Dopamine Nerve Terminals Research Articles

Severe depletion of cocaine recognition sites associated with the dopamine transporter in Parkinson's‐diseased striatum

The cocaine congener [3H]CFT, also designated [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyltropane), labels cocaine receptors associated with the dopamine transporter in primate striatum. Autoradiographic distribution of [3H]CFT binding (5 nM) in human postmortem control and Parkinson's-diseased striatal tissue sections was compared. In control tissue, high and comparable levels of [3H]CFT binding were observed in the putamen and caudate nucleus. At least 90-99% of total [3H]CFT bound was inhibited by (-)-cocaine (30 microM), suggesting that a high proportion of [3H]CFT is specifically bound. In Parkinson's-diseased tissue, binding sites for [3H]CFT were reduced by 80% in the caudate nucleus and 96% in the putamen. This pattern of depletion parallels the previously reported loss of dopamine in these brain regions (Kish, Shannak, and Hornykiewicz, New Engl. J. Med., 318:876-880, 1988). In the dorsal caudate nucleus of Parkinson's-diseased tissue, a lateral-to-medial gradient of [3H]CFT binding was observed, with the lateral caudate more severely depleted than the medial caudate. The marked decrease of [3H]CFT binding sites in Parkinson's diseased striatum supports the following conclusions: 1) the dopamine transporter is localized primarily on presynaptic nigrostriatal terminals; 2) in the caudate and putamen, cocaine recognition sites are associated primarily with the dopamine transporter; 3) the low level of nonspecific binding of [3H]CFT and the marked depletion of [3H]CFT-labeled sites suggest that radiolabeled derivatives of CFT or its congeners may be suitable imaging probes for presynaptic dopamine nerve terminals.

Effect of aging on tyrosine hydroxylase protein content and the relative number of dopamine nerve terminals in human caudate.

This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15-63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.

Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra

Interruption of the ascending dopamine neurons of the nigrostriatal pathway, by 6-hydroxydopamine (6-OHDA) lesion in rats, produced a significant loss of the dopamine transport complexes labeled with the phencyclidine derivative [ 3H]BTCP. This loss of dopamine innervation in the striatum was present at least 12 to 14 months after lesioning and was functionally manifested by ipsilateral rotation of the animals in response to amphetamine. In these same animals, in comparison to controls, there was a significant increase in the number ( B max) of [ 3H]SCH 23390-labeled D-1 receptors in the striatum (36.7%) and the substantia nigra (35.1%) and a 54.4% increase in the number ( B max) of [ 3H]sulpiride-labeled striatal D-2 receptors without an apparent change in affinity ( K d ). Ten to twelve months after the transplantation of homologous fetal substantia nigra into the denervated striatum, there was a significant decrease in amphetamine-induced turning behavior. In these animals, there was an ingrowth of dopamine nerve terminals in the striatum as demonstrated by a return of [ 3H]BTCP binding. Accompanying this reinnervation was the normalization of D-1 and D-2 receptors to control values in the striatum as well as the return of D-1 receptors to prelesion densities in the substantia nigra. In a subgroup of transplanted rats, amphetamine continued to induce ipsilateral turning. In these animals both D-1 and D-2 receptors remained supersensitive. These results support the hypothesis that the functional recovery of transplanted animals is due, in part, to reinnervation of the striatum. In addition, long-term alterations in receptor density may be related to the behavioral deficits that are associated with the 6-OHDA-lesioned rat. Furthermore, dopamine receptor plasticity may play a role in the functional recovery of substantia nigra transplanted animals and graft viability seems to be a prerequisite for behavioral recovery as well as receptor normalization.

Role of dynorphin-containing neurons in the presynaptic inhibitory control of the acetylcholine-evoked release of dopamine in the striosomes and the matrix of the cat caudate nucleus

