Axoplasmic transport of dopamine in nigro-striatal neurons

The possibility that dopamine is transported in the nigro-striatal system was investigated by the stereotaxic injection of labelled tyrosine or l -DOPA into the substantia nigra of tranylcypromine-pretreated rats. At various intervals thereafter (2-48 h), significant quantities of labelled material were recovered from the ipsilateral substantia nigra, globus pallidus and caudate-putamen, The activity in the substantia nigra consisted of DOPA, dopamine, methoxytyramine, acid metabolites and other unidentified metabolites. In the caudate-putamen, however, nearly all of the activity (85 per cent) was recovered in the dopamine fraction, the remainder being distributed among some of the metabolites. No DOPA was recovered from the caudate-putamen. On the basis of time-course studies after the injection of [¹⁴C]DOPA into the substantia nigra, we calculated the transport rate of dopamine in the nigro-striatal bundle to be 0.8 mm/h. Electrolytic lesions of the nigrostriatal bundle at the level of the lateral hypothalamus, pretreatment with 6-hydroxydopamine, or injections of [¹⁴C]DOPA dorsal to the substantia nigra each produced profound reductions in the amount of activity subsequently recovered from the caudate-putamen. These data suggest that the activity recovered from the caudate-putamen after injections of [¹⁴C]DOPA into the or substantia nigra reflected axonal transport rather than other processes such as diffusion or transport via the circulation. Pretreatment with the DOPA decarboxy-lase inhibitor, R_o 4-4602, significantly reduced the amount of activity recovered in the caudate-putamen, an indication that decarboxylation of DOPA to dopamine was a prerequisite for transport. Pretreatment with reserpine also severely reduced the transport of dopamine in the nigro-striatal bundle, an observation suggesting that dopamine was transported by binding to the amine storage granules. There was no evidence of retrograde transport of dopamine in the nigrostriatal bundle. Injections of larger than tracer quantities of labelled tyrosine into the substantia nigra did not produce the degree of transport of dopamine that was obtained after injections of DOPA, a result suggesting that the amine storage granules may not normally be filled during axonal transport.     

Studies on axonal transport in dopaminergic neurons: effect of neuropharmacologic lesions

Axonal transport of [³H]protein to the nucleus accumbens, olfactory tubercle, septal region, caudate nucleus, and hypothalamic region was investigated in rats after unilateral injection of [³H]lysine into the substantia nigra. Co-injection of 2 μg of colchicine with the [³H]lysine depressed the recovery of [³H]protein from forebrain structures by over 70 percent without altering incorporation into midbrain protein, whereas 1 or 2 μg of cycloheximide decreased the incorporation of labelled lysine into both midbrain and forebrain protein by 69 to 76 percent. Partial 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra decreased striatal dopamine levels by 78 percent and reduced axonal protein transport by 47 to 82 percent. Injecting the [³H]lysine 2 mm dorsal to the substantia nigra decreased transport by 95 percent. Unilateral kainic acid-induced lesions of the caudate, which decreased striatal glutamic acid decarboxylase activity by 44 percent and spared striatal dopamine content, did not alter the transport of [³H]protein. Thus, axonal transport of protein in dopamine-containing systems is dependent upon the site of injection of labelled precursor and upon the integrity of a 6-OHDA sensitive pathway. Further, transport is sensitive to inhibitors of both microtubule assembly and protein synthesis, and insensitive to intrastriatal kainic acid lesions.     

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum¹. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra². The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions^3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.     

Pharmacoprotective influences on different links of the mechanism underlying 6-hydroxydopamine-induced degeneration of nigro-striatal dopaminergic neurons

In this study we examined the possibility of preventing degeneration of dopaminergic nigro-striatal neurons caused in rats by a neurotoxin, 6-hydroxydopamine. We showed that an antioxidant, Trolox, an inhibitor of nitric oxide synthase, N_ω-nitro-L-arginine methyl ester, and an inhibitor of caspases, zinc chloride, are capable of preventing to a considerable extent the neurotoxin-induced apoptosis of dopaminergic neurons of the substantia nigra. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 150–156, March–April, 2006.     

