What dopamine does in the brainSolomon H. Snyder1

The Solomon H. Snyder Department of Neuroscience, Department of Pharmacology and Molecular Sciences, and Department of Psychiatryand Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom and approved September 20, 2011 (received for review September 1, 2011)

In the early 1970s, receptors for neurotransmitters acting via second messengers had not been identified biochemically nor were theredefinitive links to such messengers. The discovery by John W. Kebabian and Paul Greengard of a dopamine-sensitive adenyl cyclase,accordingly, was a giant step forward. The investigators first characterized the enzyme in sympathetic ganglia wherein dopamine-producing cells link pre- and post-synaptic neurons. Then, in the corpus striatum, the brain area enriched in dopamine, they delineated theenzyme’s properties and showed that it was inhibited by antipsychotic drugs, leading to a large body of research on dopamine as amediatorof antipsychotic drug action and putative roles for this transmitter in the pathophysiology of schizophrenia.

In the early 1970s, a reasonableamount was known about the bio-chemistry of neurotransmitters. Afew chemical transmitters were ac-

cepted—acetylcholine, norepinephrine,serotonin, and GABA. There were hintsthat certain common amino acids such asglutamate and glycine, besides their rolesin general metabolism and protein syn-thesis, might also be neurotransmitters.However, this hint was still a controversialarea. In 1970, Chang and Leeman (1)had isolated and sequenced Substance P inthe brain as a peptide that stimulates sal-ivary secretion, but there was little else toargue for peptides as transmitters. Theexplosion of peptide transmitters thatcommenced with the isolation of enke-phalins was several years in the offing (2).Gases such as nitric oxide, carbon mon-oxide, and hydrogen sulfide as endogenousbiologically active substances were noteven fantasized.The biosynthesis of acetylcholine, sero-

tonin, norepinephrine, and GABA hadbeen elucidated, and their degradativepathways had been established. Their re-lease by exocytosis was generally accepted.Synaptic inactivation by an enzyme in thecase of acetylcholine and for amines andamino acids, by reuptake into the nerveendings that had released them seemed onsolid ground. However, molecular charac-terization of the most important element ofsynaptic transmission, transmitter receptors,was still unattained, at least in the brain.It was generally assumed that neuro-

transmitters bound to receptor proteins onpostsynaptic membranes. Several inves-tigators had identified the receptor proteinfor the actions of acetylcholine in theelectric organ of the electric eel throughthe binding of radiolabeled α-bungarotoxin(3). However, the receptor in these elec-tric organs comprises 20% of membraneprotein, about 1 million times as dense aswhat would be expected in the brain. Thus,few investigators anticipated ever under-standing neurotransmitter receptors inthe brain at a biochemical level, and theidentification by ligand binding of neuro-

transmitter and drug receptors in the mid-1970s came as a surprise (4).For neurotransmitters that act by

opening ion channels, such as acetylcholineat the neuromuscular junction, transmitterrecognition was presumed to be trans-formed into an opening or closing of ionchannels. For the biogenic amines, such asnorepinephrine and serotonin, the picturewas murkier. Hence, the work of Kebabianand Greengard (5) and Kebabian et al. (6)(Fig. 1) implying that dopamine in thesuperior cervical ganglion and the brain’scaudate nucleus acts through a receptorcoupled to a cAMP-forming enzyme—ad-enylate cyclase—was a giant step forward.How did Kebabian and Greengard (5)

and Kebabian et al. (6) come to exploreadenylate cyclase as a target for the syn-aptic effects of a neurotransmitter? Thedeep background for this work comes fromthe pioneering efforts of Sutherland andRall (7) that discovered cAMP as a secondmessenger molecule for a number of hor-mones. Sutherland and Rall (7) thenidentified adenylate cyclase as a cAMP-forming enzyme, which could be activatedby hormones, each with its own distinctivereceptor coupled somehow to a unitaryadenylate cyclase.