The roles of acetylcholine and dynorphin (1–13) in the presynaptic control of the release of [ 3H]dopamine continuously synthesized from [ 3H]tyrosine were examined in a prominent striosomal enriched area and in an adjacent matrix enriched area of the cat caudate nucleus. This was achieved using microsuperfusion devices applied vertically onto coronal slices of cat brain. These devices were placed in a striosomal enriched area located in the core of the structure (acetylcholinesterase-poor zone) and in an adjacent matrix enriched area (acetylcholinesterase-rich zone). [ 3H]Tyrosine was delivered continuously to each microsuperfusion device and [ 3H]dopamine released was estimated in the superfusate. As previously shown, in the presence of tetrodotoxin (1 μM), acetylcholine (50 μM) induces a prolonged stimulation of [ 3H]dopamine release in both compartments through an interaction with muscarinic receptors. Our present study indicates that both dynorphin 1−13 (1 μM) and the selective kappa agonist trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamine (U50488) (1 μM) inhibit the tetrodotoxin-resistant acetylcholine-evoked release of [ 3H]dopamine, these effects being slightly more pronounced in the matrix than in the striosomal enriched area. Naloxone (1 μM) reversed the inhibitory effect of U50488 in both areas. These results suggest that dynorphin exerts an inhibitory presynaptic control of dopamine release through kappa opioid receptors located on dopamine nerve terminals in the striosome as well as in the matrix. However, the presynaptic cholinergic control of dopamine release is much more complex in the matrix than in the striosomal enriched area. Besides its tetrodotoxin-resistant stimulatory effect, acetylcholine exerts two opposing tetrodotoxin-sensitive effects on [ 3H]dopamine release, one facilitatory and the other inhibitory. We demonstrate here that in the superfused matrix enriched area, the indirect acetylcholine inhibitory response is mediated by dynorphin-containing neurons. Indeed, the short-lasting stimulatory effect of acetylcholine on [ 3H]dopamine release was converted into a long-lasting response in the presence of naloxone (1 μM), and, in this latter condition, the co-application of dynorphin 1–13 (1 μM) restored the short-lasting stimulatory effect.

Changes in pituitary-adrenal activity affect the apomorphine- and cholecystokinin-8-induced changes in striatal dopamine release using microdialysis

The effects of apomorphine (0.05 mg/kg, i.p.) and cholecystokinin-8 (sulphated; CCK-8) were analyzed on the levels of dopamine and its metabolites using intrastriatal microdialysis in the adrenalectomized rat with or without corticosterone replacement treatment (5 mg/kg, twice daily for 7 days, last dose given 2 h before killing). Adrenalectomy did not affect the basal release of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA). However, the apomorphine-induced decrease in dopamine release was attenuated following adrenalectomy. Furthermore, there was an enhancement of the apomorphine-induced decrease in DOPAC levels without any modulation of the apomorphine-induced effects on HVA levels. In contrast, the CCK-8-induced increase in dopamine levels was potentiated following adrenalectomy. This potentiation was fully counteracted by replacement treatment with corticosterone. These results indicate that corticosterone may be involved in the regulation of dopamine release, perhaps through glucocorticoid receptors in nigral dopamine cells controlling inter alia the synthesis of G-proteins involved in the regulation of dopamine autoreceptors and CCK-8 receptors located on dopamine nerve terminals or of the receptor proteins themselves.

Intraventricular injection of neurotensin reduces dopamine D 2 agonist binding in rat forebrain and intermediate lobe of the pituitary gland. Relationship to serum hormone levels and nerve terminal coexistence

In order to investigate neurotensin-dopamine receptor interactions in vivo, the effects of intraventricular injection of neurotensin were analyzed on S(−)[ N-propyl- 3H(N) ]propylnorapomorphine ([ 3H]NPA) binding in cryostat sections of the forebrain, hypothalamus and pituitary gland, and on serum levels of prolactin, luteinizing hormone and corticosterone in the male rat. The relationship of modulation of [ 3H]NPA binding with neurotensin-dopamine coexistence in nerve terminals was analyzed by investigating coexistence of neurotensin and tyrosine hydroxylase (TH) immunoreactive nerve terminals in various brain areas, using a double immunohistofluorescence procedure. Intraventricular injections of neurotensin (0.03–3 nmol, 30 min) reduced dose-dependently specific [ 3H]NPA binding (0.25 nM) in the caudate-putamen ( −38 ± 4%), nucleus accumbens ( −42 ± 5%), tuberculum olfactorium ( −52 ± 7%) and in the intermediate lobe of the pituitary gland ( −17 ± 2%). Coexistence of neurotensin and TH was demonstrated in nerve terminals in the prefrontal, cingulate, piriform and entorhinal cortex and in the cortical and deep nuclei of the amygdaloid cortex. It was not possible to demonstrate coexistence in the caudate-putamen, nucleus accumbens, tuberculum olfactorium and median eminence, in view of the high density of dopamine nerve terminals present in relation to the few visualized neurotensin terminals. Nor could coexistence be demonstrated in the few remaining TH-positive nerve terminals following unilateral 6-hydroxydopamine lesions (8 μg per 4 μl; one week) in spite of increased numbers of neurotensin-containing cell bodies and terminals in the ipsilateral dorsomedial caudate. Neurotensin injection markedly decreased serum prolactin levels and increased serum corticosterone levels by about 60%, whereas serum levels of luteinizing hormone were unaffected. The present study indicates that central dopamine D 2 receptors may be regulated by neurotensin in vivo and that the neurotensin involved most likely is released from nerve terminals not containing dopamine, since fibers showing coexistence were only found in prefrontal and limbic cortical areas.

