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Enzymes concerned with neurotransmitter metabolism were measured postmortem in 50 regions from the brains of 11 chronic schizophrenics, 2 patients with senile dementia, 1 depressive, and 18 controls. Enzymes studied were tyrosine hydroxylase, dopa decarboxylase, glutamic decarboxylase, choline acetyltransferase (CAT), and acetylcholinesterase. The schizophrenic group had high CAT activities in the hippocampus, caudate, putamen, and nucleus accumbens; the other patients from the same hospital did not. A compensatory response to long- or short-term drug usage is considered, but correlations are hard to establish in the group studied. An alternative hypothesis proposes that the high levels are a compensatory response to defective cholinergic receptors in the affected areas. On this hypothesis, and by analogy with chorea, dopaminergic antagonists would act in schizophrenia by helping to reestablish cholinergic-dopaminergic balance.

Nine styrylpyridine analogs were tested as inhibitors of choline acetyltransferase which had been highly purified from rat cerebrum and bovine caudate nuclei. In general, concentrations required to achieve 50% inhibition (I50 values) were in the micromolar range. For some analogs, I50 values were similar to those obtained previously by other investigators who used less purified enzyme preparations. With certain analogs, however, the measured values of I50 changed as the transferase became more purified, which may indicate the presence in the extract of other molecules which can interact with the enzyme. The methods used in purification of the enzyme suggest that the molecule which modifies the activity of CAT is probably a protein. The mode of inhibition by naphthylvinylpyridinium was found to be uncompetitive with respect to both choline and acetyl coenzyme A for both the rat and bovine transferases.

Morphological maturation of the rat's caudate nucleus and formation of cholinergic system in it were studied in postnatal ontogenesis. Under investigation were newborn rats, 7, 14, 21 day old rats and adult animals. The growth and maturation of the caudate nucleys were most intesne during the first two postnatal weeks, it was somewhat descreased by the beginning of the third week and continued in later terms as well. The structure of dendrites and axons became complicated during the first two weeks, the axo-dendritic contacts being also formed. The neuron structure in 14 days old rats was similar to that of adult animals. The activity of acetylcholinesterase in the caudate nucleus of 7 and 14 days rats was not great. It sharply increased by the 17th day and reached the level of adults with 3 weeks after birth. Possible correlations of the morpho-chemical maturation of the caudate nucleus and formation of motor activity in rats is discussed.

The potency of beta-adrenoreceptor agonists, e.g., isoproterenol, is strikingly increased by substitution of the meta catecholic hydroxyl group with the NH group of a carbostyril system. To explore the possibility that comparable potency enhancement might occur upon similar modification of the catechol ring of dopamine, a series of 5-(2-aminoethyl)carbostyril derivatives was prepared and examined for D-1 and D-2 dopamine receptor-stimulating activity. Only the parent compound, 5-(2-aminoethyl)-8-hydroxycarbostyril (2), produced measurable activation of dopamine-sensitive adenylate cyclase (29% at a concentration of 10 microM). Some of the compounds, however, did produce significant activity in tests, namely displacement of [3H]spiroperidol binding from bovine pituitary homogenate and an isolated perfused rabbit ear artery preparation, that measure interaction with D-2 receptors. Potency of the carbostyrils was enhanced by 8-hydroxylation and by appropriate substitution of the amino group of the ethylamine side chain. The most potent member of the series was 8-hydroxy-5-[2-[[2-(4-hydroxyphenyl)ethyl]-n-propylamino]ethyl] carbostyril (16b). This compound was about 3 times more effective than dopamine in the D-2 receptor tests. Clearly, the results of this study indicate that potency of dopamine receptor agonists is not increased by carbostyril replacement of the m-hydroxyl as is noted with the beta-adrenergic receptor agonists.

We studied 5'-nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane-bound enzyme were purine nucleotides, particularly 5'AMP. Effects of metal cations and chelating agents suggest that 5'-nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5'-nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane-bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X-100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosidases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex-type glycosylation, with a fucose residue on the internal N-acetyl-D-glucosamine of the pentasaccharide core.

Postmortem samples of caudate nucleus and frontal cortex from schizophrenic, schizophrenic-like, and control subjects were examined for monoamine oxidase activity using dopamine, phenylethylamine, and 5-hydroxytryptamine as substrates. There were no significant differences between the diagnostic groups with any of the three substrates. Neither was there a difference between the sexes, nor a consistent relationship of enzyme activity to age.