Why did Kebabian and Greengard (5)and Kebabian et al. (6) choose to exploredopamine rather than a better character-ized transmitter such as norepinephrine orserotonin? Dopamine is the metabolicprecursor of norepinephrine, differingonly in the absence of a hydroxyl at theβ-position, which is added by the enzymedopamine β- hydroxylase (Fig. 2). Specu-lation that dopamine might be a biolog-ically active molecule in its own right camein the late 1950s when Carlsson et al.(8) used the recently developed spec-trophotofluorometric techniques for mea-suring biogenic amines to uncover ex-tremely high concentrations of dopaminein the caudate nucleus of the brain, vastlygreater than norepinephrine levels in thisregion. Dopamine became clinically rele-vant when Hornykiewicz (9) discoveredits depletion in the caudate nucleus of

Fig. 1. Photographs of John W. Kebabian (Left) and Paul Greengard (Right) in the 1970s at about thetime of their pioneering work on dopamine-activating adenylate cyclase.

Author contributions: S.H.S. wrote the paper.

The author declares no conflict of interest.

This article is a PNAS Direct Submission.

See Classic Article “Dopamine-sensitive adenylate cyclasein caudate nucleus of rat brain, and its similarity to the‘dopamine receptor’” on page 2145 in issue 8 of volume 69.

See Classic Profile on page 18872.1E-mail: ssnyder@jhmi.edu.

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patients with Parkinson’s disease, whereasi.v. administration of L-dihydroxyphenyl-alanine, the amino acid precursor of dopa-mine, dramatically and rapidly alleviatedParkinsonian symptoms.The path of Kebabian and Greengard

(5) and Kebabian et al. (6) to character-izing synaptic actions of dopamine beganin a peripheral system, the superior cervi-cal ganglion, in which the presynapticneuron uses acetylcholine as its trans-mitter, whereas the postsynaptic neuronemploys norepinephrine. Besides rapidexcitatory transmission from presynapticto postsynaptic neuron in this ganglion,neurophysiologists had identified a slowhyperpolarizing inhibitory component thatseemed to involve an interneuron. Histo-chemical studies revealed dopamine inthese interneurons, suggesting that itmight have a transmitter function. Theinterest of Kebabian and Greengard (5)and Kebabian et al. (6) in cAMP followedthe discovery that the cAMP-dependentprotein kinase, discovered by Krebs (10)and associated as a mediator of hormoneeffects (10), also was prominent in thebrain (11).

The protein kinase was at least two stepsremoved from the neurotransmitter.Hence, to link cAMP to neurotransmitterswould require monitoring cAMP levels,which was a tedious procedure in smallganglia at that time. Because of his back-ground as a neurophysiologist, Greengardfocused on the hyperpolarizing influencesof presumed interneurons in the superiorcervical ganglion. First, he showed thatpreganglionic stimulation increases cAMPlevels several fold. At this point, he had noreason to favor dopamine over norepi-nephrine as the transmitter, because gan-glionic levels of the two catecholamines areabout the same. In slices of the ganglion,dopamine increased cAMP levels withsubstantially greater potency than norepi-nephrine. In homogenates, dopamine andnorepinephrine increased adenylate cy-clase activity to a similar extent. In theganglion, norepinephrine was well-estab-lished as the principal catecholamine in thepostganglionic cells, but histochemicalstudies had revealed that dopamine, ratherthan norepinephrine, predominated in theinterneurons. Based on these findings,Kebabian and Greengard (5) cautiouslyconcluded that “the physiologic effects of

dopamine in the ganglion, and possiblyelsewhere in the nervous system, may bemediated by stimulating the synthesis ofadenosine-3′,5′-monophosphate” (5).The superior cervical ganglion is an

ideal tissue for investigating synaptictransmission, with a single presynaptic anda single postsynaptic neuron along withsmall interneurons linking the pre- andpostsynaptic elements. Greengard thenproceeded to the more challenging caudatenucleus, the area of highest dopaminedensity in the brain (8). He used similarstrategies as in the ganglion. In homoge-nates of the caudate, dopamine stimulatedadenylate cyclase with a potency aboutthreefold greater than norepinephrine,whereas at the known α- and β-receptorsfor norepinephrine, dopamine is muchweaker. Moreover, the β-adrenergicreceptor is most potently activated byisoproterenol, which failed to stimulateadenylate cyclase in the caudate, evenat high concentrations. Apomorphine,known to mimic dopamine pharmacologi-cally, was even more potent than dopa-mine in stimulating the cyclase.In this study, Kebabian et al. (6) noted

