Dopaminergic regulation of protein phosphorylation in the striatum: DARPP-32

known to be located on the basolateral membrane. The activation of Ca2+-dependent channels, alone 6 or together with an ion cartier 5'7, accounts for the transport of salt from the blood compartment to the lumen. Water flow is thought to occur as a consequence of ion movement. Selected references 1 Birdsall, N. J. M., Hulme, E. C. and Stockton, J. M. (1984) Trends Pharmacol. Sci. Suppl. 4-8 2 Pfaffinger, P. J., Martin, J.M., Hunter, D.D., Nathanson, N. M. and Hille, B. (1985) Nature 317, 536-538 3 Breitwieser, G. E. and Szabo, G. (1985) Nature 317, 538-540 4 Marty, A. and Neher, E. (1983) in Single Channel Recording (Sakmann, B. and Neher, E., eds), pp. 107-122, Plenum Press 5 Petersen, O. H. and Maruyama, Y. (1984) Nature 307, 693696 6 Marty, A., Tan, Y. P. and Trautmann, A. (1984) J. Physiol. (London) 357, 293-325 7 Suzuki, K. and Petersen, O. H. (1985) (9. J. Exp. Physiol. 70, 437-445 8 Lundberg, J. M. and HOkfelt, T. (1983) Trends Neurosci. 6, 325-333 90'Doherty, J., Stark, R.J., Crane, S.J. and Brugge, K.L. (1983) Pfl(Jg. Arch. 398, 241-246 10 Berridge, M. J. and Irvine, R. F. (1984) Nature 312,315-320 11 Litosch, I., Wallis, C. and Fain, J. N. (1985) J. Biol. Chem. 260, 5464-5471 12 Cockcroff, S. and Gomperts, B. D. (1985) Nature 314, 534536 13 Streb, H., Irvine, R. F., Berridge, M. J. and Schulz, I. (1983) Nature 306, 67-69 14 Selinger, Z., Batzri, S., Eimerl, S. and Schramm, M. (1973) J. Biol. Chem. 248, 369-372 15 Putney, J. W., Jr (1976) J. Physiol. (London) 281,283-394 16 Keryer, G. and Rossignol, B. (1976) Am. J. Physiol. 230, 99104 17 Kanagasuntheram, P. and Randle, P.J. (1976) Biochem. J. 160, 547-564 18 Iwatsuki, N. and Petersen, O. H. (1978) J. CellBiol. 79, 533545

19 Burgen, A. S. V. (1956) J. Cell. Comp. Physiol. 48, 113-138 20 Martin, K. and Burgen, A. S. V. (1962) J. Gen. PhysioL 46, 225-243 21 Mazariegos, M. R., Tice, L. W. and Hand, A. R. (1984) J. Cell Biol. 98, 1865-1877 22 Marty, A. (1983) Trends Neurosci. 6, 262-265 23 Maruyama, Y., Gallacher, D. V. and Petersen, O. H. (1983) Nature 302, 827-829 24 Maruyama, Y., Petersen, O. H., Flanagan, P. and Pearson, G. T. (1983) Nature 305, 228-232 25 Trautmann, A. and Marty, A. (1984) Proc. NatlAcad. Sci. USA 81,611-615 26 Findlay, I. (1984) J. Physiol. (London) 350, 179-195 27 Evans, M. G. and Marry, A. (1986) J. Physiol. (London) 378, 437-460 ~8 Marty, A., Evans, M. G., Tan, Y. P. and Trautmann, A. (1986) J. Exp. Biol. 124, 15-32 29 Maruyama, Y. and Petersen, O. H. (1982) Nature 299, 159161 30 Gallacher, D. V. and Morris, A. P. (1986) J. Physiol. (London) 373, 379-395 31 Maruyama, Y. and Petersen, O. H. (1984)J. Membr. Biol. 79, 293-300 32 Findlay, I. and Petersen, O. H. (1985) PflOg. Arch. 403,328330 33 Evans, M. G. and Marty, A. (1986) Proc. NatlAcad. Sci. USA 83, 4099-4103 34 Kusano, K., Miledi, R. and Stinnakre, J. (1982) J. Physiol. (London) 328, 143-170 35 Woods, N. M., Cuthbertson, K. S. R. and Cobbold, P.H. (1986) Nature 319, 600-602 36 Maruyama, Y. and Petersen, O. H. (1982) Nature 300, 6163 37 Iwatsuki, N. and Petersen, O. H. (1978) J. Physiol. (London) 274, 81-96 38 Findlay, I. and Petersen, O. H. (1982) Cell Tiss. Res. 225, 633-638 39 Neyton, J. and Trautmann, A. (1985) Nature 317, 331-335 40 Neyton, J. and Trautmann, A. (1986) J. Physiol. (London) 377, 283-295 41 Neyton, J. and Trautmann, A. (1986) J. Exp. Biol. 124, 93114 42 Burgen A. S. V. (1956) J. Physiol. (London) 132, 20-39

Dopaminergicregulation of protein phosphorylationin the striatum: DARPP-32 H u g h C. H e m m i n g s , Jr, S. I v a r W a l a a s , C h a r l e s C. O u i m e t

