Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of l-DOPA-treated monkeys

The effects of monoamine oxidase (MAO) inhibitors on the metabolism of dopamine synthesized from exogenous l-DOPA were investigated in the striatum and substantia nigra of squirrel monkeys. Administration of a single dose of l-DOPA (methyl ester, 40 mg/kg, i.p.) caused a significant increase in the levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and in the DOPAC/dopamine ratio in the putamen, caudate and substantia nigra. These changes were more pronounced in the substantia nigra than in the striatum and within the striatum of l-DOPA-treated monkeys, levels of dopamine and its metabolites were higher in the putamen than in the caudate nucleus. When l-DOPA treatment was preceded by the injection of clorgyline or deprenyl at a concentration (1 mg/kg) which selectively inhibited MAO A or MAO B, respectively, striatal dopamine was increased while the striatal DOPAC and HVA levels and DOPAC/dopamine ratio were significantly reduced as compared to the values obtained with l-DOPA alone. The two MAO inhibitors also counteracted the increase in the DOPAC and HVA levels and DOPAC/dopamine ratio induced by l-DOPA in the substantia nigra. Thus, both MAO A and MAO B contribute to the metabolism of dopamine when higher levels of this neurotransmitter are generated from l-DOPA in the squirrel monkey. The extent of reduction of dopamine catabolism (as assessed by the decrease in DOPAC and HVA levels) in the striatum and substantia nigra was similar with clorgyline and deprenyl even if the ratio MAO A/MAO B was approximately 1 to 10. This indicates that, though catalyzed by both MAO A and MAO B, dopamine deamination following treatment with l-DOPA preferentially involves MAO A.     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway injury similar to that observed in Parkinson's disease. Many hypotheses have been proposed to explain the mechanisms underlying MPTP neurotoxicity. Previous work showed that the inhibitor of neuronal nitric oxide synthase (nNOS) might produce protection against MPTP-induced dopaminergic toxicity. To exactly test the role of NO in MPTP neurotoxicity, we examined the effect of nNOS inhibitor 7-nitroindazole, in comparison with that of nonselective NOS inhibitor (L-NAME), immunosuppressant (FK-506), monoamine oxidase (MAO) inhibitors (clorgyline and pargyline), N-methyl-D-aspartate receptor antagonist (MK-801) and Ca2+ antagonist (amlodipine). Among seven compounds, 7-nitroindazole produced dose-dependent protection against MPTP-induced depletion of striatal dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) in mice. Clorgyline and pargyline also showed a significant effect on MPTP-induced dopamine depletion in the mouse striatum. However, both compounds did not protect against MPTP-induced depletion of striatal DOPAC Our immunohistological study with tyrosine hydroxylase (TH) and microtuble-associated protein 2 (MAP 2) showed that 7-nitroindazole or pargyline can protect against MPTP-induced depletion of TH and MAP 2 immunostained neurons in the substantia nigra. Furthermore, these compounds reduced a marked increase in GFAP-positive astrocytes of the mouse striatum after MPTP treatments. The present study demonstrates that nNOS inhibitor 7-nitroindazole as well as MAO inhibitors clorgyline and pargyline can produce dose-dependent neuroprotection against the dopaminergic neurotoxicity of MPTP. However, nonselective NOS inhibitor L-NAME, immunosuppressant FK-506, NMDA receptor antagonist MK-801 and Ca2+ antagonist amlodipine did not show a beneficial effect on MPTP neurotoxicity.     