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.

CuZn superoxide dismutase (SOD) and Mn SOD activities were analyzed in hypothalamus, nucleus caudatus, hippocampus and cortex gyrus cinguli from 12 chronic alcoholics and from 16 controls. The CuZn SOD activities were slightly lower and the Mn SOD activities were slightly higher in the brain pieces from chronic alcoholics compared to the controls. The slight differences found can hardly be assigned etiological importance in the degenerative processes in brain tissue connected with chronic alcoholism.

Six weeks following complete unilateral surgical isolation of the rat caudate nucleus, activation of adenylate cyclase was reduced in response to dopamine (DA), norepinephrine (NE), 5' -guanylyl-imidodiphosphate [Gpp(NH)p], DA + Gpp(NH)p, and NaF. The low Km form of cyclic AMP phosphodiesterase was elevated in the isolated side when compared to the intact caudate. No changes in activities of guanylate cyclase or in high Km cyclic AMP phosphodiesterase (with or without the calcium-dependent regulator protein, calmodulin or CDR) were observed between the control and isolated caudate. Histologically, the neural damage to the isolated caudate was principally confined to reduced numbers of dendritic spines of the remaining intrinsic caudate neurons.

In an attempt to determine whether or not acetylcholinesterase (AChE)-containing neurons of the caudate-putamen proper were the source of striatal efferent fibers, we infused Evans Blue, a retrogradely transported fluorescent label, into the globus pallidus, entopeduncular nucleus, substantia nigra, or retrorubral area. Following microscopic analysis of the striatum for Evans Blue-labelled somata, the same brain sections were processed for AChE according to the pharmacohistochemical regimen and, after additional microscopic evaluation, were counterstained with cresyl violet. Histology for Nissl substance revealed that the areal density of cell bodies in the caudate-putamen complex proper was about 1510 somata/mm2. Striatal neurons labelled with Evans Blue, those considered to be projection cells, were medium-sized (approximate minor and major dimensions: 11 X 14 microns), had a density of roughly 833 cells/mm2, and were predominantly oval with lesser proportions being fusiform, triangular, or round. Each of the target structures received input from approximately 55% (range = 26-78%) of the total population of striatal neurons in regions where the projection cellsions: 11 X 14 microns), had a density of roughly 833 cells/mm2, and were predominantly oval with lesser proportions being fusiform, triangular, or round. Each of the target structures received input from approximately 55% (range = 26-78%) of the total population of striatal neurons in regions where the projection cellsions: 11 X 14 microns), had a density of roughly 833 cells/mm2, and were predominantly oval with lesser proportions being fusiform, triangular, or round. Each of the target structures received input from approximately 55% (range = 26-78%) of the total population of striatal neurons in regions where the projection cells were located. The two types of AChE-containing somata in the caudate-putamen complex proper--the medium-sized, lightly staining Type A and the large, intensely staining Type B cell--had densities of 14 and 15 somata/mm2, respectively. None of the AChE neurons contained Evans Blue, indicating that they were not the source of striatal efferent fibers but rather interneurons that could be categorized best as the aspiny or sparsely spined cells described in Golgi studies.