DARPP-32 is a neuronal phosphoprotein that is specifically enriched in neurons possessing D1 dopamine receptors, including the medium-sized spiny neurons of the striatum. DARPP-32 phosphorylation is regulated by dopamine acting through cyclic AMP. Biochemical studies have shown that phosphorylated DARPP-32 functions as a potent inhibitor of protein phosphatase-1 in vitro. In vivo, this inhibition may be an important component of the biochemical mechanisms by which dopamine, acting via D1 receptors, exerts its neurophysiological effects. Regulation of DARPP-32 phosphorylation may also mediate specific interactions between dopamine, acting through cyclic AMP, and glutamate (or other first messengers), acting through Ca e+. Future studies of basal ganglion-specific phosphoproteins in general, and of DARPP-32 in particular, should lead to a clearer understanding of the molecular mechanisms underlying dopaminergic regulation of neuronal function.

a n d Paul G r e e n g a r d

of specific substrate proteins by cyclic A M P dependent protein kinase is a general mechanism by which neurotransmitters produce physiological effects in specific target neurons. T w o subtypes of dopamine

TABLE I. Summary of physical and chemical properties of DARPP-32 Property Molecular weight SDS/polyacrylamide gel electrophoresis Hydrodynamic measurements Amino acid sequence Number of amino acids Structure Isoelectric point, dephospho form Number of phosphorylation sites Phosphorylated amino acid Amino acid composition

Protein phosphorylation is an important regulatory mechanism for the control of many cellular processes in the nervous system 1. Regulation of phosphorylation TINS, Vol. 10, NO. 9, 1987

© 1987,ElsevierPublications,Cambridge 0378-5912/87/$02.00

Value

Hugh C. Hemmings, Jr, 5. Ivar Walaas, CharlesC. Ouimet and Paul6reengard areat the Laboratory of Molecularand CellularNeuroscience, TheRockefeller University, New York, 10021, USA.

32 000 27 600 22 591 202 Elongated 4.7 1 Threonine High glutamate Low hydrophobic residues Low aromatic residues 377

tubercle). Within the striatum, dopamine-stimulated adenylate cyclase is associated with neurons that 40project to the substantia nigra5. Recent studies employing quanttafive autoradiographic localization of D~ receptors in rat brain support these enzyme activity measurements, showing highest densities of D~ receptors in the caudatoputamen, nucleus accumA 30bens, olfactory tubercle and substantia nigra6-9. a~ , Considerable evidence (for review see Ref. 10) ¢q supports the hypothesis 1~ that D1 receptors are 20 involved in mediating, through the action of dopamine0.. sensitive adenylate cyclase, some of the neurotr transmitter functions of dopamine in the striatum. < t:) Thus, iontophoretically applied cyclic AMP or cyclic I 10 AMP analogues are able to mimic the specific electrophysiological effects of applied dopamine on individual neurons 12. Furthermore, pharmacological studies have found that D1 receptors are involved in a a. 0 . . . . . . . . . . . . . . . . specific subset of dopamine-elicited behaviors in the rat t3. These findings demonstrate that D1 receptors and cyclic AMP mediate some of the neurot // i i i i physiological and behavioral effects of dopamine. 0 10 - 6 10 - 5 10- 4 10- 3 One way to examine the biochemical mechanism of DOPAMINE (M) dopamine action on Dl-containing neurons in the Fig. 1. Effect of various concentrations of dopamine on striatum is to identify and characterize the dopamineDARPP-32 phosphorylation. Slices of rat caudatoputamen and cyclic AMP-regulated phosphoproteins present in (0.4 mm thick) were incubated in Krebs-Ringer buffer in these cells t°. A number of cyclic AMP-regulated the presence of the indicated concentrations of dopamine. phosphoproteins are distributed unevenly throughout The amount of phospho-DARPP-32 was determined, and is presented as percent of total DARPP-32 measured in the mammalian CNS ~4. Some of these phosphocontrol sfices that had been incubated without dopamine. proteins appear to be specifically enriched in D1containing neurons, and may be involved in mediating ( M o d i f i e d from Ref. 16.) some of the specific physiological effects of dopamine receptor, distinguishable by biochemical and pharma- acting at D1 receptors. An example of this class of cological criteria, are involved in the regulation of phosphoprotein is DARPP-32, a dopamine- and cyclic cyclic AMP levels: the Dt receptor stimulates adenyl- AMP-regulated phosphoprotein, Mr=32000, which ate cyclase whereas the D2 dopamine receptor, in has been extensively characterized and serves as a some cases, inhibits adenylate cyclase2. Increased prototype for the study of other dopamine-regulated concentration of cyclic AMP, produced in response to phosphoproteins. stimulation of D1 receptors, leads to activation of cyclic AMP-dependent protein kinase and thereby to Dopamine-regulated phosphorylation of phosphorylation of specific substrate proteins. These DARPP-32 phosphoproteins are effector molecules involved in DARPP-32 was originally observed in a biochemical producing some of the biological responses to dop- study of the regional distribution of soluble cyclic amine in target cells. The state of phosphorylation of AMP-regulated phosphoproteins in the rat CNS 14. dopamine-regulated substrate proteins is regulated DARPP-32 was found to have a restricted regional not only by protein kinase activity but also by the distribution that paralleled the gross anatomical distriactivity of phosphoprotein phosphatases, enzymes butions of dopamine, dopamine-sensitive adenylate that are also subject to regulation by intracellular cyclase, and D1 receptors, suggesting that the state of second messengers, such as cyclic AMP and Ca2+ phosphorylation of DARPP-32 in these brain regions (Ref. 3). might be regulated by dopamine and cyclic AMP. Using slices prepared from rat caudatoputamen, where Dopamine and cyclic AMP DARPP-32 is highly enriched, dopamine and an anaIn the rat CNS, both D1 receptor binding sites and logue of cyclic AMP were found to increase the state of dopamine-sensifive adenylate cyclase activity (D1 phosphorylation of DARPP-32 (Fig. 1)t5,t6. This receptors coupled to adenylate cyclase) are concen- effect was time- and dose-dependent, and was trated in brain regions rich in dopamine innervation4, observed at concentrations of dopamine that had e.g. the dorsal striatum (caudatoputamen) and the previously been found to activate receptors linked to ventral striatum (nucleus accumbens and olfactory the stimulation of adenylate cyclase in rat caudatoputamen slices17. Dopamine-induced phosphorylation TABI.I: II. Amino acid sequences around the phosphorylatable threonine of DARPP-32 could be inhibited by the dopamine receptor blocker fluphenazine, and appeared to be residues of DARPP-32 and inhibitor-1 specific for dopamine insofar as several other neuroPhosphoprotein Sequence transmitters (serotonin, adenosine, and nor-Ile-Arg-Arg-Arg-Arg-Pro-Thr(P)- Pro-Ala-Met- Leu- Phe-Arg- epinephrine) did not affect DARPP-32 phosphorylaDARPP-32 a tion. These results led to the hypothesis that DARPP!nhibitor-lb -Ile-Arg-Arg-Arg-Arg- Pro -Th r( P)- Pro-AI a-Th r - Leu-Val- Le u32 may play a functional role in the central dopamine Sequence homology between DARPP-32 and inhibitor-1 is indicated by system. In an attempt to characterize further the underlining, a From Refs 21 and 28. b From Ref. 29. precise functional role of DARPP-32 in this system, a 378

