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The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in the these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.

Choline acetyltransferase (CATase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been purified from bovine caudate nucleus. The specific activity of the pure enzyme was 120-160 mumol of acetylcholine formed per mg per min. The purified enzyme separated into two bands (band A and band B) when electrophoresed in a pH 4.3 gradient gel. Both bands exhibited CATase activity. Antisera were prepared in rabbits to each form. Immunotitrations using either antisera resulted in quantitative precipitation of CATase activity from enzyme preparations at all stages of purification. Both antisera produced single immunoprecipitin lines in double-diffusion experiments when run against the enzyme at each stage of purity. Immunoprecipitin lines cut from double-diffusion gels contained CATase activity, demonstrating that the observed reaction was due to antibody interaction with the enzyme. Immunoelectrophoresis also showed a single immunoprecipitin line against the purified enzyme as well as against a crude caudate extract. These results indicated that immunochemically pure and specific antisera have been prepared to bovine CATase and that both the A and B forms of the enzyme have common antigenic sites. The antisera were utilized to localize the enzyme in bovine brain. The localization was exclusively neuronal with both antisera, and both forms of CATase were present in the same population of neurons. Subtle differences in the precise intracellular distribution of enzyme were observed with the two antisera, indicating that although the two molecular forms of CATase have antigenic sites in common they are not antigenically identical.

This study was undertaken to evaluate the levels of cAMP-regulated phosphoproteins in the striatum of patients with neurodegenerative diseases of the dopaminergic system. Postmortem samples of caudate nucleus and putamen from 24 control subjects, 23 patients with Parkinson disease, and 13 patients with progressive supranuclear palsy were studied with immunoblotting techniques. The levels of tyrosine hydroxylase were reduced in patients with Parkinson disease (levels were 24% and 10% of controls in caudate nucleus and putamen, respectively) and with progressive supranuclear palsy (levels were 11% and 6% of controls in caudate nucleus and putamen, respectively). Five phosphoproteins, which are present in striatal neurons and are likely to play a role in the postsynaptic actions of dopamine, were measured. These included ARPP-16, ARPP-19, ARPP-21 (cAMP-regulated phosphoproteins of Mr 16,000, 19,000, and 21,000, respectively), DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), and phosphatase inhibitor I. The levels of these phosphoproteins were inversely correlated with postmortem delay. In brains of patients with Parkinson disease or progressive supranuclear palsy with postmortem delays comparable to those of controls, the levels of these proteins as well as those of synaptic (synapsin I and synaptophysin) and glial (glial fibrillary acidic protein and myelin basic protein) markers were not significantly modified. We conclude that the levels of several phosphoproteins involved in signal transduction in striatal neurons are not altered in Parkinson disease and progressive supranuclear palsy. This observation supports the view that the striatal output neurons are intact in both diseases.

We analyzed the activities of acetylcholinesterase and butyrylcholinesterase, and of the metabolic enzymes enolase and lactate dehydrogenase, in the superior cervical ganglion, ciliary ganglion, dorsal root ganglion, stellate ganglion, and caudate nucleus of the cat; we found that these tissues possess very different levels of enzymic activities. The proportions of the alpha alpha, alpha gamma, and gamma gamma enolase isozymes are also quite variable. We particularly studied the molecular forms of acetylcholinesterase and butyrylcholinesterase, in normal tissues and in preganglionically denervated SCG, in comparison with earlier histochemical findings. The results are consistent with the premise that the G1 (globular monomer) forms of both enzymes are located in the cytoplasm, the G4 (globular tetramer) forms are at the plasma membranes, and the A12 (collagen-tailed, asymmetric dodecamer) form of acetylcholinesterase is at synaptic sites.

