Proc. Nati. Acad. Sci. USA Vol. 91, pp. 7772-7776, August 1994 Neurobiology High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system (bas gania/corpus siatum/nucleus accumbens/disfram/Parkluson die) PETER MCCAFFERY AND URSULA C. DRAGER Division of Developmental Neuroscience, E. K. Shriver Center, Waltham, MA 02254; and Department of Psychiatry, Harvard Medical School, Boston, MA 02115 Communicated by Ann M. Graybiel,* April 25, 1994 (receivedfor review January 15, 1994)

ABSTRACT Retinoic acid is synthesized from retinalde- fixed, and treated with 5-bromo-4-chloro-3-indolyl f3-D- hyde by several different dehydrogenases, which are arranged galactopyranoside (X-Gal), and the calorimetric reaction is in conserved spatial and developmentally regulated patterns. quantified in an ELISA reader. The cells are much more Here we show for the mouse that a class-i aldehyde dehydro- responsive to all-trans-retinoic acid than the cis isomers, and genase, characterized by oxidation and disulfiram sensitivity, a doubling in colorimetric readings indicates a 10- to 100-fold is found in the brain at high levels only In the basal forebrain. increase in retinoic acid. We did not attempt to measure It is present in axons and terminals of a subpopulation of absolute retinoic acid levels, but all measurements shown dopaminergic neurons of the mesostriatal and meimbic represent comparisons of different samples processed in system, forming a retinoic acid-generating projection from the parallel; no values are given for the calorimetric readings, as ventral tegmentum to the corpus striatiu and the shell of the all information is contained graphically in the comparisons. nucleus accumbens. In the striatum the projection is heaviest We did the following three assays based on the reporter cells. to dorsal and rostral regions, dining gradually toward Zymography bioassay. This assay (Fig. 1) involves sepa- ventral. The enzyme is expressed early in development, shortly ration of tissue homogenates in parallel lanes by isoelectric after appearance of tyrosine hydroxylase. Other d nergic focusing, cutting the lanes into thin slices, eluting the proteins neurons in the brain, as well as the chromaffin cells of the into L15 tissue culture medium in 96-well plates, and testing for retinoic acid synthesis from 50 nM retinaldehyde in the adrenal medulla, do not contain this dehydrogenase. The presence of 2 mM NAD (5). This assay probably detects all presence ofthis enzyme may be a factor in the long-term success enzymes capable of generating any form of retinoic acid. of transplants of dopaminergic cells to the corpus stratum In Enzyme lability assay. Similar-size tissue pieces were Parkinson disease, and it may play a role in parkinsonism and dissected from the striatum and olfactory bulbs of young catatonia due to disulfiram (Antabuse) neurotoxicity. adult mice and were subjected to one of two pretreatments before being assayed for retinoic acid synthesis from added Aldehyde dehydrogenases represent a diverse family of re- retinaldehyde and NAD with the reporter cells (Fig. 2a). (i) lated enzymes involved in the oxidation of exogenous and The tissues were homogenized in L15 medium without any endogenous aldehydes (1-3). They differ in a range of char- additives or with 2.5 1LM or 12.5 ,uM disulfiram and kept on acteristics including substrate selectivity: of 13 isoforms ice, or (ii) the tissues were homogenized with or without 1 identified in the mouse, only class 1 (cytosolic) aldehyde mM dithiothreitol and kept at 370C for 10 or 20 min; the dehydrogenase was found to be capable of oxidizing retinal- control here is a 20-min air exposure at 370C in the presence dehyde to retinoic acid (4). Although in vitro this isoform can of dithiothreitol. Like the effect of air exposure, the dis- oxidize a broad range of substrates, retinoic acid production ulfiram effect can be prevented by dithiothreitol. is considered its main physiological role. In addition to class Estimates of endogenous retinoic acid levels. For this 1 aldehyde dehydrogenases, several recently reported dehy- assay (Fig. 2b), similar-size samples from adult olfactory drogenases are capable of generating retinoic acid (5). Ex- bulb, striatum, and hippocampus were homogenized in L15 pression of the different retinoic acid-generating dehydroge- medium in