Role of nitric oxide synthase against MPTP neurotoxicity in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway injury similar to that observed in Parkinson's disease. Many hypotheses have been proposed to explain the mechanisms underlying MPTP neurotoxicity. Previous work showed that the inhibitor of neuronal nitric oxide synthase (nNOS) might produce protection against MPTP-induced dopaminergic toxicity. To exactly test the role of NO in MPTP neurotoxicity, we examined the effect of nNOS inhibitor 7-nitroindazole, in comparison with that of nonselective NOS inhibitor (L-NAME), immunosuppressant (FK-506), monoamine oxidase (MAO) inhibitors (clorgyline and pargyline), N-methyl-D-aspartate receptor antagonist (MK-801) and Ca2+ antagonist (amlodipine). Among seven compounds, 7-nitroindazole produced dose-dependent protection against MPTP-induced depletion of striatal dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) in mice. Clorgyline and pargyline also showed a significant effect on MPTP-induced dopamine depletion in the mouse striatum. However, both compounds did not protect against MPTP-induced depletion of striatal DOPAC Our immunohistological study with tyrosine hydroxylase (TH) and microtuble-associated protein 2 (MAP 2) showed that 7-nitroindazole or pargyline can protect against MPTP-induced depletion of TH and MAP 2 immunostained neurons in the substantia nigra. Furthermore, these compounds reduced a marked increase in GFAP-positive astrocytes of the mouse striatum after MPTP treatments. The present study demonstrates that nNOS inhibitor 7-nitroindazole as well as MAO inhibitors clorgyline and pargyline can produce dose-dependent neuroprotection against the dopaminergic neurotoxicity of MPTP. However, nonselective NOS inhibitor L-NAME, immunosuppressant FK-506, NMDA receptor antagonist MK-801 and Ca2+ antagonist amlodipine did not show a beneficial effect on MPTP neurotoxicity.     

Striatonigral projection neurons contain D1 dopamine receptor-activated c-fos

Dopamine receptor agonists which stimulate the D1 receptor have been shown to activate c-fos in the striatum ipsilateral to a 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. In the present study, striatal neurons ipsilateral to a 6-OHDA lesion of the medial forebrain bundle were retrogradely labelled by injection of the fluorescent tracer Fluoro-Gold into the substantia nigra pars reticulata. Five days later, c-fos was induced in the 6-OHDA-denervated striatum by injection of the selective D1 agonist SKF 38393. C-fos-positive nuclei were frequently found in medium-sized striatal cell bodies labelled with Fluoro-Gold. These results indicate that D1 agonists activate c-fos in medium-sized neurons that project to the substantia nigra pars reticulata.     

Non-dopaminergic nigrostriatal pathway

The nigrostriatal projection was studied with a retrograde tracing method (Evans blue, EB) combined with a technique for dopamine histofluorescence. The study, realized in control rats and in animals with 6-hydroxydopamine-induced lesions of the dopaminergic pathway, yielded the following results.

(1) In 3 control rats injected with 0.2 μl of a 10% solution of EB in thecenter of the caudate-putamen 1 mm anterior to the globus pallidus, 96% of all substantia nigra neurons retrogradely labelled with the dye contained dopamine fluorescence. The remaining ones (average 350 per brain) were devoid of dopamine fluorescence and predominantly found in the posterior 75% of the substantia nuigra. These last cells were confined to the upper-half of the pars reticulata.

(2) In a series of 6 animals, the cytotoxic agent 6-hydroxydopamine was injected in various locations in the vicinity of either the substantia nigra ir the nigrostriatal tract 12–15 days prior to the injections of 0.2 μl of EB in the same striatal locations as in the controls. Despite a reduction of up to 85% in the number of dopaminergic cell bodies, the substantia nigra of these rats contained the same average number of EB-labelled neurons devoid of dopamine fluorescence.

(3) Eight rats received smaller injections (0.1 μl) of EB in various striatal sites and in tqo further cases such injections were placed in the globus pallidus to determine more accurately the anatomical location of the dopamine-negative nigral neurons retrogradely labelled with the dye. Following the striatal injections, these cells were found mostly in the upper-half of the pars reticulata and were arranged in longitudinally oriented clusters whose mediolateral location depended on the striatal injection site.

Following the pallidal injections, retrogradely labelled neruons devoid of dopamine fluorescence were found in greater numbers and were located in all areas of the pars reticulata. The possibility of retrograde labelling of some nigrothalamic neurons was not entirely ruled out in these two cases.

(4) Finally 6 rats received 0.1 μl injections of EB in various parts of the parietal cortex. In these cases the substantia nigra did not contain any EB-positivedopamine-negative neurons.

These results are interpreted as evidence in support of the existence of a topographically organized non-dopaminergic nigrostriatal projection.

