Synthesis and Release of Dopamine in Rat Brain: Comparison Between Substantia Nigra Pars Compacta, Pars Reticulata, and Striatum

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway, but also from the dendrites in the substantia nigra. We have investigated the regulation of the DA turnover, the DA synthesis rate, and the DA release in the substantia nigra pars compacts (SNpc) and pars reticulata (SNpr) in vivo. As a measure of DA turnover, we have assessed the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid. As a measure of the DA synthesis rate, we have determined the 3,4-dihydroxyphenylalanine accumulation after inhibition of aromatic L-amino acid decarboxylase by 3-hydroxybenzylhydrazine. As a measure of DA release, we have investigated the disappearance rate of DA after inhibition of its synthesis by alpha-methyl-p-tyrosine and the 3-methoxytyramine accumulation following monoamine oxidase inhibition by pargyline. Both the DA turnover and the DA synthesis rate increased following treatment with the DA receptor antagonist haloperidol and decreased following treatment with the DA receptor agonist apomorphine in the SNpc and in the SNpr, but the effects of the drugs were less pronounced than in the striatum. gamma-Butyrolactone treatment, which suppresses the firing of the dopaminergic neurons, increased the DA synthesis rate in the striatum (165%), but had no such effect in the SNpc or SNpr. Haloperidol, apomorphine, and gamma-butyrolactone increased, decreased, and abolished, respectively, the DA release in the striatum, but the drugs had no or only slight effects on the alpha-methyl-p-tyrosine-induced DA disappearance and on the pargyline-induced 3-methoxytyramine accumulation in the SNpc or SNpr. Taken together, these results indicate that the DA synthesis rate, but not the DA release, are influenced by DA receptor activity and neuronal firing in the SNpc and SNpr. This is in contrast to the situation in the striatum, where both the DA synthesis rate and the DA release are under such control.     

3-Methoxytyramine Formation Following Monoamine Oxidase Inhibition Is a Poor Index of Dendritic Dopamine Release in the Substantia Nigra

Abstract: This study was undertaken, using microdialysis, to compare the extracellular concentration of 3-methoxytyramine and dopamine in dialysate from the striatum and substantia nigra, after pargyline (75 mg/kg), after pargyline plus amphetamine (3 mg/kg), and after pargyline plus reserpine (5 mg/kg) administration. Treatment with pargyline alone increased the extracellular dopamine concentration by 70% in the striatum and by 140% in the substantia nigra and induced in both regions a time-dependent accumulation of 3-methoxytyramine. The addition of d-amphetamine to pargyline increased the extracellular dopamine concentration, compared with pargyline-treated controls, to the same extent in both the substantia nigra (maximally by 360%) and the striatum (maximally by 400%), but the concomitant increase of 3-methoxytyramine accumulation in the dialysate was relatively smaller in the substantia nigra compared with the striatum. Reserpine treatment decreased the extracellular dopamine concentration in both regions below the detection level (<10% of basal value). When pargyline was added to reserpine, the striatal extracellular dopamine concentration increased to 50% of pargyline-treated controls and the striatal 3-methoxytyramine accumulation was less than in pargyline-treated controls. However, in the substantia nigra, the addition of pargyline to reserpine resulted in dopamine concentrations as high as after pargyline only and the 3-methoxytyramine accumulation was not changed compared with pargyline-treated controls. In summary, our results indicate that dopamine in the substantia nigra is released from reserpine-sensitive storage sites and that pargyline-induced 3-methoxytyramine accumulation is a poor indicator of the local dopamine release. The latter observation may be explained by the fact that the dopamine-metabolizing enzyme, catechol-O-methyltransferase, is located inter alia in the dopamine-containing cell bodies/dendrites in the substantia nigra, in contrast to the situation in the terminals in the striatum where catechol-O-methyltransferase is located only in nondopaminergic cells.     

Estimation of the Turnover of 3-Methoxytyramine in the Rat Striatum by HPLC with Electrochemical Detection: Implications for the Sequence in the Cerebral Metabolism of Dopamine

Abstract: A highly sensitive method for the determination of 3-methoxytyramine (3-MT) in nervous tissue is described. The method is based on a rapidly performed isolation of 3-MT on small columns of Sephadex G 10, followed by reverse-phase high-performance liquid chromatography in conjunction with a rotating disk electrochemical detector. The detection limit of the assay (0.5–1 pmol/tissue sample) is about 10% of control value for microwave-killed rats. 3-MT as well as dopamine could be quantified in the same chromatographic run. Inhibition of catechol- O -methyl transferase with tropolone resulted in an exponential decline of 3-MT. From this exponential decline a turnover rate for 3-MT of 1.9 nmol/g/h was calculated. In the same group of rats the turnover rate of homovanillic acid was 9.1 nmol/g/h. From these data it is concluded that in the rat striatum about 80% of homovanillic acid is formed from 3,4-dihydroxyphenylacetic acid and 20% from 3-MT.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Electrical Stimulation of the Substantia Nigra and Changes of 2-Phenylethylamine Synthesis in the Rat Striatum