Adenosine A 2 stimulation of tyrosine hydroxylase in rat striatal minces is reversed by dopamine D 2 autoreceptor activation

The adenosine agonist, 2-chloroadenosine, stimulated tyrosine hydroxylase activity in rat striatal minces; this effect was attenuated by activation of dopamine (DA) D 2 autoreceptors with N-n-propylnorapomorphine and antagonized by theopylline. Forskolin and 8-bromo-cAMP also increased tyrosine hydroxylase activity and their effects were not altered by 2-chloroadenosine. D 1, α, β and 5-HT agonists did not affect tyrosine hydroxylase activity. Evidently, A 2 receptors on DA nerve terminals stimulate striatal DA synthesis and this effect is negatively modulated by D 2 autoreceptors, probably via changes in intracellular cAMP levels.

In vitro progesterone modulation of amphetamine-stimulated dopamine release from the corpus striatum of ovariectomized estrogen-treated female rats: response characteristics

We have previously reported that a pulsatile infusion of progesterone directly into superfusion chambers containing corpus striatal tissue fragments of ovariectomized estrogen-treated female rats augmented amphetamine-stimulated dopamine release in vitro. In an attempt to understand some of the means by which progesterone modulates dopamine release under these in vitro conditions, we examined two characteristics of this effect of progesterone. First, to determine the threshold dose of progesterone and whether it was necessary for progesterone to be administered in a pulsatile mode, in Expt. I we examined the effect of a single, brief infusion of progesterone at doses of 2, 4 and 40 ng/ml. Second, to evaluate the temporal relationship between progesterone infusion and its capacity to augment amphetamine-stimulated dopamine release, in Expt. II we varied the interval between progesterone infusion and amphetamine challenge, with amphetamine infused at 10, 30, 50 or 90 min post-progesterone. The results of Expt. I indicate that a single 10-min infusion of progesterone at 2 ng/ml was adequate to produce an accentuated response to amphetamine infusion. Although further increases were obtained following the 4 and 40 ng/ml doses, these values failed to differ significantly from the levels obtained with the 2 ng/ml dose. From Expt. II, we observed that this effect of progesterone was relatively rapid with statistically significant increase in amphetamine-stimulated dopamine release occurring at 30 min post-progesterone and similar responses being maintained at 50 and 90 min post-progesterone. These results demonstrate that a single brief exposure to progesterone results in a rapid increase in the capacity of striatal dopaminergic terminals to respond to amphetamine and suggests the activation of a non-genomic process leading to an increase in dopamine available for release from dopamine nerve terminals.

Chronic nicotine treatment increases dopamine levels and reduces dopamine utilization in substantia nigra and in surviving forebrain dopamine nerve terminal systems after a partial di-mesencephalic hemitransection