Effects of Fetal Methylazoxymethanol Acetate Lesion on the Synaptic Neurochemistry of the Adult Rat Striatum

Abstract We used the cytotoxic properties of methylazoxymethanol acetate (MAM), which ablates mitotically active neuroblasts, to eliminate neurons in the fetal striatum to define the factors that regulate the development of the synaptic circuitry of this region. Adult rats whose mothers received a single intraperitoneal injection of 20 mg/kg of MAM on gestational days (DG) 14-17 were used in this study. MAM treatment at 14 DG caused a 49% decrease in striatal mass whereas treatment at 17 DG reduced the striatal weight by only 16%; MAM treatment on 15 or 16 DG gave intermediate results. Histologic analysis of Nissl-stained sections did not reveal an obvious disruption of striatal organization, although the region was clearly hypoplastic. The hypoplasia was associated with significant increases in the specific activities of choline acetyltransferase and tyrosine hydroxylase, although total activities of these enzymes per striatum were significantly depressed with the 14 or 15 DG treatments. In contrast, the specific activity of glutamate decarboxylase was unaffected by MAM treatment whereas the total activity of this enzyme was reduced commensurate with the degree of striatal hypoplasia. In rats lesioned at 15 DG, there was a similar 30% increase in the specific activities of all presynaptic dopaminergic markers studied. In contrast, the specific activity of the synaptosomal uptake process for [³H]choline was elevated by 60%, the specific activity of choline acetyltransferase was increased by only 30%, and the concentration of acetylcholine in the striatum was unchanged. Whereas the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]γ-aminobutyric acid ([³H]GABA) were unaffected by the 15 DG MAM treatment, the concentration of GABA was increased significantly by 20%. The specific binding of [³H]spiroperidol, [³H]quinuclidinyl benzilate ([³H]QNB). and [³H] muscimol to, respectively, dopamine, muscarinic, and GABA receptors was unchanged by the 15 DG MAM lesion. The nigral dopaminergic perikarya appeared unaffected by the 15 DG MAM lesion in that the tyrosine hydroxylase activity remained normal. Consistent with the loss of striatal GABAergic perikarya, the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]GABA were significantly reduced in the substantia nigra; however, the concentration of endogenous GABA was twofold greater than in control in this terminal region. The results of these studies indicate that the nigro-striatal dopaminergic pathway only partially compensates for the loss of neurons in its terminal field within the hypoplastic striatum. Striatal cholinergic and GABAergic neurons differ considerably in their responses to the MAM lesion, suggesting that they are derived from different neuroblast pools. Finally, the altered synaptic relationships induced by the fetal lesion may affect neurotransmitter turnover as evidenced by disparities in GABA and acetylcholine levels when compared with other presynaptic markers for the GABAergic and cholinergic neurons.     

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities

Parkinson’s disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.     

The localization of GABA-Transaminase in the striato-nigral system

The localization of gamma-aminobutyric acid transaminase (GABA-T), the degrading enzyme for γ-aminobutyric acid, was examined in the striatum and substantia nigra using biochemical techniques. Selective destruction of the nigrostriatal dopaminergic system with 6-hydroxydopamine had no effect on the activity of GABA-T in either the striatum or the substantia nigra, although striatal tyrosine hydroxylase activity was reduced by half. Intrastriatal injection of kainic acid in adult rats resulted in a significant dose-dependent decrease in GABA-T activity in both the striatum and the substantia nigra. The decrease in both of these regions was significantly correlated with the decrease in the GABA synthetic enzyme glutamate decarboxylase (GAD). The intrastriatal injection of kainic acid in ten day old rats did not affect striatal GAD or GABA-T activities, although striatal choline acetyl-transferase activity was reduced by half.It is concluded that the GABA-T activity in the striatum is predominantly localized in neuronal elements, although not, apparently, in cholinergic neurons. Some GABA-T activity is also present in the terminals of the striatonigral neurons. However, the dopaminergic nigrostriatal neurons do not appear to contain GABA-T. It is suggested that high GABA-T activity may be characteristic of GABA neurons.     