the presence of 2 mM NAD, but no retinaldehyde nases occurs in stereotypic spatial and developmentally was added. The homogenates were incubated overnight at regulated patterns that are similar in mammals, birds, and 370C, and the supernatants were plated in serial dilutions onto cold-blooded vertebrates. For instance, the embryonic retina the reporter cells. of all vertebrates tested contains in its dorsal part a class 1 Histology. All murine tissue, derived from an outbred mouse aldehyde dehydrogenase, termed AHD2 in the mouse, and in colony, was fixed in periodate-lysine-paraformaldehyde (9), its ventral part different dehydrogenases (5, 6). either by immersion (embryos) or by transcardiac perfusion MATERIALS AND (adult); the monkey tissue came from an adult rhesus macaque METHODS perfused with 4% paraformaldehyde for an unrelated experi- Retinoic Acid Assays. All determinations of retinoic acid ment. Cryostat sections were labeled with 2 different antisera shown here make use of a reporter cell line: teratocarcinoma to rodent class 1 aldehyde dehydrogenase (10, 11) and with a cells transfected with a sensitive retinoic acid-response ele- monoclonal antibody or an antiserum to tyrosine hydroxylase ment driving (3-galactosidase expression (7). These cells (TH; Boehringer Mannheim; Eugene Tech). Antibody binding specifically detect retinoic acid, the product of the irrevers- was visualized with fluorescent secondary antisera. ible dehydrogenation reaction of retinaldehyde, but they do not give information on the preceding enzymatic reaction, the RESULTS reversible oxidation of retinol to retinaldehyde, which is In the embryonic mouse eye, retinoic acid is generated by catalyzed by an alcohol dehydrogenase (8). The cells are four zymographically distinct enzymatic activities exposed to a retinoic acid-containing sample overnight, (Fig. la, Abbreviations: AHD2, murine class 1 (cytosolic) aldehyde dehydro- The publication costs ofthis article were defrayed in part by page charge genase; TH, tyrosine hydroxylase. payment. This article must therefore be hereby marked "advertisement" *Communication of this paper was initiated by W. J. H. Nauta and, in accordance with 18 U.S.C. §1734 solely to indicate this fact. after his death (March 24, 1994), completed by Ann M. Graybiel. 7772 Downloaded by guest on September 26, 2021 Neurobiology: McCaffery and DrAger Proc. Natl. Acad. Sci. USA 91 (1994) 7773

a AHD2 Vl V2 V3 b lower. Acidic dehydrogenases were detectable throughout the brain, but their compositions and levels varied: in most regions in both developing and mature brain, high activity was associated with the pia. In the adult brain the relatively highest activity was present in the olfactory bulbs, where several different enzymes were expressed, and only low u A levels were found in the hippocampus, which contained mainly a single activity (Fig. la, bottom traces). AHD2 was to N detectable in the brain at high levels only in one area: the r. striatum (Fig. la). Whereas in the adult striatum all retinoic acid synthesis was mediated by AHD2, the fetal striatum contained rather high levels ofacidic dehydrogenases similar to those in the embryonic eye; the basic AHD2 took over basioctory bulb slowly during the perinatal period (Fig. lb). In in vitro hippocampus assays with substrates other than retinoic acid, basic < > acidic the class 1 isoform is known to stand out from other mem- basic < > acidic bers of the family of nonspecific aldehyde dehydrogenases through its very high susceptibility to oxidation and to FIG. 1. (a) Zymographs of retinoic acid-generating enzymes in inhibition by the sulfhydryl reagent disulfiram (12, 13). The similar-size samples dissected from the corpus striatum, olfactory same bulb, and hippocampus of adult mice, processed in parallel with an distinction applies to a comparison of AHD2 with the embryonic day 16 (E16) eye, which serves here as a pH/enzyme novel retinoic acid-generating dehydrogenases, which prob- marker; the designations of the eye enzymes are indicated at the top ably do not belong to the nonspecific aldehyde dehydroge- (6). No numerical values are given for the colorimetric readings, as nase family (14): enzyme activity in homogenates of adult only the comparative values, depicted graphically, are of signifi- striatum was much more susceptible to