Monoclonal antibodies to choline acetyltransferase (ChAT) were used in an immunocytochemical study to characterize putative cholinergic neurons and synaptic junctions in rat caudate-putamen. Light microscopy (LM) revealed that ChAT-positive neurons are distributed throughout the striatum. These cells have large oval or multipolar somata, and exhibit three to four primary dendrites that branch and extend long distances. Quantitative analysis of counterstained preparations indicated that ChAT-positive neurons constitute 1.7% of the total neuronal population. Electron microscopy (EM) of immunoreactive neurons initially studied by LM revealed somata characterized by deeply invaginated nuclei and by abundant amounts of organelle-rich cytoplasm. Surfaces of ChAT-positive neurons are frequently smooth, but occasional somatic protrusions and dendritic spines occur. Although infrequently observed, axons of ChAT-positive neurons branch, receive synapses, and become myelinated. Unlabeled boutons make both symmetrical and asymmetrical synapses with ChAT-positive somata and proximal dendrites, but are more numerous on distal dendrites. In addition, some unlabeled terminals form asymmetrical synapses with ChAT-positive somata and dendrites that are distinguished by prominent subsynaptic dense bodies. Light microscopy demonstrated a dense distribution of ChAT-positive fibers and punctate structures in the striatum, and these structures appear to correlate, respectively, with labeled preterminal axons and presynaptic boutons identified by EM. ChAT-positive boutons contain pleomorphic vesicles, and make symmetrical synapses primarily with unlabeled dendritic shafts. Furthermore, they establish synaptic contacts with somata, dendrites and axon initial segments of unlabeled neurons that ultrastructurally resemble medium spiny neurons. These observations, together with the results of other investigations, suggest that medium spiny GABAergic projection neurons receive a cholinergic innervation that is probably derived from ChAT-positive striatal cells. The results of this study also indicate that cholinergic neurons within caudate-putamen belong to a single population of cells that have large somata and extensive sparsely spined dendrites. Such neurons, in combination with dense concentrations of ChAT-positive fibers and terminals, are the likely basis for the large amounts of ChAT and acetylcholine detected biochemically within the neostriatum.

Tyrosine hydroxylase (TH) contents in the caudate nucleus, putamen, and substantia nigra from control and parkinsonism brains were measured for the first time by a sandwich enzyme immunoassay. Both the TH protein content and TH activity (Vmax) were decreased in parallel in the parkinsonian brains as compared with those of the control brains. In contrast, TH "homospecific activity" (activity per enzyme protein) was significantly increased in the parkinsonian brains. The results indicate that the decrease of TH activity in parkinsonian brains is due to the decrease of TH protein content as a result of cell death. The increase in the "homospecific activity" of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.

The effect of hydrogen peroxide on monoamine oxidase (MAO) activity has been determined in homogenates of human brain areas taken postmortem. It could be shown that hydrogen peroxide enhances significantly the activity of MAO-B after short-term incubation (2 min), while no changes have been noted after long-term preincubation (60 min) indicating reversibility of this effect. MAO-A activity was not changed or decreased after preincubation with hydrogen peroxide. Freezing and thawing procedures did not change hydrogen peroxide stimulation of MAO in the caudate nucleus, while MAO-A activity dose dependently decreased. Inhibition of hydrogen peroxide stimulated MAO-B activity in human cortex by (-)deprenyl was found to be of similar potency compared to hydrogen peroxide free estimations. Glutathione, ascorbic acid and mannitol did not block MAO stimulation by hydrogen peroxide, while sodium azide led to a complete inhibition of hydrogen peroxide derived MAO activitation. Interorgan comparison showed increase of MAO-B activity in crude mitochondrial fractions of rat liver after preincubation with hydrogen peroxide, while with rat heart a reduction of MAO activity was detectable. As a conclusion these data indicate a possible role of hydrogen peroxide in the age-dependent increase of MAO-B in platelets and brain. Furthermore, increase of cytotoxic hydrogen peroxide via combined L-dopa therapy cannot be excluded to be of importance in the appearance of adverse reactions after long-term treatment with high doses. To reduce hydrogen peroxide production due to MAO activity, a combined treatment of L-dopa plus a selective MAO inhibitor and eventually additional administration of radical scavengers (ascorbic acid, vitamin E etc.) seems to be indicated.

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.

The activity of the dipeptidyl carboxypeptidase, angiotensin converting enzyme, was assayed in several brain regions of patients dying with Alzheimer's disease and compared to that of appropriately age-matched controls. Enzyme activity was found to be elevated by 44% and 41% in the medial hippocampus and parahippocampal gyrus, respectively, and by 27% and 29% in the frontal cortex (area 10 of Brodman) and caudate nucleus, respectively, in Alzheimer's disease patients. Converting enzyme activity did not differ from controls in the nucleus accumbens, substantia nigra, temporal cortex, anterior or posterior hippocampus, amydgala, and septal nuclei.

Acid hydrolases and lysosomal membrane properties were studied at various ages in the normal human brain. In CSF and four brain regions, the inferior olive, the cerebellar cortex, the caudate nucleus and the frontal cortex were thus beta-galactosidase, beta-glucosidase, alpha-mannosidase, hexosaminidase and acid phosphatase biochemically quantitated at ages varying between 2 and 89 years of age. Also the membrane latency for acid phosphatase was studied in these regions. No major regional quantitative differences were found with regard to the enzymes studied. Their kinetic properties were also defined. There appeared to exist a regional and intra-areal variation in lysosomal membrane permeability. There was, however, no age related increase in total enzyme contents. The possibility significance of these findings are discussed with reference to the aging process.