that two antipsychotic drugs, chlorproma-zine and haloperidol, blocked dopamine’seffects. These drugs, first introduced intopsychiatry in 1952, had revolutionized thetreatment of schizophrenia, and therefore,understanding their mechanism of actionwas of great importance. The first insightsinto how they acted came from the studiesof Carlsson and Lindqvist (12) just afew years after they identified dopamineas a putative neurotransmitter/neuro-modulator. Carlsson and Lindqvist (12)noted that the pharmacology of thesedrugs resembled reserpine, an antipsy-chotic drug that acts by depleting the brainof dopamine, serotonin, and norepineph-rine. Chlorpromazine and haloperidolfailed to influence levels of dopamine ornorepinephrine but increased metabolicproducts of dopamine fairly selectively.Carlsson and Lindqvist (12) speculatedthat the antipsychotics were causinga functional dopamine depletion byblocking receptors, leading to a feedbackaugmentation of dopamine turnover. Theincreased turnover of dopamine was con-firmed by numerous investigators, but noone had examined a receptor-linkedevent directly.In a subsequent study, Clement-Cormier

et al. (13) explored potencies of a series ofantipsychotics of the phenothiazine andbutyrophenone class and found substantialpotency in inhibiting the dopamine-sensi-tive adenylate cyclase. Among the pheno-thiazines, clinical potency paralleledinhibition of the enzymes. Thus, the verypotent antipsychotic fluphenazine in-hibited the enzyme about 10 times morepotently than chlorpromazine.

Tyrosine

DOPA

Dopamine

Norepinephrine

D2 Receptor

Tyrosine Hydroxylase

DOPA Decarboxylase

Dopamine

β-hydroxylase

(Inhibits adenylate

cyclase; blockade by

antipsychotic drugs

underlies therapeutic

efficacy)

D1 Receptor

(Stimulates

adenylate cyclase)

Fig. 2. Biosynthesis and receptor actions of dopamine.

18870 | www.pnas.org/cgi/doi/10.1073/pnas.1114346108 Snyder

Promethazine, a phenothiazine antihista-mine that is not antipsychotic, was about25-fold weaker than chlorpromazine. Thisfinding suggested that these drugs act byblocking dopamine receptors associatedwith the adenylate cyclase. However, therewere exceptions to the rule. Thus, halo-peridol and pimozide, which clinically, areone to two orders of magnitude more po-tent then chlorpromazine, were, re-spectively, 3 and 20 times weakerthan chlorpromazine.The resolution to this knotty problem

came a few years later when it was possibleto identify dopamine receptors by ligandbinding (4). Two classes of dopamine re-ceptors could be labeled with [3H]halo-peridol and [3H]dopamine (14) cor-responding, respectively, to dopamine-D2and dopamine-D1 receptors, which weresubsequently enunciated by Kebabian andCalne (15). At D1 receptors, dopamine

activates adenylate cyclase, whereas at D2receptors, the transmitter inhibits the en-zyme (Fig. 2). Antipsychotic drug poten-cies are predicted by blockade of D2receptors at which haloperidol and pimo-zide are much more potent than chlor-promazine. The dopamine-sensitiveadenylate cyclase characterized byKebabian et al. (6) corresponds to D1receptors.The studies of dopamine by Greengard,

coupled with his epic contributions toneural regulation of protein kinases, havebeen landmarks in the biochemical eluci-dation of synaptic transmission in thebrain, which was acknowledged by hiscoreceiving of the Nobel Prize in Physiol-ogy or Medicine in 2000. In regards to thequestion raised at the outset of this essay(how does one pinpoint a neurotransmitterreceptor at a biochemical level), Green-gard was cautious. The title of the publi-

cation by Kebabian et al. (6) that is thesubject of this essay, “Dopamine-sensitiveadenylate cyclase in caudate nucleus of ratbrain, and its similarity to the ‘dopaminereceptor,’” was itself a hedge. Throughoutthe paper, Kebabian et al. (6) always ad-umbrated the term in quotation marks.With the use of ligand binding techniquesjust a few years after the publication byKebabian et al. (6), it became easy to char-acterize neurotransmitter receptors in depthand investigate the linkage between thereceptor, defined as the recognition sitefor the neurotransmitter, and coupling toG proteins followed by influences onadenylate cyclase and related molecules(4). Greengard’s efforts lie at the base ofthis edifice.

ACKNOWLEDGMENTS. This work was supportedby US Public Health Service Grants MH18501and DA000266.

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