oxidation and dis- cance. For easier comparison of enzyme activities, the colorimetric ulfiram treatment than activity in olfactory-bulb homoge- reading traces are aligned and offset. The basic enzyme activity is nates (Fig. 2a). Like the effect of air exposure, the disulfiram AHD2; the acidic activities in the brain samples represent novel effect could be prevented and, at least partially, reversed by dehydrogenases resembling those in the embryonic eye. (b) Devel- the reducing reagent dithiothreitol; it is possible that both opmental changes in retinoic acid-generating enzymes in the devel- manipulations oping striatum. Similar-size samples were dissected from the stria- target the same sulfhydryl groups on AHD2. tum of E16, postnatal day 2 (P2), and P27 mice and were processed The exact location of the enzymes in the fetal striatum is in parallel by zymography. not clear; they might be associated with the germinal zone giving rise to the striatum, the ganglionic eminence, which top trace): one basic enzyme representing AHD2 localized in contaminated the dissected samples. For AHD2, however, dorsal retina, and three acidic activities, V1, V2, and V3, antisera specific for rodent class 1 aldehyde dehydrogenase mainly localized in ventral retina (V1 and V3) and the retinal allow a more detailed analysis (10, 11). Immunohistochemical pigment epithelium (V2) (5,6). We screened different regions examination of the adult striatum showed bright AHD2 of the central nervous system from forebrain to spinal cord labeling, all localized in axons and synaptic terminals (Fig. 3 for zymographically detectable retinoic acid-generating de- a and b). Cell bodies of origin were exclusively located in the hydrogenases, starting from embryonic day 8 (E8, with day nigral-ventral tegmental areas A9, A8, and A10. Double- of conception = EO.5) to adulthood. In general, levels of labeling with a monoclonal antibody to TH showed AHD2 in retinoic acid-generating enzymes were much higher in the a subpopulation of the dopaminergic neurons: most double- developing nervous system than in the mature system, and at labeled somata were found in the nigral pars compacta (A9; any given age enzyme levels varied substantially between Fig. 4 a and b), fewer in the ventral portions of A8, and only different regions-e.g., an occasional one in A10 (Fig. 4 c and d). The projections embryonic-retina and spinal-cord from these levels were very high, and brain levels were overall much neurons, relatively concentrated in rostral and ventral portions of the tegmental dopamine system, corre- a spond to the known topography of mesostriatal and mesolim- striahim nlf^dnr% h..lls bic projections (15): AHD2-containing fibers were densest in to 100%1 111 most rostral and dorsal portions of the striatum, with the density gradually declining toward ventral (Fig. 3 a and c). Of the mesolimbic projection sites, only the shell of the nucleus '~60%! U accumbens received heavy AHD2 input (Fig. 3 a and c, arrow). AHD2 projection to the accumbens core and olfac- tory tubercle was much weaker than the dopaminergic input 20 0 2.5 12.5 0 10' 20' 0 2.5 12.5 0 10' 20' (Fig. 3 a, c, and d), and to the septal area both projections disulfiram 02/370C disulfiram 0 /370C were sparse. No AHD2 input to the cortex was found. Other [AM] [PM] 2 dopaminergic cells, including the ones in the hypothalamus, olfactory bulb, retina, and adrenals, did not contain AHD2. olfactory bulb Zymographically the adrenal gland did contain low levels of striatum AHD2 (not shown); double-labeled immunohistochemical hippocampus preparations, however, showed no AHD2-like immunoreac- tivity in dopaminergic chromaffin cells, but only in some colorimetric readings adjoining cortical and in nondopaminergic medullary cells (Fig. 4 e and f). FIG. 2. (a) Comparisons of the decay of retinoic acid-generating In immunohistochemical screens of the developing murine enzyme activity in homogenates of the striatum and olfactory bulb nervous system, exposed to disulfiram or to room air. Note the much more pro- from E8 onward, expression of AHD2 in nounced activity decline in the striatum as compared with the dopaminergic neurons was found to begin about 1 day after olfactory bulb. (b) Endogenous retinoic acid content, or synthesis expression of TH (16). In double-labeled sections through from endogenous precursor, in homogenized, similar-size samples E12 mice, TH-positive cells were present in the mesenceph- from adult olfactory bulb, striatum and hippocampus. alon, but they did not contain AHD2. At E13 double-labeled Downloaded by guest on September 26, 2021 7774 Neurobiology: McCaffery and Drager Proc. Natl. Acad Sci. USA 91 (1994)