Localization of striatal and nigral tachykinin receptors in the rat

The effects of lesioning mesostriatal dopamine projections or striatal neurons on tachykinin binding in the basal ganglia were assessed in the rat. 6-Hydroxydopamine lesions of the medial forebrain bundle destroyed striatal dopamine terminals as assessed by [³H]mazindol autoradiography, but did not significantly affect the binding of NK-1 ([³H][Sar⁹, Met(O₂)¹¹]substance P) or NK-3 ([³H]senktide) tachykinin ligands in the striatum. 6-Hydroxydopamine lesions significantly reduced NK-3 binding in the substantia nigra pars compacta, but not the ventral tegmental area. In contrast, striatal quinolinic acid lesions reduced both NK-1 and NK-3 binding in the striatum, but failed to affect NK-3 binding in the substantia nigra. These findings suggest that both NK-1 and NK-3 receptors within the striatum are predominantly post-synaptic with respect to dopamine neurons, whereas nigral NK-3 receptors are located on dopaminergic neurons.     

Brain dopamine transporter: gender differences and effect of chronic haloperidol

Gender differences and the effect of chronic haloperidol on the rat brain dopamine transporter is reported. The density of striatal dopamine transporter sites labelled with [³H]GBR 12935, and of substantia nigra dopamine transporter mRNA measured by in situ hybridization were higher in female compared to male rats whereas striatal D2 specific binding labelled with [³H]spiperone was not significantly higher. Daily haloperidol treatment (1 mg/kg, i.p.) for 21 days increased striatal [³H]spiperone specific binding but left unchanged striatal [³H]GBR 12935 binding density and affinity as well as substantia nigra dopamine transporter mRNA levels. A reduce clearance rate of dopamine in the striatum after acute and chronic haloperidol was previously reported; the present results indicate that this may occur without changes in the sites of dopamine transport or in gene expression of this transporter.     

Pattern Formation in the Mammalian Forebrain: Striatal Patch and Matrix Neurons Intermix Prior to Compartment Formation

The striatum of the mammalian forebrain is divided into two compartments: the patches and the matrix. Neurons of the patch compartment in the rat striatum become postmitotic earlier in neurogenesis than neurons of the matrix compartment. The selective adhesion of patch neurons to one another has been suggested previously to be an important developmental mechanism of striatal compartmentation. We asked if the selective adhesion of patch neurons is expressed before or after the migration of the majority of the matrix neurons into the striatum. Patch neurons were labelled in vivo by a fluorescent retrograde tracer injected into the substantia nigra on embryonic day 19, which almost exclusively labelled patch neurons. Matrix neurons were labelled with a maternal injection of bromodeoxyuridine at embryonic day 18. When animals were killed at embryonic day 20, the majority of the retrogradely labelled patch neurons were intermixed with the bromodeoxyuridine-labelled matrix neurons, although there appeared to be clustering of some of the patch neurons. However, by postnatal day 2 there was a complete segregation of the clusters of the retrogradely labelled patch neurons from the bromodeoxyuridine-labelled matrix neurons in the striatum. This process was modelled in vitro. The patch and matrix compartments were labelled in vivo at embryonic day 13 and 18 respectively, with different birthdate markers ([³H]thymidine or bromodeoxyuridine). At embryonic day 20 the striatal tissue was removed, dissociated and reaggregated in suspension cultures. After 1 day in vitro, labelled patch and matrix neurons were randomly intermixed within the reaggregates. Examination of the cultures at 2.5 and 4 days in vitro revealed clumping of the labelled patch neurons towards the centres of the reaggregates. Over this same period, the labelled matrix neurons did not clump and were dispersed towards the periphery of the reaggregates. The results suggest that patch neuron adhesiveness may appear relatively soon after these neurons become postmitotic, but that this adhesiveness is unable to overcome the initial force produced by the massive migration of matrix neurons into the striatum. We hypothesize that a migratory phase of embryonic striatal development exists, when fated patch and matrix neurons intermix. After this migratory phase, patch neuron adhesiveness can produce the mature segregation of the striatal compartments.     