An excess of the tryptophan metabolite quinolinic acid in the brain has been hypothetically related to the pathogenesis of Huntington disease. Quinolinate's immediate biosynthetic enzyme, 3-hydroxyanthranilate oxygenase (EC 1.13.11.6), has now been detected in human brain tissue. The activity of 3-hydroxyanthranilate oxygenase is increased in Huntington disease brains as compared to control brains. The increment is particularly pronounced in the striatum, which is known to exhibit the most prominent nerve-cell loss in Huntington disease. Thus, the Huntington disease brain has a disproportionately high capability to produce the endogenous "excitotoxin" quinolinic acid. This finding may be of relevance for clinical, neuropathologic, and biochemical features associated with Huntington disease.

By use of a sensitive in vitro microsuperfusion method, the cholinergic prsynaptic control of dopamine release was investigated in a prominent striosome (areas poor in acetylcholinesterase activity) located within the core of cat caudate nucleus and also in adjacent matrix area. The spontaneous release of [3H]dopamine continuously synthesized from [3H]tyrosine in the matrix area was found to be twice that in the striosomal area; the spontaneous and potassium-evoked releases of [3H]dopamine were calcium-dependent in both compartments. With 10(-6) M tetrodotoxin, 5 x 10(-5) M acetylcholine stimulated [3H]dopamine release in both striosomal and matrix areas, effects completely antagonized by atropine (10(-6) M), thus showing the involvement of muscarinic receptors located on dopaminergic nerve terminals. Experiments without tetrodotoxin revealed a more complex regulation of dopamine release in the matrix: (i) In contrast to results seen in the striosome, acetylcholine induced only a transient stimulatory effect on matrix dopamine release. (ii) Although 10(-6) M atropine completely abolished the cholinergic stimulatory effect on [3H]dopamine release in striosomal area, delayed and prolonged stimulation of [3H]dopamine release was seen with atropine in the matrix. The latter effect was completely abolished by the nicotinic antagonist pempidine (10(-5) M). Therefore, in the matrix, in addition to its direct (tetrodotoxin-insensitive) facilitatory action on [3H]dopamine release, acetylcholine exerts two indirect (tetrodotoxin-sensitive) opposing effects: an inhibition and a stimulation of [3H]dopamine release mediated by muscarinic and nicotinic receptors, respectively.

The hydrolysis of [Leu]enkephalin and substance P by purified pig kidney endopeptidase (EC 3.4.24.11) and synaptic membranes prepared from pig caudate nuclei has been compared. The hydrolysis of an enkephalin analogue (Tyr-D-Ala-Gly-Phe-Leu) at the Gly-Phe bond was completely inhibited by phosphoramidon. The IC50 concentration (8 nM) was similar to that reported for [Leu]enkephalin hydrolysis by the purified endopeptidase [Fulcher, I. S., Matsas, R., Turner, A. J. & Kenny, A. J. (1982) Biochem. J. 203, 519-522]. Seven peptides were produced when substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) was hydrolyzed by the kidney endopeptidase. These were formed by cleavage at bonds Gln-Phe (positions 6 and 7), Phe-Phe (positions 7 and 8), and Gly-Leu (positions 9 and 10). Synaptic membranes generated peptides with the same HPLC retention times and hydrolysis of substance P by either preparation was inhibited completely by 10 microM phosphoramidon. The most susceptible bond appeared to be Gly-Leu (positions 9 and 10). A specific polyclonal antibody raised in rabbits to purified pig endopeptidase inhibited the hydrolysis of [Leu]enkephalin and substance P by detergent-solubilized kidney microvilli or synaptic membranes; the titration curves were essentially identical. We conclude that the endopeptidase, which we suggest should be designated "endopeptidase-24.11," is present in caudate synaptic membranes and could play an important role in the hydrolysis of neuropeptides.