FIG. 3. Coronal sections through the striatum of young adult mice double-labeled with antisera to rodent class 1 aldehyde dehydrogenase (10, 11) (a-c) and a monoclonal antibody to TH (d). (a) Low-power view ofone cerebral hemisphere to illustrate the dorsoventrallygraded AHD2 distribution. In addition to the nigrostriatal system, both class-i antisera label the brain surface/pia; as zymographs show no AHD2 here, this probably represents a dehydrogenase that is immunologically similar to AHD2. (b) High-power view ofthe striatum edge showing AHD2-positive fibers ending in bright specks, presumably synaptic terminals. (c and d) Partial view of the striatal-limbic transition, double-labeled for AHD2 (c) and TH (d). Note the relatively homogeneous TH distribution; by contrast, the AHD2 input is weak to most of the limbic regions except for heavy input to the shell of the nucleus accumbens (arrow). AHD2 is detected by fluorescein isothiocyanate immunofluorescence, and TH is detected by rhodamine isothiocyanate. [Bars = 1 mm (a), 50 inm (b), and 300 pim (c and d).] cells were found, and a few AHD2-positive fibers could demonstrate such afunction in the striatum. A sensitive assay already be detected in the beginning medial forebrain bundle for endogenous retinoic acid synthesis in small samples is (Fig. 5a, arrows); by E14 this projection was quite pro- overnight coculture of test tissues in tight contact with the nounced (Fig. Sb, arrows). The dopaminergic cell bodies of retinoic acid reporter cells (7). Because it was not possible to origin in the mesencephalon were very brightly labeled with dissect the few AHD2-containing cell bodies clean enough the AHD2 antiserum, as illustrated for E16 (Fig. Sc). Al- from other tegmental cells to assure good contact and be- though at E16 the AHD2 projection to the striatum was quite cause overnight culture of disrupted terminals embedded obvious immunohistochemically (not shown), it was not yet between live cells in the striatum was problematic, the detected by the less-sensitive zymography assay (Fig. lb). reporter cells were used to assay homogenized tissue. No Preliminary examinations ofsections through the substantia retinaldehyde is added in this assay, and all detected activity nigra from an adult monkey (Macaca fasciculars), double- reflects either endogenous retinoic acid content or synthesis labeled with the same antibodies, revealed a similar selective from endogenous retinoid precursors. In embryonic tissues, class 1 aldehyde dehydrogenase labeling ofa subpopulation of assays ofhomogenates correlate well with tissue explants (5). dopaminergic neurons, with double-labeled cells concentrated Fig. 2b shows such comparative retinoic acid estimates for in ventral regions (not shown). This makes it likely that the similar-size samples from striatum, olfactory bulbs, and pattern described here for the mouse extends to primates. hippocampus. Consistent with zymographically detectable Although retinoic acid synthesis is considered the main enzyme activities (Fig. la), retinoic acid levels were very low role of class 1 aldehyde dehydrogenase, it was necessary to in the hippocampus and considerably higher in both striatum Downloaded by guest on September 26, 2021 Neurobiology: McCaffery and DrAger Proc. Nati. Acad. Sci. USA 91 (1994) 7775

FIG. 4. Comparisons of AHD2 (Left) and TH (Right) in double-labeled specimens. (a and b) Coronal section through the substantia nigra (A9) and adjoining A8. (c and d) Coronal section through the ventral tegmental area, showing A10 Qeft cell cluster, above the arrow in c, which indicates the brain midline), and A8 (right cluster). Note the prevalence ofAHD2-containing neurons among the ventral dopaminergic population. (e andf) Section through adrenal gland; note the absence of AHD2 labeling from the bright TH-positive chromaffin cells. (Bar = 300 jm.) and olfactory bulb. These assays demonstrate retinoic acid formed synaptic inputs, and low levels in the hippocampus. synthesis in