In vivo synthesis of substance P in the corpus striatum of the rat and its transport to the substantia nigra

Incorporation of [³⁵S]methionine into substance P in the striatum of the rat and the subsequent transport of the labelled peptide to the substantia nigra has been demonstrated in vivo. After a 4-h infusion of [³⁵S]methionine into the corpus striatum and an additional interval of 4 h radiolabelled substance P was found in the striatum and the substantia nigra of the animals. The criteria for concluding that the labelled product was substance P were: (a) gel chromatography and subsequent ion exchange chromatography of an acetic acid extract of the infused striatum or the ipsilateral substantia nigra yielded a peak of radioactivity co-eluting with endogenous immunoreactive substance P or a sample of synthetic substance P; (b) the radioactive material from this peak also co-chromatographed with synthetic substance P on high-voltage paper electrophoresis or high-pressure liquid chromatography; and (c) bound specifically to the substance P antibody. Intracisternal injection of colchicine (70 μg, i.c.) completely suppressed the appearance of radiolabelled substance P immunoreactive material in the substantia nigra.The data indicate that synthesis of substance P occurs in nerve cell bodies located in the corpus striatum and that substance P is transported to the substantia nigra by a colchicine sensitive mechanism.     

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO‐B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO‐B radioligand. The highest uptake was observed in the striatum, one of the MAO‐B‐rich regions, with a peak at approximately 2–3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO‐B inhibitor, L ‐deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO‐B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO‐B activity. Synapse, 2012. © 2011 Wiley Periodicals, Inc.     

Deprenyl induces GFAP immunoreactivity in the intact and injured dopaminergic nigrostriatal system but fails to counteract axotomy-induced degenerative changes

There is increasing evidence of a trophic-like mechanism for some effects ascribed to deprenyl therapy in the central nervous system. For that, we studied the effect of chronic treatment with deprenyl in an animal model of Parkinson's disease induced by unilateral knife transection of the medial forebrain bundle (MFB) in adult rats. The experimental conditions included a 3-week pretreatment with deprenyl before stereotaxic transection of the MFB. Following surgery, deprenyl treatment was maintained for 3 weeks. Neurochemical and immunohistochemical procedures were used to study the dopaminergic system and reactive astrocytes in the nigrostriatal system. Deprenyl treatment failed to counteract the axotomy-induced degenerative changes of the nigrostriatal dopaminergic system. However, it was effective in increasing the density of reactive astrocytes in terms of glial fibrillary acidic protein (GFAP) immunoreactivity in the intact contralateral substantia nigra and also in further enhancing the axotomy-induced increase of GFAP immunolabeled astrocytes in the lesioned substantia nigra. This deprenyl-induced effect on GFAP immunoreactivity was confined to substantia nigra without effect in striatum. In addition, we found a medial to lateral gradient decrease in the distribution pattern of GFAP immunolabeled astrocytes. Axotomy increased the number of reactive astrocytes in either striatal area examined, but yet the preferential distribution pattern of reactive astrocytes in striatum was still evident. GLIA 21:204–216, 1997. © 1997 Wiley-Liss, Inc.     

Convergent Synaptic Input From the Neostriatum and the Subthalamus Onto Identified Nigrothalamic Neurons in the Rat