Sodium-Sensitive Cocaine Binding to Rat Striatal Membrane: Possible Relationship to Dopamine Uptake Sites

Abstract: In rat striatal membranes, NaCl induced a twofold increase in the maximal number of cocaine binding sites but did not alter the affinity of these sites for cocaine. This effect was concentration-dependent, specific to sodium ions, and occurred in membranes prepared from corpus striatum but not from other brain regions. Lesions with 6-hydroxydopamine but not with kainic acid eliminated the sodium-induced increase in binding and produced a decrease in the B_max of binding measured in the presence of NaCl. The capacity of a series of drugs to interfere with Na⁺–dependent cocaine binding correlated well with their capacity to inhibit [³H]dopamine uptake into rat striatal synaptosomes. The present results suggest that Na⁺–dependent cocaine binding sites are localized presynaptically on dopaminergic nerve terminals in corpus striatum, and may be related to dopamine uptake sites.     

ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS

Abstract— In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE), further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl-β-methyl-choline (AcβMeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with AcβMeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-response curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide, inhibited the enzyme in dopaminergic neurons only at concentrations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS.     

Dopaminergic neurons in explants of substantia nigra in culture

Explants of substantia nigra and corpus striatum obtained from newborn rats were maintained in tissue culture for up to six days. Explants of substantia nigra exhibited a net increase in the ability to take up H³-dopamine, a process associated with the dopaminergic neurons; in contrast, the explants of corpus striatum showed a rapid loss in this ability to accumulate H³-dopamine. After three days in culture, the specific activity of tyrosine hydroxylase and monoamine oxidase had decreased 50% in explants of substantia nigra. A medium including fetal calf serum and chick embryo extracts was necessary for the increase in H³-dopamine uptake, and nerve growth factor had an inhibitory effect. Histofluorescent examination of nigral explants cultured for three days indicated morphologically normal dopaminergic neurons.     

Effect of Intranigral Administration of 6-Hydroxydopamine and 5, 7-Dihydroxytryptamine on Rat Brain Tryptamine

Earlier experiments have shown that unilateral electrolytic lesions of the substantia nigra result in significant reductions in the rate of accumulation of rat striatal tryptamine. For elucidation of the type of neuronal degeneration that is associated with tryptamine depletion, the effects of intranigral injections of 6-hydroxydopamine or 5,7-dihydroxytryptamine, which would affect, respectively, dopamine- or indoleamine-containing neurons, have been assessed. Nigral 6-hydroxydopamine lesions resulted in an ipsilateral reduction in the rate of accumulation of striatal tryptamine, but no changes were observed after nigral injections of 5,7-dihydroxytryptamine. The present results suggest that decreases in the pargyline-induced accumulation of striatal tryptamine may be associated with lesions of the nigral dopamine-containing cell bodies. Alternatively, there may exist specific tryptamine-containing neurons that are damaged by 6-hydroxytryptamine and unaffected by 5,7-dihydroxytryptamine.     

Neostriatal dopamine receptor loss and behavioral deficits in the senescent rat

This study was carried out to evaluate the behavioral implications of previously reported declines in striatal dopamine receptors sensitive to [³H]-neuroleptic specific binding. Rotational behavior was examined following right intrastriatal dopamine (DA) injections in nialamide pretreated rats that had been previously unilaterally lesioned in the left substantia nigra with 6-hydroxydopamine. Results showed that following DA injections old rats exhibited significant deficits in rotational behavioral response strength when compared to young rats. Results are discussed in terms of relating behavioral alterations in stereotypic behavior that occur with senescence to changes in striatal D₂ receptors.     

Axonal transport of glycerophospholipids following intracerebral injection of glycerol into substantia nigra or lateral geniculate body