Unilateral radiofrequency lesions or chemical lesions with 6-hydroxydopamine were produced in the substantia nigra of rat brain in order to destroy dopaminergic innervations to caudate nucleus and thereby to produce functional denervation supersensitivity. Both types of lesions resulted in enhanced stimulation of caudate adenylate cyclase (EC 4.6.1.1) activity by dopamine at all dopamine concentrations tested, with more marked enhancement at the lower concentrations. Response to another dopamine agonist, 1-(3,4-dihydroxybenzyl)-4-(20pyrimidinyl) piperazine (S584) was also enhanced. 6-Hydroxydopamine lesions resulted in selective enhancement of the dopamine-stimulated component of adenylate cyclase, whereas radiofrequency lesions resulted also in a marked decrease in basal activity. It is postulated that the basal activity of caudate represents primarily an adenylate cyclase distinct from that stimulated by dopamine and destroyed only by the less selective radiofrequency lesion. The enhancement of dopamine-sensitive adenylate cyclase after lesions serves as indirect evidence for a significant role of this system in the transmitter function of dopamine and indicates, furthermore, that it is directly involved in dopamine receptor supersensitivity in vivo produced by denervation.

The manner in which dyskinesia and intermittency of neurological control had emerged late in the therapy of Parkinsonism with L-3,4-dihydroxyphenylalanine (levodopa) had suggested to us that this drug can imprint on the brain a chemical memory of its passage. The majority of authors ascribed these events to denervation hypersensitivity caused by the nigral and other lesions of the disease. By feeding levodopa to mice, however, we induced a state that simulated denervations hypersensitivity, including hyperreaction to single injections of levodopa and increased dopamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in homogenates of caudate nuclei. These phenomena were not caused by actual denervation, because the hypersensitivity declined and disappeared some weeks after the dietary levodopa was stopped.

The distribution of dopamine-containing processes in the striatum of fetal and neonatal cats was studied by immunohistochemical and glyoxylic acid histofluorescence methods and compared to the distribution of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) observed by thiocholine histochemistry in the same or serially adjoining sections. Both methods for demonstrating the dopamine innervation revealed the characteristic patchwork of dopamine "islands" in the caudoputamen, in which catecholamine histofluorescence or tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]-like immunoreactivity was concentrated into 0.2- to 0.6-mm-wide patches. Both methods also demonstrated a high degree of patterning of the dopamine innervation in the ventral striatum, including the nucleus accumbens septi. A detailed and striking match was found between these configurations and the compartmental distribution of acetylcholinesterase observed in the caudoputamen and ventral striatum of the same brains. The correspondence between the dopamine and acetylcholinesterase figures was most obvious in the fetal brains, in which the background acetylcholinesterase staining was lightest, but matches between the dopamine islands and acetylcholinesterase patches could still be seen in the kittens. There was no clear alignment of striatal cell bodies stained for acetylcholinesterase with either the dopamine or the acetylcholinesterase-positive patches. Nor was there an obvious correspondence between dopamine and acetylcholinesterase in the striatal background matrix. We conclude that, at least during ontogenesis, it is the clustered arrangements of dopamine and acetylcholinesterase that are, in particular, tightly linked, and we suggest that information about the maturation of these clusters may be crucial in assessing the functions of striatal dopamine and acetylcholinesterase in the adult.

Adenylate cyclase (EC 4.6.1.1), selectively stimulated by low concentrations of dopamine, has been found in the olfactory tubercle, the nucleus accumbens, and the caudate nucleus of several mammalian species. Several different classes of drugs effective in the treatment of schizophrenia (antipsychotic drugs) were potent inhibitors of the stimulation by dopamine of the enzyme from these various regions. The drugs studied included representatives of the phenothiazine, butyrophenone, and dibenzodiazepine classes. The inhibition by these antipsychotic drugs was competitive with respect to dopamine. The most potent of the antipsychotic agents tested was fluphenazine, which had a calculated inhibition constant (K(i)) of about 5 x 10(-9) M. For each of several drugs tested, the K(i) for the enzyme from the olfactory tubercle was similar to that for the enzyme from the caudate nucleus. Several compounds closely related structurally to the psychoactive phenothiazines, but which have little or no antipsychotic or extrapyramidal actions clinically, had low relative potencies as inhibitors of dopamine-stimulated adenylate cyclase activity. The results, considered together with other data, raise the possibility that the therapeutic effects, as well as the extrapyramidal side effects, of these antipsychotic agents may be attributable, at least in part, to their ability to block the activation by dopamine of specific dopamine-sensitive adenylate cyclases in the human brain.