The increase of 5-hydroxytryptophan after treatment with the decarboxylase inhibitor Ro 4/4602 was 30 times greater in the raphe nuclei (which contain serotonergic cell bodies) than the hippocampus or caudate (which contain serotonergic terminals). Pretreatment with chlorimipramine reduced the 5-hydroxytryptophan accumulation in the median but not dorsal raphe. The large amount of tryptophan hydroxylase in cell bodies appears to be active in vivo. The serotonin rapidly turning over there may have a functional role.

Electron cytochemistry was used to study the distribution of adenylate cyclase in the rat brain cortex and nucleus caudatus. In neurons and glial cells, the enzyme was localized on the outside surface of plasma membrane, within the perinuclear space, in the cisterns of endoplasmic reticulum and the Golgi apparatus. In capillaries, it was detected on the luminal surface of endothelial cells and along the basal layer. The addition of NaF and isoproterenol to the incubation medium led to the increased adenylate cyclase activity.

Cholinergic neurons in the monkey neostriatum were examined at the light and electron microscopic level by immunohistochemical methods in order to localize choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. At the light microscopic level a sparse distribution of cholinergic neurons was identified throughout the caudate nucleus. Neurons had large (25-30 microns) somata, eccentric invaginated nuclei, primary dendrites of unequal diameters, and varicosities on distal dendritic branches. Ultrastructural study showed that the cholinergic cells had a cytoplasm abundant in organelles. Within dendritic branches, mitochondria and cisternae were localized primarily to varicosities. Synaptic inputs were distributed mostly to the dendrites and at least four types that formed symmetric or asymmetric synapses were observed. Immunoreactive fibers were relatively numerous within the neuropil and exhibited small diameters (0.1-0.15) micron) and swellings at frequent intervals. Cholinergic boutons that formed synapses were compared to unlabeled terminals making asymmetric synapses with dendritic spines. Results showed that ChAT-positive axons had significantly smaller cross-sectional areas, shorter synaptic junctions, and a higher density and surface area of mitochondria than the unlabeled boutons. Cholinergic axons formed symmetric synapses mostly with dendritic spines (53%) and the shafts of unlabeled primary and distal dendrites (37%). A relatively small proportion of the boutons contacted axon initial segments (1%) and cell bodies (9%) that included medium-sized neurons with unindented (spiny) and indented (aspiny) nuclei. The majority of dendritic spines contacted by cholinergic axons were also postsynaptic to unlabeled boutons forming asymmetric synapses. The results suggest that cholinergic neurons in the primary neostriatum belong to a single morphological class corresponding to the large aspiny (type II) interneuron identified in previous Golgi studies. Present results along with earlier Golgi-electron microscopic observations from this laboratory suggest that neostriatal cholinergic cells integrate many sources of intrinsic and extrinsic inputs. The observed convergence of ChAT-immunoreactive boutons and unlabeled axons onto the same dendritic spines suggests that intrinsic cholinergic axons modulate extrinsic inputs onto neostriatal spiny neurons at postsynaptic sites close to the site of afferent input.

The partially purified soluble guanylate cyclase (GTP pyrophosphatelyase(cyclizing), EC 4.6.1.2) from human caudate nucleus is stimulated from 2 to 4-fold by metal chelating agents. EDTA (K 1/2 - 4.8 microM) is more potent than CDTA (K 1/2 = 13.2 microM) or EGTA (K 1/2 = 21.8 microM) at stimulating activity. Stimulation by chelating agents is apparently not due to removal of inhibitory divalent cations which contaminate the enzyme or reaction mixture. EDTA increases guanylate cyclase activity in part by increasing the affinity of the enzyme for the substrate (MgGTP) 10-fold. Dopamine inhibits partially purified guanylate cyclase in the presence or absence of EDTA. Dopamine increases the Ka of guanylate cyclase for the activator, free Mn2+, more than 50-fold, from 3 to 150 microM.