[2-³H]Glycerol was injected into one substantia nigra of adult rats. Incorporation of radioactivity into lipids at the injection site was maximal by 2 hr, after which it declined. Rapidly transported³H-labeled lipids were just beginning to accumulate in the primary projection site, the ipsilateral corpus striatum by 2 hr, as evidenced by 20-fold higher levels of lipid radioactivity in the projection site relative to control regions. However, the bulk of labeled lipid arrived between 6 hr and 3 days postinjection, suggesting either a prolonged period of release of rapidly transported lipids from the nerve cell bodies or a slow rate of transport for the later arriving lipids. Colchicine applied locally to the fibers of this tract blocked the axonal transport of lipids to the striatum almost completely. Choline and ethanolamine phosphoglycerides were the major transported lipids, accounting for approximately 60% and 25%, respectively, of the total. Similar results were obtained in studies of [2-³H]glycerol-labeled lipids synthesized in the lateral geniculate body and transported to the visual cortex. The rapid axonal transport of lipids labeled with [³²P]phosphate (injected simultaneously with [2-³H]glycerol) could also be demonstrated in both tracts. However, in contrast to [2-³H]glycerol, considerable amounts of³²P soluble label were present in the projection sites, and colchicine only partially blocked the accumulation of³²P-labeled lipid. These results demonstrate the relative utility of [2-³H]glycerol as a lipid precursor for examination of axonal transport in intrabrain tracts. Characteristics of lipid axonal transport in these two intrabrain tracts are similar to each other and are also similar to those previously described for retinal ganglion cells, indicating a common requirement for the axonal transport of these membrane constituents to axons and nerve endings in widely divergent CNS tracts.Presented in part at the 11th meeting of the American Society for Neurochemistry, Houston, Texas, March 1980.     

Lipopolysaccharide-Induced Striatal Nitrosative Stress and Impaired Social Recognition Memory Are Not Magnified by Paraquat Coexposure

Systemic inflammation triggered by lipopolysaccharide (LPS) administration disrupts blood–brain barrier (BBB) homeostasis in animal models. This event leads to increased susceptibility of several encephalic structures to potential neurotoxicants present in the bloodstream. In this study, we investigated the effects of alternate intraperitoneal injections of LPS on BBB permeability, social recognition memory and biochemical parameters in the striatum 24 h and 60 days after treatments. In addition, we investigated whether the exposure to a moderate neurotoxic dose of the herbicide paraquat could potentiate LPS-induced neurotoxicity. LPS administration caused a transient disruption of BBB integrity, evidenced by increased levels of exogenously administered sodium fluorescein in the striatum. Also, LPS exposure caused delayed impairment in social recognition memory (evaluated at day 38 after treatments) and increase in the striatal levels of 3-nitrotyrosine. These events were observed in the absence of significant changes in motor coordination and in the levels of tyrosine hydroxylase (TH) in the striatum and substantia nigra. PQ exposure, which caused a long-lasting decrease of striatal mitochondrial complex I activity, did not modify LPS-induced behavioral and striatal biochemical changes. The results indicate that systemic administration of LPS causes delayed social recognition memory deficit and striatal nitrosative stress in adult mice and that the coexposure to a moderately toxic dose of PQ did not magnify these events. In addition, PQ-induced inhibition of striatal mitochondrial complex I was also not magnified by LPS exposure, indicating the absence of synergic neurotoxic effects of LPS and PQ in this experimental model.

     

The neurotoxicity of DOPAL: behavioral and stereological evidence for its role in Parkinson disease pathogenesis

Background

The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival.

Methods/Principal Findings

A total of 800 nl/rat of DOPAL (1 µg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p<0.001) on the side ipsilateral to the DOPAL injections when compared to the non-injected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p<0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p = 0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra.

Conclusions

The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the “catecholaldehyde hypothesis” as an important link for the etiology of sporadic PD.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X_1–7 and metabotropic P2Y_{1,2,4,6,11–14} receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y_11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X_5,6 and P2Y_1,6,14 in striatum), medium (P2X₃ in striatum and substantia nigra, P2X_6,7 and P2Y₁ in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y_2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X_1,3,4,6 on GABAergic neurons and P2Y₄ on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease.     