the adult striatum, mediated by AHD2, the only Maybe, in the adult striatum retinoic acid is involved in some retinoic acid-generating enzyme detectable there. The exact continuing structural adjustments necessary for acquisition levels of endogenous retinoic acid remain to be determined of motor patterns. with an alternate method, as the reporter cells respond poorly The observation that the retinoic acid-generating enzyme to the 9-cis-retinoic acid isomer (22), which is likely to form in the basal ganglia is present in axons and synaptic terminals a fraction of the retinoid content in the striatum, because of projecting over a long distance from the midbrain reveals an the high concentration of a 9-cis-retinoic acid-binding recep- unusual form of information transmission. While the princi- tor (RXRy) here (18). ple function of neuronal projections, at least in the adult, is thought to be the transfer of electrical information, axonal DISCUSSION transport of the retinoic acid-producing enzyme indicates a The importance ofretinoic acid as a transcriptional regulator way by which the mesencephalic neurons can directly exert in the corpus striatum is evident from the extensive array of an influence on gene transcription in the forebrain. This retinoic acid receptors and binding proteins expressed in this represents a mechanism for a long-distance trophic or induc- region (18, 19). More than 100 genes are known to be tive neuronal interaction. regulated by retinoic acid (20), including that encoding the Although we describe a retinoic acid-synthesizing alde- neurotrophin receptor TrkB (21). Retinoic acid levels are hyde dehydrogenase in the meso-telencephalic dopamine generally much higher in the embryonic than adult nervous system, nonspecific aldehyde dehydrogenase activity has system, and high levels seem to correlate with times and been demonstrated in dopaminergic regions throughout the regions of reduced morphogenetic cell death, neuronal dif- brain (23). This is attributed to the aldehyde dehydrogenase ferentiation, and process outgrowth, but not with electric involved in dopamine metabolism. Dopamine degradation is activity (6, 22). This interpretation is consistent with rela- catalyzed mainly by a mitochondrial isoform but not the tively high retinoic acid levels in the olfactory bulb, which is cytosolic class-1 enzyme (24), and the mitochondrial enzyme unique in the adult brain for having to accommodate newly is presumably present in all dopaminergic regions, whereas Downloaded by guest on September 26, 2021 7776 Neurobiology: McCaffery and DrAger Proc. Natl. Acad. Sci. USA 91 (1994)

FIG. 5. Coronal sections through heads of embryonic day E13 (a), E14 (b), and E16 (c) mice labeled for AHD2; relative planes of sections (a > c) move from rostral to caudal. Arrows point to the medial forebrain bundle, which at E13 (a) contains only a few AHD2-positive axons (single fibers are visible at higher magnification; not shown), and is far overshadowed by the heavy retinal labeling. At E14 (b), AHD2-positive axon in the medial forebrain bundle are already pronounced as compared with the AHD2-labeled optic axons in the chiasm and around the di/mesencephalon. As in the nigrostriatal system, AHD2 is expressed in axons and growth cones ofretinal ganglion cells, but here the expression is transient, limited to outgrowing axons and disappearing later (17). (c) Cell bodies oforigin in the E16 nigrotegmental region. Not shown here is TH labeling, which is present only in the nigrotegmnental cells and their projections but not in the retina or optic axons. (Bar = 500 inm.) AHD2 is highly expressed only in a subpopulation of dopa- 2. Lindahl, R. (1992) Crit. Rev. Biochem. Mol. Biol. 27, 283-335. minergic neurons. Nevertheless, in addition to 3. Manthey, C. L., Landkamer, G. J. & Sladek, N. E. (1990) Cancer synthesizing Res. 50, 4991-5002. retinoic acid, AHD2 could function as a back-up for dopa- 4. Lee, M.-O., Manthey, C. L. & Sladek, N. E. (1991) Biochem. mine metabolism in these cells. Pharmacol. 42, 1279-1285. One of the outstanding and conserved characteristics of 5. McCaffery, P., Lee, M.-O., Wagner, M. A., Sladek, N. E. & class 1 aldehyde dehydrogenases is their oxidation sensitivity, Driger, U. C. (1992) Development 115, 371-382. 