was based on the method of Krishna et al, 13. The incubation medium (0.1 ml) contained the following components in the final concentrations indicated: 3.6 mM (0.5 /tCi) [8-14C]ATP, 5.0 mM MgC12, 1.0 mM cyclic AMP, 40 mM Tris-HC1 (pH 7.3 at 37 °C), 10 mM phosphoenolpyruvate, 4 /~g pyruvate kinase, 15 mM (NH4)eSO4 (added with pyruvate kinase), 20 mM caffeine, 0.1 mg bovine serum albumin, 0.2 mM EGTA, 5 #g phosphatidylserine and 50-1130
59 /zg brain protein. Incubation was conducted in air for 5 rain at 37 °C. Protein was measured by the method of Lowry et al. 14. Biogenic amines, psychotropic drugs and all related compounds used in these investigations were of the highest purity obtainable. Sigma supplied L-norepinephrine hydrochloride, dopamine hydrochloride, serotonin creatinine sulfate, and propranolol hydrochloride. The following compounds were obtained from Sandoz: D-LSD, L-lysergic acid diethylamide (L-LSD), 2-bromo-D-lysergic acid diethylamide bitartrate (BOL), 1-methyl-D-lysergic acid diethylamide (MLD), 1-methyl-D-lysergic acid butanolamide (UML), thioridazine hydrochloride and N,N-dimethyl-4-hydroxytryptamine (psilocin). California Corp. for Biochemical Research was the source of N,Ndimethyltryptamine monooxalate and N,N-dimethyl-5-hydroxytryptamine monooxalate hydrate (bufotenine). Mescaline monooxalate was obtained from Aldrich Chemical Company, cyproheptadine hydrochloride from Merck, Sharp and Dohme, chlorpromazine and trifluoperazine hydrochlorides from Smith, Kline and French, haloperidol from McNeil Labs and promethazine hydrochloride from Wyeth Labs. RESULTS
Previous investigations from this laboratory revealed that adenylate cyclase systems in cell-free preparations from cerebral cortex of the adult rat were highly responsive to norepinephrine and dopamine, but were quite insensitive to serotonin 22, 2.5. Nevertheless, the serotonin-blocking agents, D-LSD and BOL, in 10/JM concentration, completely abolished the stimulation of cerebral adenylate cyclase activity by 10/zM norepinephrine plus I 0/zM dopamine 26. Comparable results were obtained in the present studies when serotonin antagonists were tested against equimolar amounts of either norepinephrine or dopamine at concentrations of these amines which produced maximal stimulation of adenylate cyclase activity in particulate preparations from adult rat cerebral cortex (see ref. 22). Norepinephrine (1130/zM) and dopamine (IC0/~M) resulted in increases in cerebral adenylate cyclase activity of approximately 60% and 50 ~o, respectively (Table I). The addition of D-LSD, BOL, or UML, each in 100/~M concentration, obliterated or markedly reduced these responses. Catecholamine-sensitive adenylate cyclase systems in particulate preparations from the hippocampus of adult rats were also strongly inhibited by D-LSD. The cerebral and hippocampal adenylate cyclase systems closely resembled one another in their responses to the catecholamines. Parallel studies from this laboratory demonstrated that D-I_SD was capable of producing a small stimulation of adenylate cyclase activity, as well as partially inhibiting the response to serotonin, in particulate preparations from newborn rat colliculi23, 24. These collicular preparations appeared to be unusually rich in serotonin receptors and poor in catecholamine receptors for adenylate cyclase24. However, o-LSD evoked a considerably larger increase in adenylate cyclase activity in particulate preparations from corpus striatum of adult rats 26. These striatal preparations contain adenylate cyclase systems which respond to norepinephrine or dopamine with large increases in activity but are relatively unresponsive to serotonin (see below). Adenylate
60 TABLE 1 INFLUENCE OF LYSERG1C ACID DERIVATIVES ON ACTIVATION OF ADENYLATE CYCLASE B' t CATECHOLAMINFS IN PARTICULATE PREPARATIONS FROM CEREBRAL CORTEX AND HIPPOCAMPUS OF A D U L T RATS
']7he particulate preparation from the cerebral cortex was a crude mitochondrial fraction obtained by centrifuging the 1000 i< g supernatant fraction from the cerebral homogenate at 10,000 ;;i g for 20 min 22. The preparation from the hippocampus was obtained by centrifuging directly the hippocampal homogenate at 10,000 ;~: g for 20 min. Values for cyclic AMP formed are means J_ S.E. for triplicate samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the lysergic acid derivative. P values were calculated for the difference between the increase in cyclic AMP formed with only the catecholamine added and the increase with both the catecholamine and the lysergic acid derivative added. The catecholamines and lysergic acid derivatives were tested at 100 ltM concentration.