In rats pretreated with deprenyl (2 mg/kg), electrical stimulation of the left substantia nigra produced an increase in the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the left striatum by 57 and 45%, but the levels of 2-phenylethylamine and p-tyramine decreased by 22 and 41%, respectively, as compared with those in the right striatum. The administration of alpha-methyl-p-tyrosine (1.25 mg/kg, i.p.), a tyrosine hydroxylase inhibitor, 1 h before nigral stimulation, did not affect the concentration of 2-phenylethylamine in unstimulated striata but prevented the stimulation-induced decrease in the concentration of 2-phenylethylamine. Neither stimulation nor alpha-methyl-p-tyrosine affected the activity of monoamine oxidase A or B, and stimulation did not produce any change in striatal blood flow, a finding demonstrating that the changes in the rate of accumulation of 2-phenylethylamine were not due to changes in catabolism or removal of 2-phenylethylamine from the brain. These experiments demonstrate that the rate of synthesis of striatal 2-phenylethylamine is decreased following nigral stimulation and that this effect is blocked after partial inhibition of tyrosine hydroxylase. This suggests that 2-phenylethylamine is present in tyrosine hydroxylase-containing neurons and therefore supports the coexistence of 2-phenylethylamine and dopamine in the nigrostriatal pathway.     

Low Selenium Diet Affects Monoamine Turnover Differentially in Substantia Nigra and Striatum

Abstract: Turnover of dopamine, noradrenaline. serotonin, and their metabolites has been measured in striatum and substantia nigra of adult female rats that were fed control or selenium-deficient diets for 15 days. In addition, the glutathione peroxidase activity has been studied. The most striking result was the increase of dopamine turnover (63%) and 3- methoxytyramine turnover (55%) in substantia nigra between control and experimental animals. On the other hand, no changes were found in the turnover rate of dopamine and its metabolites in the striatum. Likewise, no changes were found in noradrenaline turnover in substantia nigra. In the striatum, there was a significant increase of serotonin turnover versus no change for 5-hydroxy-3-indoleacetic acid. However, in the substantia nigra, serotonin turnover did not show significant changes, whereas 5-hydroxy-3-indoleacetic acid turnover decreased. At the same time, glutathione peroxidase activity significantly decreased in both structures after selenium-deficient diets. These results suggest that a selenium-deficient diet for a short period of time decreases brain protection. principally in the substantia nigra, against oxidative damage.     

Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin-500–750 mouse lethal doses (MLD)-injected into the SN 6 h before preparing the slices significantly reduced the calcium-dependent, potassium-evoked release of [³H]GABA. Endogenous GABA levels in the SN and [³H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium-dependent, potassium-evoked release of [¹⁴C]GABA and also [³H]dopamine, but had no effect on the K⁺-evoked release of [³H]5-hydroxytryptamine or [¹⁴C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.     

Conjugated Dopamine in Superfusates of Slices of Rat Striatum

Abstract: An acid-hydrolyzable conjugate of 3,4-dihydroxyphenylethylamine (dopamine, DA) was detected in superfusates from slices from rat striatum. The concentrations of endogenous free and conjugated DA, and of the acid metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in superfusates were measured using HPLC with electrochemical detection. Conjugated DA in superfusates represented 10–20% of the free DA under basal conditions and during release evoked by p -tyramine (5 × 10⁻⁶ M to 5 × 10⁻⁴ M ); much smaller amounts of conjugated DA overflowed into superfusate when DA was released by equimolar concentrations of β-phenylethyl-amine. Surprisingly, inhibition of monoamine oxidase by the inhibitors N -methyl- N -propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochlo-ride (clorgyline) or N -methyl- N -2-propynylbenylamine (pargyline) had little effect on the amounts of conjugated DA present in superfusate. Under basal conditions, the amounts of conjugated DA in superfusate were always less than the amounts of DOPAC but quite similar to the amounts of HVA. However, during release of DA evoked by p -tyramine the concentrations of conjugated DA in superfusate showed much more pronounced increases than those of the acidic metabolites.     