In human caudate nucleus and adrenal medulla dopamine-stimulated adenylate cyclase was measured. At a concentration of 100 microM dopamine, liver cirrhosis, carcinoma and therapy resistent parkinsonian patients showed decreased stimulation or even a reduction compared to controls. Therapy responding parkinsonian patients showed a decreased stimulation compared to controls, but this stimulation was significantly higher than in drug resistent patients.

The ultrastructural localization of choline acetyltransferase in various regions of rat's CNS is studied. It is established that the reaction product is located in some structures of the neuronal perikarya--on the outer surface of the rough endoplasmic reticulum sacs, along the vesicular profiles and in the mitochondria. Electron dense material is found in the axon terminal as well as, where enzyme activity is detected in the cytoplasm, on the membrane of spherical and flat synaptic vesicles and in the mitochondria. These findings, the control results and literature data are discussed.

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.

Measurements on human brain samples of some enzymes concerned with neurotransmitter synthesis suggest serious losses with age. The most severe loss found was that in striatal tyrosine hydroxylase activity, the rate-controlling enzyme in the synthesis of dopamine. Cell counts in the substantia nigra where this dopaminergic tract originates suggest that the decrease in enzyme activity is partly due to cell loss, but must largely reflect decreased activity of residual cells. It is possible that this loss may account for some of the difficulties in movement seen in aged individuals and that it might be less if pigment formation in these cells could be inhibited.

The distribution of glycerylphosphorylcholine diesterase in the rat brain has been examined. The enzyme was evenly distributed throughout the brain but was localized in the synaptosome (nerve ending) fraction which was prepared by ultracentrifugation.

Administration to rats of chronic electroconvulsive shock (ECS) or chronic desipramine (DMI, 10 mg/kg daily i.p. for 3 weeks) did not affect either basal or forskolin-stimulated adenylate cyclase activity in membranes prepared from the caudate nucleus. In cerebellar membranes prepared from rats which had received chronic ECS, forskolin-stimulated activity was significantly increased compared to activity in sham- or single ECS treated rats. Forskolin-stimulated adenylate cyclase was increased in both hippocampal and cerebellar membranes from rats which received chronic DMI, compared to saline-treated animals. In cerebellar membranes, increases comparable to those with forskolin were also obtained with guanylyl-5'-imidodiphosphate (GppNHp) after both treatments, while with Mn2+ ions, either alone or in the additional presence of forskolin, the changes observed were similar to those previously reported in cortical membranes. A possible mechanism for these effects was investigated by studying antidepressant-induced and in vitro desensitization of the cyclic AMP response in slice preparations from the various brain areas. In slices from caudate nucleus, chronic DMI did not alter stimulation of cyclic AMP formation by either noradrenaline or forskolin, while in cerebellar slices the noradrenaline response was significantly reduced, and in hippocampal slices both responses were reduced (heterologous desensitization). In vitro incubation of cortical slices with noradrenaline also resulted in a reduction in the response to both agents. However, in membranes prepared from the desensitized cortical slices, there was no change in the degree of activation of adenylate cyclase by either NaF or forskolin. Thus, the increase in these activities, observed in certain brain areas after chronic antidepressant treatment may not necessarily be related to beta-adrenoceptor desensitization.

Carboxylesterase activity of primate brain (Macaca mulatta) was determined using phenyl valerate (PV) as substrate. Eight carboxylesterases of primate brain were characterized in respect to PV-hydrolysing activity and to their inhibition rate constants for the reaction with organophosphorus compounds. Carboxylesterase III was identified as neurotoxic esterase (NTE). Organophosphate inhibition data of primate acetylcholinesterase (EC 3.1.1.7) and of primate cholinesterase (EC 3.1.1.8) were determined and are compared to corresponding data of primate brain carboxylesterases. Physiological functions, clinical and toxicological significance of primate brain carboxylesterases are discussed.

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.

A highly sensitive assay for acetylcholinesterase (AChE) activity was devised by high-performance liquid chromatography with electrochemical detection. It is based on the separation of acetylcholine and choline on an octadecylsilane reversed-phase column, followed by their enzymatic conversion into hydrogen peroxide through the post-column reaction with immobilized AChE and choline oxidase. The system is highly sensitive, and the relationship between the peak height and the amount of choline is linear over the range 5 pmol to 5 nmol. When homogenate of bovine caudate nucleus was used as enzyme, the Michaelis constant of the enzyme for acetylcholine was 0.4 mM. The regional distribution of AChE activity in rat brain was examined, and the order of the activity from the highest to the lowest agreed with the reported brain distribution of AChE: striatum, thalamus plus hypothalamus, pons plus medulla oblongata, cerebral cortex, olfactory bulb, and cerebellum.