Lysergic acid derivative
Cyclic AMP Jbrmed (nm:~le~/mgprotein/h) Basal
bwrease with norepinephrine
Stimulation ( %)
Increase w i t h dopamine
Stimulation (%)
5.64 1.48 0.49 2.47
63 16 6 29
4.21 0.78 0.11 0.93
47 9** 1"* 11'*
49 1"**
2.85 ± 0.73 0.08 ~ 0.53
Cortex None D-LSD BOL UML
8.91 9.13 8.02 8.66
± ± ± ±
0.04 0.29 0.29 0.10
± 0.12 ± 0.30* :~::0.43* ± 0.11'
{_ 0.45 ± 0.44 ± 0.34 A: 0.40
Hippocampus None D-LSD
7.43 ± 0.67 8.53 ± 0.52
3.64 :L 0.69 0.08 ~_ 0.58
38 l***
* P < 0.001. ** P < 0.01. *** P << 0.02.
cyclase activity in particulate preparations from corpus striatum of the adult rat was quite sensitive to the stimulatory action of D-LSD, but was unaffected by even high concentrations of L-LSD (Table I I, exp. 1). Significant activation of the striatal enzyme system was obtained with 0.1 # M D-LSD (Fig. 1). Half maximal activation was obtained with about 0.5 # M D-LSD and maximal activation with 5-10 # M o-LSD. The maximal degree of stimulation of striatal adenylate cyclase activity was about 30~. The sensitivity of adenylate cyclase to dopamine in cell-free fractions from the rat corpus striatum has been well documented12,15, 25. In the present investigations, adenylate cyclase activity in particulate preparations from striatal tissue of the adult rat was stimulated about 5 0 ~ by l0 # M dopamine (Table II). Equimolar concentrations of the dopamine-blocking agents, haloperidol (exp. 2) and chlorpromazine (exp. 3) eradicated this response. Other dopamine-blocking agents (e.g., trifluoperazine or thioridazine) had similar actions (see also, ref. 7). However, the response of adenylate cyclase to l0 # M dopamine was also eliminated or markedly reduced by equimolar concentrations of D-LSD and several other serotonin antagonists, e.g., BOL, MLD and cyproheptadine (exp. 4). The dopamine-blocking potency of these serotonin antagonists was similar to that of the phenothiazine antagonists.