TINS, Vol. 10, No. 9, 1987

series of biochemical and anatomical studies was undertaken.

TABLE III. Regional distribution of DARPP-32 in rat CNS determined by radioimmunoassaya

Biochemical characterization of DARPP-32 DARPP-32 from bovine caudate nucleus cytosol was purified to homogeneity and characterized (Table I) 18-2]. It exists in solution as a highly elongated and extremely acidic monomer. Purified DARPP-32 is phosphorylated with favorable kinetic parameters in vitro by cyclic AMP-dependent protein kinase on a single threonine residue located on the carboxyterminal side of four consecutive arginine residues (Table II)2°'2L It is also phosphorylated by cyclic GMP-dependent protein kinase, but not by Ca2+/ calmodulin-dependent protein kinase I, Ca2+/ calmodulin-dependent protein kinase II or protein kinase C. DARPP-32 is dephosphorylated most efficiently in vitro by protein phosphatase-2B ]9'22, also known as calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. This observation is of particular interest since the substrate specificity of protein phosphatase-2B had been reported to be relatively limited 23. Protein phosphatase-2B is concentrated in many of the same brain regions as DARPP-32, including the striatum24; within the caudatoputamen, it appears to be contained within medium-sized spiny neurons 25, as is DARPP-32 (see below). Enzymological studies have established a biochemical function of DARPP-32. In the initial characterization of DARPP-3218, several similarities were noted between its physicochemical properties and those of protein phosphatase inhibitor-1 (inhibitor-I), a potent non-competitive inhibitor (in its phosphorylated form) of the catalytic subunit of the enzyme protein phosphatase-126'27. Determination of the complete amino acid sequence of DARPP-32 revealed marked homology with the sequence surrounding the threonine residue of inhibitor-1 that can be phosphorylated (Table II), as well as with other regions of inhibitor-1 thought to be essential for its inhibition of protein phosphatase-128-3°. By investigating the effect of DARPP-32 on protein phosphatase-1 activity in vitro ]9, phosphorylated, but not dephosphorylated, DARPP-32 was found to act as a potent noncompetitive inhibitor, with an ICso of approximately 10-9 M (Fig. 2). Thus, the striatum contains a regionspecific neuronal phosphoprotein, namely DARPP-32, whose phosioo
Brain region

O Distribution of DARPP-32 The distribution of DARPP-32 in T 6 0 various brain regions, peripheral fit tissues, neuronal cell types, and O animal species was studied by use O of a phosphorylation assay TM, as " 4 0 well as by immunocytochemistr~ x 8 ¢.and radioimmunoassay32. In each of these studies, the distribution of .=-- 2 0 DARPP-32 in the rat CNS was ~D found to parallel the distribution of dopaminergic synapses. More specifically, the distribution of DARPP-32 correlated strongly with the distribution of cells pos-