SK&F 89124 (4-[2-(N,N-di-n-propylamino)ethyl]-7-hydroxy-2(3H) indolone) can be considered as a derivative of N,N-di-n-propyldopamine (DPDA) in which the meta-hydroxyl is replaced by a cyclic amide function. SK&F 89124 is at least one order of magnitude more potent than DPDA as an agonist at peripheral inhibitory prejunctional dopamine receptors (DA2 receptors) in the isolated perfused rabbit ear artery. A potent agonist action of SK&F 89124 at the DA2 receptor can also be demonstrated by inhibition of radioactive overflow from prelabelled canine coronary artery or saphenous vein, and in the anesthetized dog as an inhibition of the tachycardia induced by cardioaccelerator nerve stimulation or the increase in hind-limb perfusion pressure induced by stimulation of the lumbar sympathetic chain. SK&F 89124 is a potent inhibitor of the binding of [3H]spiroperidol to D2 receptors in bovine pituitary homogenates. High concentrations of SK&F 89124 do not activate the adenylate cyclase D1 receptor in rat caudate homogenates, nor produce activation of alpha 2-adrenoceptors or H2-histamine receptors in the guinea pig atrium. Although some alpha 1-adrenoceptor mediated vasoconstriction is produced in the rabbit ear artery and rabbit aorta, the concentrations required are several orders of magnitude higher than those active at the DA2 receptor. From these data it is evident that this structural modification can increase both the potency and selectivity of DPDA as a DA2 receptor agonist. The potency and selectivity of SK&F 89124 make this agent a useful tool for determination of the functional role of the DA2 receptor.

The activity of phosphate-activated glutaminase was reduced throughout the brain of cases with longstanding illnesses (agonal controls) compared to cases dying suddenly. The reduction was less marked in cortical than sub-cortical areas, with the caudate nucleus occupying an intermediate position. In control brains succinic dehydrogenase and pyruvate dehydrogenase were little affected by the ante-mortem clinical state. Of 9 brain areas studied, only the caudate nucleus showed a reduction of phosphate-activated glutaminase and succinic dehydrogenase in Huntington's disease greater than in agonal controls. The levels of succinic dehydrogenase and pyruvate dehydrogenase were highly correlated in frontal cortex and in caudate nucleus of Huntington's disease and control brains. There was a significant reduction in pyruvate dehydrogenase mean activity and a significant increase in gamma-glutamyl transpeptidase mean activity in Huntington's disease caudate nucleus. The level of pyruvate dehydrogenase significantly decreased and the level of gamma-glutamyl transpeptidase significantly increased with increasing duration of illness, possibly due to a progressive loss of neurons and increase in the density of glia in Huntington's disease caudate nucleus.

Tryptophan hydroxylase (TPH) activity was measured in various rat brain regions after administering large doses of methamphetamine (METH). After four sequential doses of METH (15 mg/kg), given every 6 hr, TPH activity was decreased (to approximately 10% of control) in both the neostriatum and hippocampus. The depression of enzyme activity persisted for at least 30 days. When compared with the depression of neostriatal tyrosine hydroxylase activity, the depression of neostriatal and hippocampal TPH activity occurred sooner and was more pronounced. The depression of TPH activity was dependent on the number of doses and the amount of drug administered. Five days after one to two doses of METH, a transient recovery was observed but when four doses were given, the enzyme was depressed. No decrease in TPH activity was observed in brain areas containing serotonergic cell bodies. Agents which prevent the METH-induced decrease of neostriatal tyrosine hydroxylase activity, i.e., haloperidol, alpha-methyl-p-tyrosine and gamma-aminobutyric acid transaminase inhibitors also prevented the decrease in TPH activity caused by METH. In addition, fluoxetine, an inhibitor of 5-hydroxytryptamine re-uptake, prevented the METH-induced decrease in neostriatal and hippocampal TPH activity but did not alter the decrease in nenostriatal tyrosine hydroxylase activity.