The two major afferents of the substantia nigra pars reticulata are the subthalamic nucleus and the striatum. Stimulation of these afferents has opposing physiological effects on the output neurons of the substantia nigra pars reticulata. In order to better understand the role of these afferents in the flow of information through the basal ganglia and to better understand the ways in which they might interact, experiments have been performed to test the possibility that single-output neurons of the substantia nigra pars reticulata receive convergent synaptic input from the subthalamic nucleus and the neostriatum. To address this, rats received iontophoretic deposits of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the subthalamic nucleus, injections of the anterograde tracer biocytin in the neostriatum and injections of the retrograde tracer horseradish peroxidase conjugated to wheat-germ agglutinin in the ventral medial nucleus of the thalamus. Following appropriate survival times the animals were perfusion-fixed and sections of the substantia nigra were processed to reveal the transported tracers and prepared for electron microscopy. Light microscopic examination revealed that the substantia nigra contained rich plexuses of anterogradely labelled subthalamic and striatal terminals, as well as many retrogradely labelled nigrothalamic neurons. The anterogradely labelled terminals were often seen apposed to the retrogradely labelled neurons. In the electron microscope the subthalamic terminals were seen to form asymmetrical synaptic contacts (subthalamic type 1) with the identified nigrothalamic neurons as well as unlabelled perikarya and both proximal and distal dendrites. In confirmation of previous findings, the striatal terminals made symmetrical synaptic contact with the nigrothalamic neurons as well as unlabelled neurons. In areas of overlap between the two classes of terminals, identified nigrothalamic neurons and unlabelled nigral neurons were found to receive convergent synaptic input from the subthalamic nucleus and the neostriatum. In addition to the anterogradely labelled subthalamic terminals that formed asymmetrical synaptic specializations, a second, much rarer class was also observed (subthalamic type 2). These terminals were much larger and formed symmetrical synapses; several lines of evidence suggest that they originated not in the subthalamic nucleus but in the globus pallidus. These terminals were found to make synaptic contacts with identified nigrothalamic neurons and non-labelled neurons and to form convergent synaptic contacts with subthalamic type 1 terminals and striatal terminals. It is concluded that the topographical and synaptic organization of the so-called direct (striatum to substantia nigra pars reticulata) and indirect pathways (i.e. pathways involving the subthalamic nucleus andlor the globus pallidus) of information flow through the basal ganglia underlies the inhibition and excitation of the output neurons of the substantia nigra pars reticulata that occur following stimulation of the striatum.     

Sprouting of dopaminergic fibers from spared mesencephalic dopamine neurons in the unilateral partial lesioned rat

A unilateral partially lesioned rat model of Parkinson's disease was developed following selective lesioning of the dopamine neurons of the substantia nigra pars compacta by stereotactic injection of the neurotoxin 6-hydroxydopamine. In this animal model the dopamine neurons of the ventral tegmental area and medial substantia nigra are spared. The neuronal loss in such partial lesioned models mimics more closely that seen in human mid-stage parkinsonism. Cografts of adrenal medullary cells and sciatic nerve to the partially lesioned striatum induced a sprouting response in grafted animals that was confirmed by immunocytochemical staining with antibodies to tyrosine hydroxylase (TH) and by quantification of the high affinity dopamine uptake complex using [³H]GBR 12935 binding. Enhanced TH fiber immunostaining was evident even in the presence of poor cograft survival. The origin of the TH-like immunostained fibers in the striatum was determined using Lucifer yellow retrograde axonal transport. Following discrete tracer injections into the striatum adjacent to a cograft, neurons in the medial substantia nigra and ventral tegmental area (areas A9 and A10, respectively) were labelled with Lucifer yellow. These labelled neurons displayed a morphology characteristics of dopamine neurons and, in double-labelling experiements, also immunostained for TH. These results support the utility of unilateral partially lesioned rat models of Parkinson's disease for studies investigating a host sprouting or upregulation response and confirm that the immunostained striatal fibers originate from spared dopamine neurons in the ventromedial midbrain.     

The MAO-B inhibitor deprenyl, but not the MAO-A inhibitor clorgyline, potentiates the neurotoxicity ofp-chloroamphetamine

The effect of co-administration of MAO inhibitors together with a low dose of the neurotoxic amphetaminep-chloroamphetamine (pCA) on neurotoxicity was examined. Neurotoxicity was assessed by measuring decreases in the binding of [³H]cyanoimipramine to serotonin uptake sites using quantitative autoradiography. By itself, a low dose of pCA (2 mg/kg) did not produce any alterations in radioligand binding, measured 7 days after drug administration. However, co-administration of the MAO-B selective inhibitor deprenyl (1 mg/kg) or the non-selective inhibitor pargyline (50 mg/kg) produced significant ecreases in radioligand binding. Measurements of the effects of these drugs on body temperature ruled out the possibility that deprenyl and pargyline were increasing neurotoxicity by producing a drug-induced hyperthermia. In contrast to the effects of deprenyl and pargyline, co-administration of the MAO-A selective inhibitor clorgyline (1 mg/kg) did not alter binding. By themselves none of the MAO inhibitors produced neurotoxic effects. There are a number of possible explanations for these results. Administration of deprenyl or pargyline, together with pCA, itself a MAO-A inhibitor, will lead to inhibition of both MAO-A and MAO-B activities. This will likely lead to an enhanced release of dopamine and serotonin compared with the release following administration of pCA alone or pCA together with clorgyline. Elevation of the extracellular levels of either or both of these monoamines could lead to enhanced neurotoxicity. Whatever the mechanism involved, our results show that the co-administration of a type-B MAOI enhances the neurotoxic effects of pCA on serotonin neurons.     