TINS, Vol. 10, No. 9, 1987

Substantia nigra Caudatoputamen Globuspallidus Olfactory tubercle Nucleus accumbens Thalamus Cerebellum Cerebral cortex Hippocampus Retina Frontal cortex Hypothalamus Amygdala Septum Pons/Medulla Olfactory bulb Spinal cord a

Immunoreactive DARPP-32 (pmol mg-I total protein) 133.6 + 11.0 129.5 + 9.4 112.3 + 7.0 77.3 + 14.6 60.3 + 12.4 16.5 + 3.2 14.8 + 2.6 8.0-+ 3.2 7.4 + 2.1 6.3 + 3.0 6.0 _+ 1.0 5.0 & 1.0 4.3 & 1.2 3.4 & 0.8 2.2 + 0.6 1.1 + 0.6 0.6 + 0.4

From Ref. 32.

sessing D1 receptors, and showed no apparent correlation with the distribution of cells possessing only D2 receptors. The regional distribution of DARPP-32 in the rat CNS as determined by radioimmunoassay is shown in Table III. The highest concentrations of immunoreactive DARPP-32 were found in substantia nigra, caudatoputamen and globus pallidus, whereas slightly lower concentrations were found in olfactory tubercle and nucleus accumbens. More detailed information was obtained by immunocytochemistry; the results are summarized schematically in Fig. 3. Strong DARPP-32 immunoreactivity was observed within cell bodies and dendrites of neurons in the caudatoputamen (Fig. 4), nucleus accumbens and olfactory tubercle. In contrast, strong DARPP-32 immunoreactivity was observed within axon terminals in the globus pallidus and substantia nigra pars reticulata (Fig. 4) 3]. In the caudatoputamen, nucleus accumbens and olfactory tubercle, DARPP-32 was localized to the medium-sized spiny neurons, which directly receive most of the dopamine input to the neostriatum a3,a4 and comprise about 96% of the neurons in this brain region 35. Electron micro-

Fig. 2, Inhibition of the catalyticsubunit of purified protein phosphatase-1 by various concentrations of phospho-DARPP-32 (o-o) or dephosphoDARPP-32 (A-A). Proteinphosphatase activity was determinedby measurin&the release of [32P]phosphate from 32p-labelled phosphorylase-a. (Modifiedfrom Ref.

19.)

10-7

(M) 379

scopic immunocytochemistry has shown that DARPP-32 is a cytosolic protein present throughout the cytoplasm of the cell bodies of the medium-sized spiny neurons of the caudatoputamen, and in their dendrites, axons and axon terminals36. In contrast, DARPP-32 was found to be absent from the nigrostriatal and mesolimbic dopaminergic neurons and their axon terminals. The distribution of DARPP-32 in rat brain was in excellent agreement with the distribution of D1 receptors as determined by quantitative receptor

Fig. 3. Diagrams of coronal sections through the rat brain at the following levels: (A) forceps minor; (B) rostral caudatoputamen; (C) septum; (D) globus pallidus; (E) arcuate nucleus; (F) substantia nigra. Only the strongly immunoreactive cells and processes are represented. Stippled areas, labelled neuronal cell bodies and processes; • (E only), labelled glial cells; densely packed lines, high density of labelled axons and puncta; less densely packed lines (F only), low density of axons and puncta. Abbreviations: A, nucleus accumbens; AC, anterior commissure; ACe, central amygdaloid nucleus; ALP, lateral posterior amygdaloid nucleus; AR, arcuate nucleus; CC, corpus callosum; CP, caudatoputamen; GP, globus pallidus; HI, hippocampus; IC, internal capsule; ICa, islands of Calleja; IP, interpeduncular nucleus; LOT, lateral olfactory tract; LV, lateral ventricle; A4G, medial geniculate nucleus; MH, medial habenula; OT, olfactory tubercle; PC, substantia nigra, pars compacta; PR, substantia nigra, pars reticulata; Rh, rhinal fissure; SC, superior colliculus; VP, ventral pallidum. DARPP-32 immunoreactivity was very strong in neuronal cell bodies and dendrites in those brain regions that are enriched in dopamine-containing axon terminals: the caudatoputamen, nucleus accumbens septi, olfactory tubercle, dorsal part of the bed nucleus of the stria terminalis, and portions of the amygdaloid complex. Within the caudatoputamen, immunoreactivity was present in medium-sized (10-15 i~m) neuronal cell bodies, dendrites, spines and axons. A small number of axon terminals in the caudatoputamen were also immunoreactive, and these typically formed synapses on immunoreactive elements. In parasagittal sections, immunoreacrive fiber bundles were traced from the caudatoputamen to the globus pallidus, entopenduncular nucleus, and pars reticulata of the substantia nigra; within these regions, the labelled axons gave rise to immunolabelled axon terminals. (Taken, with permission, from Ref. 31.) 380

autoradiography6-9. Thus, DARPP-32 may represent a useful molecular marker for dopaminoceptive cells within the mammalian CNS that possess D1 receptors. Recently, the ontogenetic development of DARPP32 has been investigated in the CNS of the prenatal and newborn rat by immunocytochemistry37. DARPP32 immunoreactivity first appeared in the rat brain at day 14 of gestation in the anlage of the caudatoputamen, with the number of immunoreactive cell bodies increasing rapidly over the next two days. The arrival of tyrosine hydroxylase-immunoreactive axon terminals followed the appearance of DARPP-32immunoreactive cell bodies by at least two days. Thus, the development of DARPP-32 did not appear to depend on the prior appearance of dopaminergic innervation.