61
~= o
6
(aE •-= -~
3
v
2
I O.I
I 0.3
I I
I 2 LSD
I 5
I I0
I I 2030
I 50
I IOO
(/~M)
Fig. 1. Dose-response curve for adenylate cyclase activity in the 10,000 × g particulate preparation from corpus striatum of adult rats in the presence of D-LSD. Results are expressed as increase in cyclic AMP formation in response to various concentrations of D-LSD. Each value represents the mean :~ S.E. for triplicate samples. Basal adenylate cyclase activity was 24.73 ~ 0.43 nmoles/mg protein/h. Compounds other than D-LSD which blocked the response of adenylate cyclase to dopamine in particulate preparations of striatal tissue from adult rats also inhibited the response to D-LSD (Table II). These substances included the antipsychotic dopamine-blocking agents, haloperidol, chlorpromazine, thioridazine and trifluoperazine, as well as the serotonin antagonists, cyproheptadine, BOL and MLD. The fl-adrenergic blocking agent, propranolol, even when present in 10-fold greater concentration than dopamine or D-LSD, had no effect on the activation of striatal adenylate cyclase by these compounds. Promethazine and L-LSD, which have no antipsychotic action, also failed to block activation of this enzyme system by dopamine and D-LSD. No blocking agent other than D-LSD was in itself capable of stimulating striatal adenylate cyclase. Serotonin, i n concentrations of 10 or 100 # M , prod uced about l 0-15 % increase i n the activity of striatal adenylate cyclase and partially inhibited stimulation of this enzyme system by 10/~M dopamine but had no effect on the activation by 10 # M D-LSD (Table Ill, exp. 1). A very high concentration of serotonin (10C0 # M ) was required to produce a strong inhibition of the dopamine stimulation (exp. 2). Several other drugs related to serotonin which have hallucinogenic properties were also tested in this system. Mescaline, psilocin, dimethyltryptamine and bufotenine were all without effect on basal adenylate cyclase activity in particulate preparations from the corpus striatum of adult rats. However, like serotonin per se, the indoleamines, dimethyltryptamine and bufotenine, appeared to inhibit partially the response to dopamine in these preparations.
62 TABLE lI INFLUENCE OF BLOCKING AGENTS ON ACTIVATION OF ADENYLATE CYCLASE BY DOPAMINE AND
D-LSD
IN PARTICULATE PREPARATIONS FROM CORPUS STRIATUM OF A D U L T RATS
The particulate fractions were prepared by centrifuging striatal homogenates at 10,000 > g for 20 rain. Values for cyclic A M P formed are means i S.E. for 3-5 samples in representative experiments. Where indicated, basal values shown were obtained in the presence of the blocking agent. P values were calculated for the difference between the increase in cyclic A M P formed with only 10 ~tM dopamine or 10/~M D-LSD and the increase with the blocking agent added as well.
Agent
Cyclic' AMP Jbrmed Basal Cnmoles/mg protein~h)
Increase with dopamine
Increase with D-LSD