Axonal transport of proteins and glycoproteins in the rat nigro-striatal pathway and the effects of 6-hydroxydopamine

Following stereotaxic injection of [35S]methionine into the substantia nigra of adult rats, there was rapid local incorporation of radioactivity into acid-insoluble material. Incorporation peaked by 4 h and then decreased. In contrast, acid-precipitable radioactivity in the corpus striatum (the major projection site of the substantia nigra) rose markedly between 1 and 8 h followed by a plateau period and another even more marked increase between 24 h and 6 days. Experiments involving injection of [3H]fucose gave similar results except that most of the acid-precipitable radioactivity in the striatum appeared in an early wave. In each case radioactivity in the contralateral striatum was less than 11% of that on the ipsilateral side. Stereotaxic injection of colchicine (20 microgram) into the nigrostriatal pathway (within the median forebrain bundle) blocked transport of [35S]protein and [3H]glycoprotein by 90% and 50%, respectively. In animals treated with 6-hydroxydopamine (6-OHDA; treated neonatally or as adults) the accumulation of striatal [35S]protein was reduced to 7 to 26% of control levels; striatal [3H]glycoprotein was also reduced, but not as much (29% to 73% of control). In control experiments, [3H]DOPA wa injected into the substantia nigra, and [3H]dopamine was measured in corpus striatum; 6-OHDA treatment reduced the amounts of striatal [3H]dopamine recovered to 3% of control values. The failure of colchicine or 6-OHDA to block transport of incorporated fucose as effectively as the transport of incorporated methionine is possible due to greater diffusion of fucose away from the injection site to non-dopaminergic nuclei projecting to the striatum. The molecular weight distribution of radioactive proteins at the substantia nigra and corpus striatum was analyzed by polyacrylamide gel electrophoresis. For both [35S]methionine and [3H]fucose, the gel electrophoretic pattern of radioactive proteins in the injection site (substantia nigra) was complex and did not change greatly between 2 h and 6 days. At the projection site (striatum) the electrophoretic distribution pattern was initially different from that of the substantia nigra, and changed markedly over the course of several days. In 6-OHDA-treated animals (treated neonatally or as adults), the bulk of proteins transported in nigro-striatal non-dopaminergic neurons appears to be very similar to that transported in the intact pathway in control rats. However, in striata of 6-OHDA-treated animals, a consistent reduction in striatal 35S- and 3H-radioactivitiy was observed in proteins with molecular weight from about 67,000 to 77,000. Assuming that the 6-OHDA treatment did not substantially affect the non-dopaminergic neurons, we interpret this to mean that some of the proteins in this molecular weight range are transported primarily by dopaminergic neurons.     

Characterization of monoamine oxidase activity during early stages of quail embryogenesis

Catecholamines and other biogenic amines may play a role in early embryogenesis in addition to functioning as neurotransmitters after neuronal differentiation. Regulation of amine levels is mediated by several different parameters including activity levels of degradative enzymes. Since monoamine oxidase (EC 1.4.3.4) is the primary degradative enzyme for these biogenic amines, we have begun to characterize MAO activity during quail embryogenesis. Our results demonstrate that MAO activity is present at all stages of development examined (stages 2–22) and that the MAO specific activity levels are highest during the earliest stages (stages 2–6). Two types of MAO activity similar to adult avian and mammalian MAO-A and MAO-B have been demonstrated by differential clorgyline sensitivity of tryptamine deamination. In addition, SDS-PAGE of embryonic quail [³H]pargyiine-labeled MAO demonstrates that the quail MAO-A and MAO-B flavin-containing subunits have apparent molecular weights of 63,000 and 62,000 respectively.We have begun to assess the functional significance of embryonic quail MAO activity by daily injection of MAO inhibitors (clorgyline or clorgyline plus deprenyl) into fertilized eggs. Clorgyline injection selectively and completely inhibited MAO-A activity, while injection of clorgyline and deprenyl inhibited both MAO-A and MAO-B activities when embryos were assayed after either 2 or 7 days of embryonic development. This paradigm will allow a detailed examination of the effects of MAO inhibition on the developing embryo.     