Possible physiological roles of DARPP-32 The observation that dopamine acting at D1 receptors increases the state of phosphorylation of DARPP-32 in intact nerve cells suggests that DARPP-32 acts as an intracellular 'third messenger' for dopamine. As such, it is presumably involved in the regulation of one or more specific dopamineregulated physiological processes in Drcontaining cells. DARPP-32 probably mediates some of the effects of dopamine through its ability to act as a protein phosphatase inhibitor. Fig. 5 illustrates one possible mechanism by which the inhibition of protein phosphatase-1 by phosphoDARPP-32 may regulate some of the physiological effects produced by dopamine. Regions of the brain that contain DARPP-32, most notably the striatum, also contain many other substrates for cyclic AMPdependent protein kinase TM. It is likely that many of these substrates can be dephosphorylated by protein phosphatase-1, since protein phosphatase-1 has a relatively broad substrate specificity3'23. The phosphorylation and activation of DARPP-32 in these brain regions could therefore inhibit the dephosphorylation of these other substrates for cyclic AMP-dependent protein kinase, and thereby amplify the effects of cyclic AMP by a positive feedback mechanism. Inhibition of protein phosphatase-1 by phosphoDARPP-32 may also allow interactions between dopamine and other neurotransmitters acting on D1containing cells. Since protein phosphatase-1 most likely dephosphorylates phosphoproteins that are substrates for a variety of other protein kinases3'23, phosphorylation and activation of DARPP-32 could allow dopamine, acting through cyclic AMP, to modulate the phosphorylation state of substrates for other second messenger-regulated protein kinases. By this mechanism, dopamine and cyclic AMP would be able to interact with other first and second messenger systems at the level of protein phosphorylation by regulating the state of phosphorylation of some of the same substrate proteins. This effect is illustrated schematically in Fig. 6 for dopamine interacting with glutamate. In Fig. 6, dopamine is shown to regulate the phosphorylation of DARPP-32 (through cyclic AMP), while glutamate is shown as an example of a neurotransmitter that may produce some of its effects through the elevation of intracellular Ca2+ concentration. The biochemical mechanisms discussed above TINS, Vol. 10, No. 9, 1987

may underlie the electrophysiological interactions between dopamine and glutamate in the striatum, where dopamine has been observed to antagonize the excitatory effect of glutamate on striatal neuronsaS, apparently through a postsynaptic mechanism. The synaptic organization of the striatum supports this suggestion. The caudatoputamen receives a large excitatory projection from the cerebral cortex, consisting primarily of glutaminergic fibersa9, which together with nigrostriatal dopaminergic axon terminals make synaptic contact with dendritic spines of medium-sized spiny striatal neurons in the caudatoputamen33'34. Moreover, the corticostriatal fibers terminate with asymmetric (Gray type I) synapses on the distal heads of these dendritic spines. This type of synapse is characterized by the postsynaptic density (PSD), a proteinaceous organeUe associated with the postjunctional membrane, which has been found to be selectively associated with both the NMDA (N-methyl-D-aspartate) and quisqualate types of glutamate receptors4° and which contains both Ca2+/calmodulin-dependent protein ldnase I141-4a and protein phosphatase-144. Glutamate-induced changes in intracellular Ca2+ concentrations resulting fl-om membrane depolarization and the activation of voltage-dependent Ca ~+ channels45, from the activation of NMDA receptor-coupled Ca2+ channels46 or from the activation of glutamate receptors coupled to the formation of inositol 1, 4, 5-trisphosphate 47, may be involved in producing some of the physiological effects of glutamate on neurons. The CaZ+-mediated effects of glutamate may interact with the cyclic AMPmediated effects of dopamine at the level of protein phosphorylation/dephosphorylation. At least two mechanisms for the observed inhibitory action of dopamine on glutamate excitability, involving DARPP-32 inhibition of protein phosphatase-1, are possible. First, a protein (X in Fig. 5) phosphorylated in response to a dopamine-induced elevation of cyclic AMP, may have a direct inhibitory effect on glutamate excitability, which would be potentiated by DARPP-32 phosphorylation if this phosphoprotein (phospho-X) were a substrate for protein phosphatase-1. Second, the inhibition of protein phosphate-1 resulting from the dopamine- and cyclic AMP-stimulated phosphorylation of DARPP-32 may lead to the decreased dephosphorylation of a protein (Z in Fig. 6) phosphorylated in response to a glutamate-induced elevation of Ca2+; this phosphoprotein would then exert an inhibitory effect (negative feedback) on glutamate excitability 48. The interaction of dopamine and cyclic AMP with other first and second messenger systems at the level of protein phosphorylation is also possible by another mechanism. Given the probable cellular co-localization of DARPP-32 and protein phosphatase-2B, and the observation that DARPP-32 is a particularly effective substrate for this protein phosphatase in vitro, a physiological role for protein phosphatase-2B in catalysing the dephosphorylation of DARPP-32 in vivo is likely19'22. The Ca2+-induced dephosphorylation of DARPP-32 by protein phosphatase-2B provides a possible mechanism by which Ca2+, acting as a second messenger, may antagonize some of the effects of the dopamine-induced cyclic AMP signal in dopaminoceptive neurons. Further biochemical and electrophysiologial studies TINS, Vol. 10, No. 9, 1987