nmoles/mg protein/h
%
nmoles/mg protein/h %
25.19 4- 0.52 31.08 A: 0.73 24.53 4- 0.40
12.41 4- 0.69 1.59 4- 0.95* 12.61 4- 0.71
49 5 51
5.89 4- 0.90
23
6.45 4- 0.68
26
24,06 30.35 24.59 23.95 24.20 25.64
~: 0.37 4- 0.33 + 0.90 4- 0.91 4- 0.53 4- 0.63
10.66 2.26 4.63 0.05 12.75 10.58
~ 1.12 4- 0.42** ± 1.02"** ± 1.00"* 4- 0.74 4- 0.75
44 7 19 2 53 41
6.29 £ 0.49
26
5.30 2.05 7.98 5.10
22 9 33 20
18.93 23.16 24.43 18.23 17.78 19.54 18.95 20.09 20.33 21.37 19.12
:t: 0.23 4- 0.30 4- 0.31 ± 0.24 4- 0.27 4- 0.50 4- 0.34 i 0.63 4- 0.56 i 0.47 4- 0.39
9.62 4.22 0.72 5.95 1.45 3.60 1.59 1.87 -- 0.81 8.06 8.75
i 0.28 ± 0.86** i 0.76* :t: 0.42** 4- 1.00"* 4- 0.62* 4- 0.42* 4- 0.67* ± 0.61" i 0.89 4- 0.50
51 18 3 33 ;3 I8 8 9 -- 4 38 46
23.87 29.09 23.35 22;3t 22.58 21.06 23.88 23.09
i 0.46 ± 0.27 4- 0.36 + 0.55 4- 0.32 4- 0.29 :t: 0.23 4- 0.16
11.89 1.93 0.51 0.48 1.28 1.46 1.14 1.20
4- 0.65 4- 0.40* 4- 0.51" 4- 1.07"* 4- 0.71" 4- 0.45* _4- 0.23* 4- 0.25*
50 7 2 2 6 7 5 5
Experiment 1 None o-LSD, 10 # M L-LSD, 80/~M
Experiment 2 None D-LSD, 10 ArM Haloperidol, 1 # M Haloperidol, 10 # M Propranolol, 10/~M Propranolol, 100/~M
± 4± 4-
0.97 1.18~ 0.90 0.86
Experiment 3 None D-LSD, 1/~M D-LSD, 10 # M Chlorpromazine, 1/~M Chlorpromazine, 10 p M Thioridazine, I / ~ M Thioridazine, 10 p M Trifluoperazine, 1 / z M Trifluoperazine, 1 0 / t M Promethazine, 1 p M Promethazine, 1 0 / t M
5.50 =3:0.39
29
5.49 2.42 5.38 1.42 3.84 1.08 4.61 6.61
30 I4 28 7 19 5 22 35
4- 0.57 4- 0.31"* 4- 0.68 ± 0.70** ± 0.67 4- 0.57** 4- 0.71 _% 0.57
Experiment 4 None D-LSD, 10 # M BOL, 10 p M BOL, 100 p M Cyproheptadine, 10 p M Cyproheptadine, 100/zM MLD, 10 p M MLD, 100 p M *
P < 0.001.
** P < 0.01. *** P < 0.02. P < 0.05.
5.22 ~ 0.53 3.19 0.94 1.85 1.72 4.31 1.54
=~: 0,41 ± 0.92"* :L 0.38** 4- 0.40** ± 0.38 4- 0.50~
22 !4 .... 4 8 8 8 7
63 TABLE III INFLUENCE OF SEROTONINON ACTIVATIONOF ADENYLATE CYCLASEBY DOPAMINEIN PARTICULATE PREPARATIONS FROM CORPUS STRIATUMOF ADULT RATS
The particulate fractions were prepared by centrifuging striatal homogenates at I0,000 x g for 20 rain. Values for cyclic AMP formed are means ± S.E. for 3 4 samples in a representative experiment. Where indicated, basal values shown were obtained in the presence of se, otonin. P values were calculated for the difference between the increase in cyclic AMP formed with only 10 ttM dopamine or D-LSD added and the increase with both 10 I~M dopamine or D-LSD and serotonin added.
Serotonin (/~M)
Basal (nmoles/mg protein/h)
Cyclic AMP formed Increase with D-LSD
Increase with dopamine nmoles/mg protein/h
%
nmoles/mg protein/h
%
23.87 ~ 0.46 27.59 ± 0.83 27.23 -E 0.52
11.89 ::E 0.65 10.18 ± 0.88 8.67 ± 0.61"
50 37 32
5.22 -3- 0.53 4.37 ±. 0.97 6.31 ± 0.85
22 16 23
23.84 26.33 25.82 25.79
14.60 12.52 11.83 3.31
61 48 46 13
Experiment 1 0 10 100
Experiment 2 0 10 100 1000
~ 0.48 ± 0.29 ± 0.49 :k: 0.22
:~: 0.68 ± 0.67 ± 0.57** ± 0.26***
* P < 0.05. ** P <~ 0.01. *** P <~ 0.001.