ANALYSIS OF PROTEINS UNDERGOING AXONAL TRANSPORT IN NIGRO-STRIATAL NEURONS IN THE RAT

Abstract— An analysis of proteins undergoing axonal transport in nigro-striatal neurons, after the stereotaxic injection of [³H]leucine into the substantia nigra of rat brain was performed. As early as 6 h after the injection [³H]proteins appeared in the caudate-putamen. The maximum accumulation was at 5 days and there was still residual protein radioactivity present at 30 days. About 70 per cent of the total radioactive protein in the caudate-putamen was solubilized by homogenization in 0–5%, (v/v) Triton X-100 and remained in the supernatant on centrifuging for 1 h at 100,000 g. The supernatant fraction, when chroma-tographed on a DEAE-cellulose column, was resolved into four protein peaks (A, B. C and D) which were found to be labelled differently as a function of time after the injection of [³H]leucine. Peak A was substantially labelled in a first phase (6–24 h) and reached its maximum in a second phase (5 days). The proteins comprising this peak appeared to undergo both fast and slow axonal transport. Although some labelling in peak B was evident at 6 h, maximal activity did not occur until 5 days. No radioactivity could be detected in peaks C and D at 6 h. Maximal labelling of these two peaks also occurred at 5 days. These data suggest that the proteins of peaks B, C and D were transported primarily by slow axoplasmic flow. The radioactive protein peaks A and B from the second phase of the transport were excluded from a Sephadex G-200 column, pointing to their high molecular weights (13,000–200,000). Peak B. which had the highest specific radioactivity (c.p.m./mg protein) at 5 days, contained a significant level of tyrosine hydroxylase, an important component of dopaminergic neurons.     

Neuronal localization of the neutral endopeptidase ''enkephalinase'' in rat brain revealed by lesions and autoradiography.

The cellular localization of rat brain enkephalinase was studied after induction of selective unilateral lesions using in vitro quantitative autoradiography of the specific binding of the enzyme inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly). Twenty-one days following injection of kainic acid in the caudate-putamen (CP) [3H]HACBO-Gly binding was locally decreased by 52% with a concomitant reduction of 67 and 78% in the ipsilateral substantia nigra (SN) and globus pallidus (GP), respectively. Inhibition of axonal transport in the CP by unilateral stereotaxic injection of colchicine induced a large (30-60%) and progressive decrease in enkephalinase labelling within the ipsilateral GP and SN. Taken together these results strongly suggest that in the CP a large fraction of enkephalinase is localized on intrinsic striatal neurones, and that the enzyme present both in the GP and the SN is partly localized on nerve terminals originating from neurones in the CP. No change in [3H]HACBO-Gly binding was observed in the CP following injection of 6-hydroxydopamine in the nigrostriatal bundle, contrasting with the 30% depletion in opioid receptors. This would indicate that enkephalinase is present in only very low amounts, if at all, on striatal dopaminergic nerve terminals.     

Exercise Promotes Axon Regeneration of Newborn Striatonigral and Corticonigral Projection Neurons in Rats after Ischemic Stroke

Newborn striatal neurons induced by middle cerebral artery occlusion (MCAO) can form functional projections targeting into the substantia nigra, which should be very important for the recovery of motor function. Exercise training post-stroke improves motor recovery in clinic patients and increases striatal neurogenesis in experimental animals. This study aimed to investigate the effects of exercise on axon regeneration of newborn projection neurons in adult rat brains following ischemic stroke. Rats were subjected to a transient MCAO to induce focal cerebral ischemic injury, followed by 30 minutes of exercise training daily from 5 to 28 days after MCAO. Motor function was tested using the rotarod test. We used fluorogold (FG) nigral injection to trace striatonigral and corticonigral projection neurons, and green fluorescent protein (GFP)-targeting retroviral vectors combined with FG double labeling (GFP⁺ -FG⁺) to detect newborn projection neurons. The results showed that exercise improved the recovery of motor function of rats after MCAO. Meanwhile, exercise also increased the levels of BDNF and VEGF, and reduced Nogo-A in ischemic brain. On this condition, we further found that exercise significantly increased the number of GFP⁺ -FG⁺ neurons in the striatum and frontal and parietal cortex ipsilateral to MCAO, suggesting an increase of newborn striatonigral and corticonigral projection neurons by exercise post-stroke. In addition, we found that exercise also increased NeuN⁺ and FG⁺ cells in the striatum and frontal and parietal cortex, the ischemic territory, and tyrosine hydroxylase (TH) immunopositive staining cells in the substantia nigra, a region remote from the ischemic territory. Our results provide the first evidence that exercise can effectively enhance the capacity for regeneration of newborn projection neurons in ischemic injured mammalian brains while improving motor function. Our results provide a very important cellular mechanism to illustrate the effectiveness of rehabilitative treatment post-stroke in the clinic.