Fig. 4 Low power photomicrograph illustrating the distribution of DARPP-32 immunoreactivi~ in a horizontal section of rat brain. DARPP-32 was stained using mouse monoclonal antibodies to purified bovine DARPP-32 with immunoperoxidase detection. Strong immunoreactivity can be seen throughout the caudatoputamen (CP), and in the globus pallidus (GP) and substantia nigra (SN). At higher magnification, and through the use of electron microscopy, it was found that DARPP-32 immunoreactivity is located predominantly in neuronal cell bodies and dendrites in the caudatoputamen, and in axon terminals in the globus pallidus and substantia nigra (Ouimet, C. C., Hemmings, H. C., Jr and Greengard, P., unpublished observations).

physiological effects

Fig. 5. Schematic diagram illustrating a hypothetical positive feedback mechanism by which DARPP-32 may be involved in regulating some of the physiological effects of dopamine acting on Dl-dopaminoceptive cells. The first messenger dopamine, by interacting with DI dopamine receptors, activates adenylate cyclase and thereby elevates intracellular cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Cyclic AMP-dependent protein kinase then stimulates the phosphorylation of DARPP-32 and of various other substrate proteins in target cells. The phosphorylation of DARPP32 converts it to an active inhibitor of protein phosphatase-1. PhosphoDARPP-32 decreases the dephosphorylation of some of the other proteins (represented by X) that are substrates for both cyclic AMP-dependent protein kinase and protein phosphatase-1. By increasing the state of phosphorylation of X, which is involved in producing the physiological effects of dopamine acting at D~ dopamine receptors, phospho-DARPP-32 represents a positive feedback signal through which some of the actions of dopamine may be amplified. 381

Selected references

~1~

~MP

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Z

Z

Ii

IXOt~

, i'~lNll~$

ohosDho DARPP-32

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Fig. 6. Schematic diagram illustrating a hypothetical mechanism by which

DARPP-32 may be involved in mediating interactions between dopamine and another neurotransmitter (glutamate in the example given) at the level of protein phosphorylation. Glutamate stimulation produces membrane depolarization, activates Ca2+ channels and/or stimulates inositol 1,4,5-trisphosphate formation. This leads to increased intracellular Ca2+ concentrations and thereby to the activation of Ca2+-dependent protein kinase(s) and to the phosphorylation of specific substrate proteins (represented by Z). These proteins are in turn involved in mediating some of the physiological effects of glutamate, inducting negative feedback on glutamate excitation. By inhibiting protein phosphatase1 activity and decreasing the dephosphorylation of Z, phospho-DARPP-32 represents an intracellular signal through which dopamine, acting via cAMP and cAMP-dependent protein kinase, may modulate the action of glutamate. This scheme provides a possible mechanism to explain the inhibitory effects of dopamine on glutamate excitability.

should help to elucidate the precise molecular mechanisms involved in mediating the physiological responses of neurons to dopamine as well as the interactions between dopamine and other neurotransmitters. The discussion above describes some potential interactions between the cyclic AMP and Ca2+ second messenger systems, which may be mediated through the regulation of the state of phosphorylation of DARPP-32 and the concomitant control of protein phosphatase-1 activity. These interactions provide molecular mechanisms by which a protein phosphatase inhibitor could mediate either synergistic or antagonistic effects between two first messengers through the second messengers cyclic AMP and Ca2+. A synergistic effect of cyclic AMP on the effects of Ca2+ could occur by the cyclic AMPdependent phosphorylation of DARPP-32, leading to the inhibition of the dephosphorylation of specific substrate proteins for Ca2+-dependent protein kinases. Alternatively, by activating protein phosphatase-2B, Ca2+ may antagonize the effects of cyclic AMP by causing the dephosphorylation of DARPP-32 and possibly other substrate proteins for cyclic AMPdependent protein kinase. It is clear that there is great potential for a variety of types of interactions between neurotransmitters at the level of neuronal protein phosphatase regulation. It is likely that these interactions occur in many different types of neurons and between various pairs of neurotransmitters. 382