DISCUSSION
Earlier investigations from this laboratory revealed that D-LSD and several other serotonin antagonists significantly but incompletely blocked stimulation of adenylate cyclase activity by serotonin in cell-free preparations from the colliculus of newborn rats 23,24. The dopamine-blocking agent, chlorpromazine, had a similar effect in this serotonin-sensitive system. The present results indicate that serotonin antagonists block even more strongly the stimulation of adenylate cyclase activity by norepinephrine or dopamine in cell-free preparations from areas of the adult rat brain which are sensitive to these catecholamines; e.g., cerebral cortex, hippocampus and corpus striatum. Palmer and Burks 17 previously reported that D-LSD and BOL were potent blocking agents for the increase in cyclic AMP induced in rat brain slices by norepinephrine. The physiological significance of the blocking action of serotonin antagonists on catecholamine receptors for adenylate cyclase in the central nervous system is supported by the results of electrophysiological studies which have indicated that the effects of norepinephrine on central neural pathways can be inhibited by D-LSD 3. In our earlier investigations, several of the compounds which produced partial blockade of serotonin-induced stimulation of adenylate cyclase activity in particulate
64 fractions from rat colliculus were themselves capable of stimulating adenylate cyclase activity in these preparations z4. These compounds included chlorpromazine as well as the serotonin antagonists, D-LSD and cyproheptadine. In contrast, only D-LSD stimulated adenylate cyclase activity in catecholamine-sensitive areas of the rat brain in the present studies (see also ref. 26). The greatest stimulation was observed with cellfree preparations from the corpus striatum. This area of rat brain is replete with dopaminergic neurons 11 and contains adenylate cyclase systems which are markedly stimulated by dopamine 12,t~,'~5. These considerations and the fact that activation of adenylate cyclase by D-LSD in striatal preparations could be blocked by the dopamineblocking agents, chlorpromazine and haloperidol, but not by the norepinephrineblocking agent, propranolol, suggest that this stimulation was specifically mediated through dopamine receptors. This conclusion was reenforced by the finding that serotonin itself did not appear to interact strongly with catecholamine receptors tbr adenylate cyclase in rat brain. A high concentration of this indoleamine (1 mM) was required to produce a strong inhibition of the stimulation of striatal adenylate cyclase activity by dopamine. Weiss and Costa 27 earlier reported that this concentration of serotonin completely inhibited the stimulation of adenylate cyclase by norepinephrine in rat pineal homogenates. The present observations strongly suggest that central catecholamine receptors do not have the specificity formerly attributed to them. It is not possible from these results to decide whether more than one site of interaction exists on these receptors. However, the fact that stimulation of adenylate cyclase by D-LSD in dopaminesensitive areas of the brain was eradicated by dopamine-blocking agents, as well as by serotonin-blocking agents, suggests that dopamine and D-LSD interact with the same receptor at the same site. The capacity of D-LSD to stimulate adenylate cyclase activity in brain was first suggested by the findings of Uzunov and Weiss 21. These investigators reported that D-LSD increased cyclic AMP levels in rat brain in vivo. The possibility that the stimulatory action of D-LSD was mediated by catecholamine receptors was supported by the observation that this action was prevented by administration of the phenothiazine tranquillizer, trifluoperazine21. Trifluoperazine was also capable of inhibiting the norepinephrine-induced accumulation of cyclic AMP in rat brain slices and pineal homogenates~°, as well as the stimulation of adenylate cyclase activity by dopamine in homogenates of rat corpus striatum 7,15 (this paper). The present investigations provide direct evidence that D-LSD can stimulate dopamine receptors and may thereby activate dopaminergic mechanisms in the brain. This conclusion is consistent with the observation of Fogs that D-LSD produced stereotypy in rats, a condition associated with dopaminergic stimulation6. In addition, our results indicate that D-LSD is capable of blocking the interaction of dopamine and norepinephrine with their specific receptors. These investigations, along with earlier findings from this laboratory 24,28, suggest that the psychotomimetic actions of D-LSD may be related to complex interactions of this drug with central receptors for serotonin, dopamine and norepinephrine, rather than a single action on one neurotransmitter system.
65 ACKNOWLEDGEMENTS This research was supported Foundation
by research grants from the National
(GB-30300) and the National
Institute of Neurological
Science
Diseases and
Stroke, U.S. Public Health Service (NS-07869).
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