In order to further study the previously demonstrated protective action of chronic nicotine treatment on lesioned meso-striatal dopamine (DA) pathways, the following study was carried out on DA utilization in these lesioned neurons. Male Sprague-Dawley rats were partially hemitransected at the meso-diencephalic junction and treated with nicotine (0.125 mg.kg-1.h-1) by means of Alzet minipumps implanted subcutaneously for 2 weeks. The overall serum nicotine level obtained was 64.6 +/- 2.7 ng.ml-1. The results demonstrated that partial di-mesencephalic hemitransections produced a marked reduction of DA fluorescence (quantitative histofluorimetry) on the lesioned side in the nucleus caudatus putamen, anterior nucleus accumbens and posterior lateral tuberculum olfactorium. No significant effects were observed on the intact side. Furthermore, studies on changes in DA utilization as evaluated 2 h after tyrosine hydroxylase inhibition showed an augmentation in the alpha-methyl-(+/-)-p-tyrosine methyl ester (alpha-MT)-induced depletion of the DA stores on the hemitransected side in comparison with the operated side of the sham-operated animals. On the hemitransected side chronic nicotine treatment increased DA stores in the DA nerve terminals of the nucleus caudatus putamen and the posterior lateral tuberculum olfactorium. No significant effects were observed on the intact side. Following chronic nicotine treatment a marked and preferential attenuation of the alpha-MT-induced depletion of DA stores was seen in the various DA nerve terminal systems of the forebrain on the hemitransected side. In the substantia nigra reduced DA levels (HPLC) were demonstrated on the hemitransected side, while no effects on the non-operated side were observed. Also an increase of the alpha-MT-induced depletion of the DA stores was seen on the hemitransected side in comparison with the operated side of the sham-operated animals. In contrast, on the non-operated side an attenuation of the alpha-MT-induced depletion of the DA stores was found. Following chronic nicotine treatment the lesion induced reduction of the nigral DA stores on the hemitransected side was counteracted, as was the lesion induced increase in the alpha-MT-induced depletion of DA stores, which was replaced by a reduction of the alpha-MT-induced depletion of the nigral DA stores. However, on the non-operated side an increased DA depletion was observed after alpha-MT treatment in rats treated chronically with nicotine. Chronic nicotine treatment under the present conditions did not significantly alter serum levels of corticosterone and reduced prolactin serum levels in sham-operated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Striatal L-dopa metabolism studied in vivo in rats with nigrostriatal lesions

We have used cerebral dialysis to monitor striatal metabolism of exogenously administered L-dopa (L-dihydroxyphenylalanine) in rats with unilateral lesions of the substantia nigra. The concentration of extracellular dopamine (DA) increased in both striata following L-dopa administration but the increase was markedly attenuated in the lesioned striatum. The formation of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the major DA metabolites, was also reduced in the lesioned striata following L-dopa administration; however, the reduction was not as great as was that of DA formation. A significant metabolism of exogenous L-dopa to 3-O-methyldopa occurred in both striata. L-dopa administration transiently increased extracellular levels of 5-hydroxyindoleacetic acid (5 HIAA) in both the lesioned and intact striata. These results suggest that the striatum with a reduction in DA nerve terminals is deficient both in the capacity to synthesize DA and in the storage mechanisms necessary to protect the newly synthesized DA from oxidative metabolism.

Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (−)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40–50% inactivation of G i and G o proteins, which is evident from the reduction of ADP ribosylation with [ 32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.

Regulation of dopamine and serotonin synthesis by acute administration of cocaine

Because cocaine effectively increases extracellular levels of both dopamine (DA) and serotonin (5HT), it might be expected that this agent would inhibit transmitter biosynthesis in these monoamine neurons by activation of autoregulatory feedback pathways. This possibility was tested by measuring the effect of cocaine on 3,4-dihydroxyphenylalanine accumulation (DA synthesis) and 5-hydroxytryptophan accumulation (5HT synthesis) in vivo and in vitro after inhibition of aromatic amino acid decarboxylase with NSD-1015. In vivo, cocaine suppressed both DA and 5HT synthesis in a dose-dependent (10-60 mumols/kg, i.p.) and time-dependent fashion (maximum 60 min after administration, recovery by 120-150 min). Inhibition of DA and 5HT synthesis ranged from 35% to 60% depending on the brain region and was apparent in dopaminergic fields such as the medial prefrontal cortex, nucleus accumbens, piriform cortex, striatum, and in noradrenergic fields, such as the hippocampus and temporal cortex. Inhibition of DA, but not 5HT, synthesis was blocked by the D2 antagonist sulpiride in brain areas containing DA nerve terminals. Procaine (30 mumols/kg) did not inhibit DA or 5HT synthesis and prior treatment with reserpine diminished the effectiveness of cocaine in the medial prefrontal cortex, but not in the striatum. Cocaine did not reverse the gamma-butyrolactone-induced increase in striatal DA synthesis nor did cocaine block the ability of the D2 agonist quinpirole to reverse the increase. In vitro, cocaine inhibited DA synthesis in depolarized (K+ = 30 mM) striatal brain slices, an effect that was reversed by the D2 antagonist eticlopride. These results suggest that DA and 5HT neurons compensate in situ for cocaine-induced increases in synaptic transmitter levels by a transient inhibition of transmitter biosynthesis. Acute suppression of transmitter synthesis (and release) in mesoprefrontal DA neurons may represent the principal compensatory mechanism in this group of neurons.