6. McCaffery, P., Posch, K. C., Napoli, J. L., Gudas, L. & Drfiger, a property that in several other enzymes is known to form the U. C. (1993) Dev. Biol. 1S8, 390-399. basis for a redox regulatory mechanism of enzymatic activity 7. Wagner, M., Han, B. & Jessell, T. M. (1992) Development 116, (25). It is intriguing that this oxidation-sensitive enzyme is 55-66. selectively found in a neuronal system commonly linked to 8. Duester, G., Shean, M. L., McBridge, M. S. & Steward, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646. oxidative stress (reviewed in ref. 26). A possible pharmaco- 9. McLean, I. W. & Nakane, P. K. (1974) J. Histochem. Cytochem. logical correlate ofthe oxidation sensitivity is the susceptibil- 22, 1077-1083. ity to the sulfhydryl reagent disulfiram: in in vitro assays, 10. Lindahl, R. & Evces, S. (1984) J. Biol. Chem. 259, 11991-11996. disulfiram acts as a specific inhibitor of class 1 aldehyde 11. Russo, J. E. & Hilton, J. (1988) Cancer Aes. 48, 2963-2968. 12. Feldman, R. I. & Weiner, H. (1972) J. Biol. Chem. 247, 260-266. dehydrogenases (13). Although in vivo the drug is likely to 13. Vallari, R. C. & Pietruszko, R. (1982) Science 216, 637-639. have additional actions due to formation of metabolites, its 14. Zhao, D., McCaffery, P., Neve, R. L., Hogan, P., Chin, W. W. & central neurotoxic effects seem to target the TH/AHD2- DrAger, U. C. (1993) Soc. Neurosci. Abstr. 19, 218. containing system: disulfiram, given as an alcohol deterrent 15. Fallon, J. H. & Loughlin, S. E. (1985) in The Rat Nervous System lead to ed. Paxinos, G. (Academic, New York), pp. 353-374. (Antabuse), can extrapyramidal disturbances- 16. Specht, L. A., Pickel, V. M., Joh, T. H. & Reis, D. J. (1981) J. parkinsonism, catatonia-and basal-ganglia lesions (27-29). Comp. Neurol. 199, 233-253. The loss of dopaminergic innervation to the basal ganglia 17. McCaffery, P., Tempst, P., Lara, G. & DrAger, U. C. (1991) in Parkinson disease is not diffuse but follows a topographic Development 112, 693-702. pattern that is mimicked by the dorsoventrally graded de- 18. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., in the weaver mutant a for Ong, E. S., Oro, A. E., Kakizuka, A. & Evans, R. M. (1992) Genes generation pattern mouse, model Dev. 6, 329-344. parkinsonism (30). The weaver pattern resembles the distri- 19. Ruberte, E., Friederich, V., Chambon, P. & Morriss-Kay, G. (1993) bution of the AHD2 projection, except for the shell of the Development 118, 267-282. nucleus accumbens, which is not affected in weaver. As 20. Chytil, F. & ul-Haq, R. (1990) Eukaryotic Gene Expression 1, 61-73. transplants of adrenal chromaffin cells, which in the mouse 21. Kaplan, D. R., Matsumoto, K., Lucarelli, E. & Thiele, C. J. (1993) do not contain AHD2, are less successful than fetal tegmental Neuron 11, 321-331. 22. McCaffery, P. & DrAger, U. C. (1994) Proc. Natl. Acad. Sci. USA transplants in Parkinson disease, presence of the enzyme 91, 7194-7197. could be a determining factor. Transfection of chromaffin 23. Zimatkin, S. M. (1991) J. Neurochem. 56, 1-11. cells with a construct expressing high levels of human class 24. Tank, A. W., Deitrich, R. A. & Weiner, H. (1986) Biochem. Phar- 1 aldehyde dehydrogenase (31) may improve the long-term macol. 35, 4563-4569. transplant success. 25. Brigelius, R. (1985) in Oxidative Stress, ed. Sies, H. (Academic, London), pp. 243-272. This paper is dedicated to the memory of W. J. H. Nauta. We 26. Olanow, C. W. & Calne, D. (1992) Neurology 42, Suppl. 4, 3-26. thank M. Wagner and T. Jessell for the retinoic-acid reporter cells, 27. Fisher, C. M. (1989) Arch. Neurol. 46, 798-804. R. Lindahl and J. Hilton for aldehyde dehydrogenase antisera, G. 28. Krauss, J. K., Mohadjer, M., Wakloo, A. K. & Mundinger, F. Blasdel for the monkey tissue, and D. Cardozo and N. Marsh- (1991) Movement Disord. 6, 166-177. Armstrong for valuable suggestions. This work was supported by 29. Laplane, D., Attal, N., Sauron, B., de Billy, A. & Dubois, B. (1992) Grant R01 EY01938 from the National Eye Institute and a gift from J. Neurol. Neurosurg. Psychiatry 55, 925-929. Johnson & Johnson. 30. Roffler-Tarlov, S. & Graybiel, A. M. (1984) Nature (London) 307, 62-66. 1. Greenfield, N. J. & Pietruszko, R. (1977) Biochim. Biophys. Acta 31. Hempel, J., von Bahr-Lindstrom, H. & Jdrnvall, H. (1984) Eur. J. 483, 35-45. Biochem. 141, 21-35. Downloaded by guest on September 26, 2021