In contrast to antipsychosis drugs which inhibit the dopamine-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of caudate nucleus, dopaminergic drugs for treatment of Parkinson's disease stimulate this cyclase. Stimulants and inhibitors of cholinergic neurons inhibited this adenylate cyclase activity competitively and specifically. Thus, the mechanism by which dopaminergic medications ameliorate the effects of Parkinson's disease includes activation of the dopamine-sensitive adenylate cyclase. Excessive activation might be present during the psychotic episodes seen in patients with parkinsonism who are overtreated. The enzymatic effects of the drugs that affect cholinergic mechanisms seem to be generally in keeping with the pharmacological reciprocity between psychoses and extrapyramidal function, except for the anticholinergic ones which inhibited this cyclase although they can be hallucinogenic.

The distribution of acetylcholinesterase activity (acetylcholine acetylhydrolase, EC 3.1.1.7) in the developing human striatum has been studied histochemically in autopsy material from fetal brains of estimated gestational ages 16-29 weeks (180-1000 g) and from the brains of infants 2 days to 4 months old. The findings provide evidence that striatal acetylcholinesterase activity in the human fetus and neonate is concentrated in a network of densely stained zones that appear in cross section as variably shaped 0.5- to 1.0-mm-wide dark patches distributed in a lighter background matrix. An orderly arrangement of the patches seemed well established in the putamen by the 16th-18th week of gestation (crown-rump length 14-15 cm) but in the caudate nucleus the pattern was still not fully elaborated at these ages. The lateroventral part of the caput was mainly dark and its rostromedial margin, though rich early on in pseudocholinesterase activity, was still without strong acetylcholinesterase activity as late as 20-21 weeks (crown-rump length 16-20 cm). The ganglionic eminence at these ages was sharply divided into a dorsal part with little cholinesterase activity and a ventral part with a high content of pseudocholinesterase. Little information was gained about striatal development during late stages of gestation, but in three 5- to 7-month fetal specimens not only dark patches but also patches with dark perimeters and pale centers were present. Clumping of cholinesterase activity appeared at birth and up to the third month of postnatal life. The patches in both caudate nucleus and putamen were dark and of fairly uniform tint in the striatum of the young infant and the matrix staining was darker than in the fetuses. Around the fourth postnatal month hints of the mature pattern were present, with zones of low cholinesterase activity appearing against a dark background in the caudate nucleus and (in one case) a nearly homogeneous staining pattern appearing in the putamen.

We obtained 12 groups of mice with widely different neurological responses to levodopa by selecting them from different strains and submitting some of them to pretreatments. We scored the symptoms evoked by a standardized dose of levodopa in one subgroup from each group. We tested another subgroup for activation of an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by a standardized dose of dopamine added to homogenates of the caudate nuclei of the brains of these mice. Both sets of tests were performed randomly. When the accruing two sets of data were plotted against each other there emerged a straight line which fitted the data with a coefficient of correlation of 0.97 (P less than 0.0001). The dopamine-dependent activity of the adenylate cyclase of the brain was thus shown to be a determinant of the neurological responses of intact animals to a dopaminergic drug.

An adenylate cyclase that is activated specifically by low concentrations of dopamine has been demonstrated in homogenates of caudate nucleus of rat brain. A half-maximal increase in the activity of the enzyme occurred in the presence of 4 muM dopamine. Concentrations of dopamine as low as 0.3 muM stimulated the activity of the enzyme. The adenylate cyclase activity of the homogenates was also stimulated by low concentrations of apomorphine, a substance known to mimic the physiological and pharmacological effects of dopamine. The stimulatory effect of dopamine was blocked by low concentrations of either haloperidol or chlorpromazine, agents known to block the actions of dopamine in mammalian brain. The results suggest that dopamine-sensitive adenylate cyclase may be the receptor for dopamine in mammalian brain. The isolation of this enzyme from caudate nucleus should facilitate the search for new therapeutic agents useful in the treatment of extrapyramidal diseases.