Striatal opiate mu-receptors are not located on dopamine nerve endings in the rat

In rat striatal slices, the autoradiographic analysis of [ 3H]naloxone binding allows one to define highly labelled patches corresponding to the striosomes and representing about 17% of the total striatal volume, surrounded by a poorly labelled zone, the matrix. Previous studies have shown that the density of these mu-opiate receptor binding sites is decreased by about 28% following destruction of the striatal dopamine innervation suggesting a partial localization of these receptors on dopamine presynaptic nerve endings. These results were confirmed but, in addition, we have shown that a chronic (30 days) blockade of dopamine transmission obtained by treatment of the animals with a long acting neuroleptic induces a similar decrease of mu binding sites. Further experiments made with d-Pen 2, d-Pen 5-[tyrosyl-3-5(n)- 3-H] enkephalin, a selective delta opiate receptor agonist, have revealed that the density of delta opiate binding sites is decreased (30%) in rats with striatal dopamine denervation but not in those treated with the long acting neuroleptic. These data indicate that part of these delta receptors is located on dopamine nerve terminals but are not in favour of the presence of mu receptors on these nerve terminals. The decrease in [ 3H]naloxone binding sites induced by prolonged interruption of dopamine transmission can be attributed to postsynaptic events.

Electrophysiological basis of the reinforcing effects of cocaine

The meteoric rise in cocaine abuse over the past decade has led renewed interest in the mechanisms by which this drug produces it potent reinforcing effects. Animal studies have clearly implicated the mesoaccumbens and mesocortical dopamine (DA) neuronal systems as integral brain sites involved in mediating self-administration of cocaine. Biochemical studies have determined that the most reward-relevant action of cocaine within the brain is the inhibition of reuptake of synaptic DA which occurs as a result of cocaine binding to a site on the DA transporter. In this review, electrophysiological studies regarding the effects of cocaine on mesoaccumbens and mesocortical DA systems are presented and a comprehensive picture of how cocaine influences both individual neurons and total neurotransmission within these reward-relevant systems is offered. It is concluded that the powerful rewarding effects of cocaine may be related to its preferential actions at DA and serotonin nerve terminals within the nucleus accumbens and a relatively poor compensatory reduction of impulse generation in mesoaccumbens DA cells. The possibility that this hypothesis can be generalized to account for the rewarding effects of other drugs of abuse is also discussed.

Identification of a potentially neurotoxic pyridinium metabolite of haloperidol in rats

In vivo metabolic studies have revealed that haloperidol is converted to the corresponding pyridinium metabolite which has been characterized in both urine and brain tissues isolated from haloperidol treated rats. Unlike the corresponding conversion of the structurally related Parkinsonian inducing agent MPTP to the ultimate neurotoxic pyridinium metabolite MPP +, the oxidative biotransformation of haloperidol is not catalyzed by MAO-B. Microdialysis studies in the rat indicate that intrastriatal administration of this pyridinium metabolite is about 10% as effective as MPP + in causing the irreversible depletion of striatal nerve terminal dopamine. The results point to the possibility that some of the neurological disorders observed in experimental animals and man during the course of chronic haloperidol treatment may be mediated by this pyridinium metabolite.

Tyrosine Hydroxylase Content of Residual Striatal Dopamine Nerve Terminals Following 6‐Hydroxydopamine Administration: A Flow Cytometric Study

Fluorescence-activated cell sorting based on immunolabeling with a monoclonal antibody to tyrosine hydroxylase and a fluorescein-conjugated secondary antibody was used to identify striatal synaptosomes derived from nigrostriatal dopamine nerve terminals. The amount of tyrosine hydroxylase immunoreactivity in dopaminergic striatal synaptosomes prepared from control rats was compared to the amount in dopaminergic synaptosomes prepared from rats that had received intraventricular injections of 6-hydroxydopamine. Although the absolute number of dopaminergic synaptosomes was decreased in lesioned animals, those residual dopamine terminals present contained more tyrosine hydroxylase than did dopamine terminals from control rats. Both the decrease in the absolute number of dopamine terminals and the increase in tyrosine hydroxylase immunoreactivity in residual terminals were proportional to the extent of the lesion, as determined by measurement of striatal dopamine levels. These results suggest that an increase in the amount of tyrosine hydroxylase protein in residual terminals may represent one compensatory mechanism by which residual dopamine neurons maintain normal striatal function after partial destruction of the nigrostriatal dopamine projection.