Up- and down-expression of the dopamine transporter by plasmid DNA transfer in the rat brain

The functional role of the dopamine transporter (DAT) in central dopaminergic neurotransmission was assessed further by investigating the consequences on dopamine (DA) turn-over of up- and down-regulation of this protein induced by a non-viral gene transfer approach. For this purpose, expression plasmids containing the sense or antisense coding sequence of DAT complexed with the cationic polymer, polyethylenimine (PEI), were injected into the rat substantia nigra, the brain region containing the majority of DA cell bodies. Before in vivo injection, the efficacies of the different DNA constructs were assessed by transfection studies into LLC-PK1 cells. Stereotaxic administration of the sense plasmid complexed to PEI induced, 3 days later, a significant increase in the immunoautoradiographic labelling by anti-DAT antibodies of the substantia nigra and various DA projection areas. These effects were associated with a significantly enhanced capacity of striatal synaptosomes to take up [³H]-DA and lasted up to 14 days postinjection. In contrast, 7 days after intranigral administration of the antisense plasmid complexed to PEI, we observed a significant decrease of DAT immunolabelling in the substantia nigra and [³H]-DA uptake by striatal synaptosomes. Whereas DA turnover in the striatum was unaltered 3 days after intranigral administration of the sense plasmid, it was increased 7 days after intranigral administration of the antisense construct. These data indicate that non-viral transfer of the sense or antisense coding sequence of DAT can be used as a novel approach to induce long-term changes in central DA neurotransmission.     

Inhibition of striatal energy metabolism produces cell loss in the ipsilateral substantia nigra

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 μmol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.     

Neuronal protective and rescue effects of deprenyl against MPP+ dopaminergic toxicity

Summary Intranigral infusion of 1-Methyl-4-phenylpyridinium ion (MPP⁺, 2.1–16.8 nmol) dose-dependently injured nigral neurons as reflected by reduced dopamine levels in the ipsilateral striatum four days after the infusion of this toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Coadministration of deprenyl (4.2 nmol) with MPP⁺ into the substantia nigra protected against MPP⁺-induced moderate (20–50%) but not severe (over 70%) nigral injury as reflected in striatal dopamine reductions. However, supplementary treatment with deprenyl (0.25 mg/kg, s.c., twice daily for 4 days) after intranigral infusion of MPP⁺ significantly rescued nigral neurons from more severe damage caused by a higher MPP⁺ does (8.4 nmol) manifested by a lesser striatal dopamine decrease (–31%) compared to the non-deprenyl treated group (–70%). Thus, in addition to the blockade of bioactivation of MPTP, deprenyl can protect and/or rescue nigral neurons from MPP⁺-induced dopaminergic neurotoxicity. These in vivo data add further evidence to suggest that deprenyl, a putative and clinically unproven neuroprotective agent, may be of value in slowing the progressive nigral degeneration in early Parkinson's disease, but may prove to be less so in its terminal stages.     

Manganese neurotoxicity: effects of L-DOPA and pargyline treatments

Single, monolateral injection into rat substantia nigra of manganese chloride produced within two weeks from its administration a loss of dopamine in the striatum ipsilateral to the injected side. The effect was dose-dependent and was not extended to serotoninergic terminals present in this brain area, whose content in serotonin and 5-hydroxyindoleacetic acid was not affected. When L-DOPA + carbidopa or pargyline were given to these animals the decrease of striatal dopamine was more marked. Moreover, rats treated two weeks before with a dose of manganese chloride that produced a 70-80% drop in striatal dopamine concentrations, rotated ipsilaterally to the dopamine-depleted striatum when injected with apomorphine, suggesting that in these animals the stimulatory effects of apomorphine were more relevant in striatum where presynaptic dopaminergic neurons were not affected by manganese chloride. These data indicate that the alterations of dopaminergic postsynaptic receptors may be different in parkinsonian and in manganese-intoxicated patients and that current therapy used for Parkinson's disease could be a hazard in treating manganese poisoning.