Push-pull cannulae were implanted acutely in both substantiae nigrae and both caudate nuclei of the rabbit under urethane anesthesia. Direct infusion of d-amphetamine (10(-6) M) to one substantia nigra evoked a release of acetylcholinesterase and aminopeptidase not only locally, but also from the contralateral caudate nucleus and substantia nigra. The spontaneous release of acetylcholinesterase was reduced in the ipsilateral caudate nucleus. Gel electrophoresis showed that only one molecular form of each enzyme was released. Since the electrophoretic mobilities of these two enzymic activities were different, the released acetylcholinesterase and aminopeptidase belong to separate molecular species. As amphetamine is known to modify dopamine metabolism the evoked release of these two enzymes is probably related to the dopamine-containing nigrostriatal system.

(1) The activities of choline acetyltransferase (ChAc) (EC 2.3.1.6) and acetylcholinesterase (AChE) (EC 3.1.1.7) were determined in about one hundred regions and subregions of baboon brain. The activities and distributions of these enzymes were in comparable to those found previously in the brains of other species. (2) ChAc activity was highest in the interpeduncular nucleus, where it was about twice that in the putamen, the region previously thought to be the richest in this enzyme. The caudate nucleus, the substantia perforata, the nucleus basalis, the central part of the amygdala and the oculomotor nucleus also had high activities. The activities in the cerebral and cerebellar cortex were less than one twentieth of that in the interpeduncular nucleus. (3) The distribution of AChE activity was not entirely in parallel with that of ChAc.

A comparative study was made of the enzyme choline acetyltransferase (ChAT) from normal and Alzheimer (senile dementia of the Alzheimer type) brain. The number of molecular weight and charge forms of the enzyme were determined in the caudate region of both brains. Efficient purification of active ChAT was achieved using immuno-affinity purification. It was shown that the purified enzyme was identical in both cases, exhibiting a single charge (apparent pI approximately 8.2) and a single molecular weight (mol. wt. = 68,000). The idea of a selective loss of one particular isoform to explain the reduced levels of ChAT observed in Alzheimer's disease can be ruled out.

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 density of D-1 and D-2 dopamine receptors in human caudate nucleus and putamen, obtained postmortem, were studied throughout the adult lifespan using [3H]fluphenazine as the dopamine receptor ligand. The D-1 subtype increased progressively with age in both regions, while the D-2 subtype declined in caudate nucleus. The ratio of D-1/D-2 Bmax in both regions increased from approximately 1 at age 20 to 2 by age 75. The dopamine content in putamen declined with age and was inversely correlated with D-1 receptor density. We suggest that D-1 receptor density is up-regulated by loss of dopamine during aging. The D-2 receptor density in caudate nucleus was positively correlated with choline acetyltransferase activity, suggesting that loss of intrastriatal neurons with age may contribute to the decrease in D-2 sites. These divergent changes in dopamine receptor subtypes with age result in an altered complement of dopamine receptors in older humans and may provide a basis for selective pharmacotherapy in disorders of the basal ganglia.

The effects of several ergosines and different dopamine agonists and antagonists on the activity of dopamine-sensitive adenylate cyclase in synaptosomal membranes of the bovine caudate nucleus were comparatively studied. Among ergot alkaloid derivatives used, ergosinine was the most active in stimulating adenylate cyclase activity. Ergosine, bromoergosine, dihydroergosine, dihydroergocryptine and lisuride also stimulated this enzyme. Dihydroergosinine, bromodihydroergosine and bromoergocryptine did not affect adenylate cyclase activity. Saccharino derivatives of both ergosine and ergosinine were inactive. When used in higher concentrations, ergosine, ergosinine, dihydroergocryptine and lisuride inhibited dopamine-stimulated adenylate cyclase whereas other ergot alkaloid derivatives examined did not. If the extent of dopamine-sensitive adenylate cyclase stimulation is considered as a measure of dopaminergic activity, examination of the structure/dopaminergic activity relationship showed that modifications of ergot alkaloid molecules such as isomerization in position 8, hydrogenation of delta 9(10)-double bond, or introduction of bromine into position 2 of the molecule, lead to a significant decrease of stimulatory effects of adenylate cyclase. Introduction of a saccharino group into position 2 of the molecule caused a total loss of stimulatory activity of both ergosine and ergosinine, probably because of the size of the saccharino residue.