1 Nestler, E. J. and Greengard, P. (1983) Nature 305,585-588 2 Stoof, J. C. and Kebabian, J. W. (1984) Life Sci. 35, 22812296 3 Cohen, P. (1982) Nature 296, 613-620 4 Miller, R. J. and McDermed, J. (1979) in The Neurobiologyof Dopamine (Horn, A. S., Korf, J. and Westerink, B. H. C., eds), pp. 159-177, Academic Press 5 Spano, P. F., Govoni, S. and Trabucchi, M. (1976) J. Neurochem. 27, 1565-1568 6 Boyson, S. J., McGonigle, P. and Molinoff, P.B. (1986) J. Neurosci. 6, 3177-3188 7 Dawson, T. M., Gehlert, D. R., McCabe, R.T., Barnett, A. and Wamsley, J. K. (1986) J. Neurosci. 6, 2352-2365 8 Dubois, A., Savasta, M., Curet, O. and Scatton, B. (1986) Neuroscience 19, 125-137 9 Savasta, M., Dubois, A. and Scatton, B. (1986) Brain Res. 375, 291-301 10 Hemmings, H. C., Jr, Walaas, S.I., Ouimet, C.C. and Greengard, P. in Receptor Biochemistry and Methodology (Vol. 9: Structure and Function of Dopamine Receptors) (Creese, I. and Fraser, C. M., eds), Alan R. Liss (in press) 11 Kebabian, J. W., Petzold, G. L. and Greengard, P. (1972) Proc. Natl Acad. 5ci. USA 69, 2145-2149 12 Siggins, G. R., Hoffer, B. J., Bloom, F. E. and Ungerstedt, U. (1976) in The Basal Ganglia (Yahr, M. D., ed.), pp. 227248, Raven Press 13 Molloy, A. G. and Waddington, J. L. (1984) Psychopharmacology 82,409-410 14 Walaas, S. I., Nairn, A.C. and Greengard, P. (1983) J. Neurosci. 3, 302-311 15 Walaas, S. I., Aswad, D. W. and Greengard, P. (1983) Nature 301, 69-71 16 Walaas, S. I. and Greengard, P. (1984)J. Neurosci. 4, 84-98 17 Forn, J., KruEger, B. K. and Greengard, P. (1974) Science186, 1118-1120 18 Hemmings, H. C., Jr, Nairn, A.C., Aswad, D.W. and Greengard, P. (1984)J. Neurosci. 4, 99-110 19 Hemmings, H. C., Jr, Greengard, P., Tung, H.Y.L. and Cohen, P. (1984) Nature 310, 503-505 20 Hemmings, H. C., Jr, Nairn, A. C. and Greengard, P. (1984) J. BioL Chem. 259, 14491-14497 21 Hemmings, H. C., Jr, Williams, K. R., Konigsberg, W. H. and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490 22 King, M. M., Huang, C.Y., Chock, P.B., Nairn, A.C., Hemmings, H. C., Jr, Chan, K-F. J. and Greengard, P. (1984) J. Biol. Chem. 259, 8080-8083 23 Ingebritsen, T. S. and Cohen, P. (1983) Science 221, 331338 24 Wallace, R. W., Tallant, E.A. and Cheung, W.Y. (1980) Biochemistry 19, 1831-1837 25 Goto, S., Matsukado, Y., Mihara, Y., Inoue, N. and Miyamoto, E. (1986) Brain Res. 397, 161-172 26 Huang, F. L. and Glinsmann, W. H. (1976) Eur. J. Biochem. 70, 419-426 27 Nimmo, G. A. and Cohen, P. (1978) Eur. J. Biochem. 87, 341-351 28 Williams, K. R., Hemmings, H. C., Jr, LoPresti, M. B., Konigsberg, W. H. and Greengard, P. (1986) J. BioL Chem. 261, 1890-1903 29 Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 126, 235-246 30 Aitken, A. and Cohen, P. (1982) FEB5Left. 147, 54-58 31 Ouimet, C. C., Miller, P. E., Hemmings, H. C., Jr, Walaas, S. I. and Greengard, P. (1984)J. Neurosci. 4, 111-124 32 Hemmings, H. C., Jr and Greengard, P. (1986) J. Neurosci. 6, 1469-1481 33 Pickel, V. M., Beckley, S. C., Joh, T. H. and Reis, D. J. (1981) Brain Res. 225, 373-385 34 Freund, T. F, Powell, J. F. and Smith, A. D. (1984) Neuroscience 13, 1189-1215 35 Pasik, P., Pasik, T. and DiFiglia, M. (1979) in The Neostriatum (Divac, I. and Oberg, G. E, eds), pp. 5-36, Pergamon Press 36 Ouimet, C. C., Hemmings, H. C., Jr and Greengard, P. (1983) 5oc. Neurosci. Abstr. 9, 82 37 Foster, G. A., Schultzberg, M., H6kfelt, T., Goldstein, M., Hemmings, H.C., Jr, Ouimet, C.C., Walaas, S.I. and Greengard, P. J. Neurosci. (in press) 38 Brown, J. R. and Arbuthnott, G. W. (1983) Neuroscience10, 349-355