Pharmacology of electrically evoked dopamine release studied in the rat olfactory tubercle by in vivo electrochemistry

Dopamine (DA) release was evoked by electrical stimulation of the dopaminergic pathway. Electrically evoked DA release was monitored in the olfactory tubercle of anesthetized rats using differential pulse amperometry (DPA) combined with a treated carbon fiber electrode. Systemic injection of α-methyl-para-tyrosine (AMPT 250 mg/kg), NSD-1015 (50 mg/kg), reserpine (5 mg/kg), d,l-apomorphine (400 μg/kg) decreased evoked DA release while pargyline (75 mg/kg), nomifensine (4 and 20 mg/kg), methylphenidate (10 mg/kg), amphetamine (0.5, 2 and 5 mg/kg), sulpiride (20 mg/kg) and haloperidol (50 μg/kg) enhanced it. Thus, evoked DA release as measured indirectly by DPA is dependent upon synthesis, catabolism and vesicular processes. It can be also affected by DA releasers, DA re-uptake inhibitors, DA agonists and DA antagonists. These results suggest that under our experimental conditions all the drugs tested act directly on DA nerve terminals to modulate in vivo the evoked DA release.

Functional damage of dopamine nerve terminals following intrastriatal kainic acid injection

The release of [ 3H] dopamine ([ 3H]DA) previously taken up into rat striatal slices was studied one week after a monolateral intrastriatal injectuion of kainic acid (KA). Different releasing stimuli (electrical pulses, veratrine, high-K +) were applied. The electrically evoked release in the KA-lesioned striata was drastically reduced with respect to the unlesioned contralateral striata. In contrast, KA had no effect on the release of [ 3H]DA evoked by veratrine or high-K +. In unlesioned striatal slices, depolarized with 15 mM KCl, apomorphine reduced and (−)sulpiride increased the release of [ 3H]DA. The effect of apomorphine was antagonized by (−)sulpiride indicating the presence of an autoreceptor system similar to that seen in unlesioned striata stimulated electrically. However, the effects of apomorphine and of (−)sulpiride were dramatically reduced in K +-depolarized slices prepared from KA-lesioned striata. The results suggest that the axon terminals in KA-treated areas remain intact in several of their properties but may be damaged in some critical processes.

Manganese catalyzed auto-oxidation of dopamine to 6-Hydroxydopamine in vitro

Manganese (Mn) is an essential trace element which, upon excessive exposure, produces a neurological syndrome similar to chronic Parkinson's disease in animals and humans. Previous work demonstated that Mn was more potent than other transition metals in stimulating dopamine (DA) auto-oxidation. In these experiments, DA was incubated under physiological conditions in the presence and absence of Mn for up to 60 min. 6-Hydroxydopamine (6-OHDA) was produced in the presence of Mn, while the incubation mixture without Mn showed no DA oxidation. 6-Hydroxydopamine is a neurotoxicant which exerts its effects by destroying DA nerve terminals in the CNS. Therefore, this work suggests that the Mn catalyzed increase in DA auto-oxidation could be linked mechanistically to the appearance of Mn-induced neurotoxic effects.

Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum

Fluorescence-activated cell sorting (FACS) was used to identify and isolate permeabilized dopaminergic nerve terminals from rat striatum based on immunofluorescent labeling of tyrosine hydroxylase (TH). Striatal synaptosomes were permeabilized by fixation with modified Zamboni fluid. A highly fluorescent subpopulation of particles was detected by FACS following sequential incubation with a monoclonal antibody to TH (LNC 1) and a fluorescein-conjugated secondary antibody. After correcting for nonsynaptosomal particles present in the synaptosomal fraction, analysis of these data suggested that approximately 12-15% of striatal synaptosomes were dopaminergic, consistent with previous estimates. Specific labelling by LNC 1 was decreased if synaptosomes were prepared from rats that had received intraventricular injections of 6-hydroxydopamine. The decrease in labeling was highly correlated with the extent of the lesion as determined by measurement of striatal dopamine levels, suggesting that LNC 1-labeled synaptosomes were derived from nigrostriatal dopamine terminals. In order to verify that LNC 1-labeled synaptosomes were enriched for TH, equal numbers of labeled and control synaptosomes were isolated by FACS and analyzed by SDS-PAGE. LNC 1-labeled synaptosomes were shown by Western blot techniques to be enriched 6-fold for TH compared with control synaptosomes, suggesting that the labeled population consisted almost entirely of dopaminergic synaptosomes.