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39 Divac, I., Fonnum, F. and Storm-Mathisen, J. (1977) Nature 266, 377-378 40 Fagg, G. E. and Matus, A. (1984) Proc. Natl Acad. Sci. USA 81, 6876-6880 41 Kelly, P. T., McGuinness, T. L. and Greengard, P. (1984) Proc. Natl Acad. Sci. USA 81,945-949 42 Kennedy, M. B., Bennett, M. K. and Erondu, N. (1983) Proc. Natl Acad. Sci. USA 80, 7357-7361 43 Goldenring, J. R., McGuire, J.S., Jr and DeLorenzo, R.J. (1984) J. Neurochem 42, 1077-1084

44 Shields, S. M., Ingebritsen, T.S. and Kelly, P.T. (1985) J. Neurosci. 5, 3414-3422 45 Sonnhof, U. and Biihrle, C. (1981) Adv. Biochem. Psychopharmacol. 27, 195-204 46 MacDermott, A. B., Mayer, M. L., Westbrook, G. L., Smith, S. J. and Barker, J. L. (1986) Nature 321,519-522 47 Sladeczek, F., Pin, J-P., R~casens, M., Bockaert, J. and Weiss, S. (1985) Nature 317, 717-719 48 Nestler, E. J., Walaas, S. I. and Greengard, P. (1984) Science 225, 1357-1364

books Control of Human Voluntary Movement

the book concerns mechanisms operating at the spinal and peripheral level. Observations in by John C. Rothwell, Croom Helm, normal human subjects and in patients with disordered motor 1987. £17.50 pbk/£35.00 hbk (xfi + 325 pa&es) ISBN 0 7099 4229 X systems are discussed with the perspectives derived from fundamental studies in animals, but the Robert E. Burke The jacket description states that emphasis throughout is clearly on Laboratoryof Neural 'This book aims to present a the latter. For anyone not already Control,NINCD5, single text on motor control that convinced, the book is an excelNationallnsitutesof is at a level intermediate between lent example of the absolute Health,Bethesda,MD 20892, USA. basic physiology texts and ex- necessity of animal studies to an haustive research volumes. Thus, understanding of the human some background knowledge . . . condition. will be assumed.' This is a quite Rothwell has organized his accurate summation - a great material along traditional anadeal of territory is covered in a tomical lines, ascending the little over 300 pages and some motor system hierarchy from initial familiarity with the termin- muscle to basal ganglia (the first ology and conventional notions and last chapters, respectively), of motor system hierarchies will beginning each chapter with enhance the reader's enjoyment. morphological considerations and The latter term is used advisedly, then proceeding to physiological because Rothwell writes with observations and functional interclarity and style. It also helps, of pretations. The narrative has course, to take the middle road - enough detail for clarity and logic neither pedagogical text nor but sufficient momentum to carry minutely documented monograph. the general reader along - a This allows an author to select, balance not easily achieved. The explore, and compare ideas with- book is well illustrated with figures out dulling the reader with elab- chosen from original papers. orate footnotes and caveats. Rothwell's approach fails only in Within the organizational frame- the chapter on ascending and work he has chosen, Rothwell descending pathways in the spinal succeeds to a remarkable degree cord, which is a routine listing of in covering many of the major anatomical facts, reflecting the issues in motor control, with ex- relative paucity of compelling plicit attention to a very wide functional data directly associated range of the current literature. with this topic. Each chapter is followed by useful Throughout the book, one lists of reviews and original papers, finds refreshing attention to quite about half of them dated between recent data and to interpretations 1980 and 1985. that deviate from the convenThe scope of this book is tional wisdom of formal textbroader than implied by its title. It books. Rothwell provides lucid is neither primarily focused on discussion of recent viewpoints human subjects nor on voluntary on such issues as: the mechanmovement. Instead, at least isms underlying orderly motor three-quarters of the material unit recruitment (admittedly a deals with results from animal personal favorite of the reviewer); experiments and more than half the possible roles of proprioTINS, Vol. 10, No. 9, 1987

ceptors in the control of force, length, stiffness, etc.; the important implications of convergence of afferent and descending input systems onto 'reflex' pathways in the spinal cord; the existence and potential routing of 'transcortical reflexes'; and the role of the cerebellum and its output nuclei in the initiation of voluntary movement. By and large, Rothwell's choices of material convey a useful overview of current ideas in motor systems physiology. I do have one complaint in addition to the inevitable (but scattered) typos and misreferences. The choice of the anatomical schema to organize the material leaves no convenient place for unified consideration of the topic of posture and locomotion control, as the author indeed notes in the 'Foreword'. It is unfortunate to have no explicit discussion of central pattern generation and its interaction with sensory information and voluntary commands. Material on postural mechanisms is scattered through the chapters on spinal, cortical, and cerebellar organization, instead of in one clearly defined section. A general reader needs a clearer view of the fact that 'movements' necessarily begin and end with 'postures', and that control of body attitude and center of gravity are inextricable components of every voluntary movement. I raise these points because, overall, I like this book. It will be valuable for both clinical and research students, as well as for practising clinicians' wishing to have a convenient and concise overview of much current thinking in motor control. Among the recent summary treatments of motor systems physiology, this deserves attention from both neuroscientists and clinicians. 383