Relationship Between Catechol-O-Methyltransferase and Phenolsulfotransferase in the Metabolism of Dopamine in the Rat Brain

The relationship between phenolsulfotransferase (PST) and catechol-O-methyltransferase (COMT) in the metabolism of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain was studied. In rats not pretreated with a monoamine oxidase (MAO) inhibitor a huge increase of free DA in the brain, following an intraperitoneal injection of L-3,4-dihydroxyphenylalanine (L-DOPA) or an intraventricular injection of free DA, did not lead to any noticeable change in DA sulfate or 3-methoxytyramine (3-MT), which remained undetectable by the present HPLC method. However, in rats previously treated with the MAO inhibitors pargyline or tranylcypromine, the same L-DOPA or free DA treatment resulted in significant increases in both 3-MT and DA sulfate in the hypothalamus, brainstem, and striatum. This response of COMT and PST was not affected by prior treatment of the rats with 6-hydroxydopamine, which suggests that O-methylation and sulfoconjugation occur outside adrenergic neurons not destroyed by the neurotoxin. Inhibition of COMT activity did not lead to any increase in DA sulfate, which showed that despite their common mode of action (both enzymes react preferentially at the same hydroxyl group in the DA molecule), the two enzymes are not competitive. After MAO inhibition there were strong correlations between an increase in DA sulfate and 3-MT on the one hand, and between free DA and 3-MT on the other. Because 3-MT is a marker of central DA release, these data suggest that inhibition of MAO activity not only affects DA metabolism by this enzyme but also influences DA release in the rat brain.     

Changes in Levels of Dopamine and Tyramine in the Rat Caudate Nucleus Following Alterations of Impulse Flow in the Nigrostriatal Pathway

Following electric stimulation of the substantia nigra for 1 h there was a substantial increase in dopamine (DA) turnover in the rat caudate nucleus evidenced by an increase in its acid metabolite homovanillic acid (HVA). Concurrently there was an increase in striatal m-tyramine (mTA) and a substantial decrease in p-tyramine (pTA). Lesioning the substantia nigra to decrease impulse flow resulted in a buildup of striatal DA and mTA, but again a decrease in pTA. Following pretreatment with a tyrosine hydroxylase inhibitor, the effects of stimulation of the nigra on mTA were reversed, there being a significant decrease in this amine. The decrease of pTA in response was partially prevented by tyrosine hydroxylase inhibition. The effects of stimulation or substantia nigra lesions on pTA levels were reversed, however, by tyrosine hydroxylase inhibition, a significant increase in this amine being recorded. mTA and DA levels were largely unaffected by a combination of lesion and tyrosine hydroxylase inhibition. The results provide insight into the possible biosynthetic interrelationships between DA and the tyramine isomers in the rat caudate nucleus.     

Characterization and Pharmacological Responsiveness of Dopamine Release Recorded by Microdialysis in the Substantia Nigra of Conscious Rats

The extracellular concentration of dopamine (DA) and 3,4-dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5 mumol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1 mumol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolactone (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1 mumol/L) of the D2 agonist (-)-N-0437 [2-(n-propyl-N-2-thienylethylamino)-5-hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (-)-N-0437 was suppressed in the presence of nomifensine. Infusion (1 mumol/L) as well as systemic administration (40 mg/kg) of sulpiride caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (-)-N-0437 or (-)-sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Various Centrally Acting Drugs on the Efflux of Dopamine Metabolites from the Rat Brain

The influence of chlorpromazine, haloperidol, morphine, chloral hydrate, gamma-butyrolactone, probenecid, kainic acid, oxotremorine, pargyline, yohimbine, (+)-amphetamine, and cocaine on the efflux rate of 3,4-dihydroxyphenylacetic acid (DOPAC) from four brain areas was studied. All drugs studied except pargyline and morphine had an effect on the transport of DOPAC and homovanillic acid (HVA) from the brain. Nine drugs inhibited the efflux of DOPAC and HVA, whereas (+)-amphetamine stimulated this transport. These data suggest that most centrally acting drugs can interfere with the elimination of 3,4-dihydroxyphenylethylamine (DA or dopamine) metabolites from the brain. These effects are heterogeneously distributed throughout the brain and are probably related to indirect nonspecific drug effects. This implies that drug-induced changes in DA metabolite concentrations, especially when these changes are slight to moderate, cannot directly be translated to changes in the production rate of these metabolites. By studying five control groups, we concluded that formation and transport of DOPAC are not synchronized in the various brain areas.     

Study of Dopamine Turnover by Monitoring the Decline of Dopamine Metabolites in Rat CSF After α-Methyl-p-Tyrosine

CSF was continuously withdrawn from the third ventricle of anesthetized rats. CSF 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid concentrations were determined every 15 min by liquid chromatography coupled with electrochemical detection. Acute tyrosine hydroxylase inhibition [with alpha-methyl-p-tyrosine (alpha-MPT)] induced an exponential decline in levels of DOPAC and HVA in CSF. The decline in DOPAC and HVA concentrations was identical in CSF and forebrain but was much slower in the striatum, suggesting that CSF metabolites of 3,4-dihydroxyphenylethylamine (dopamine) reflect whole forebrain metabolites. The decay in CSF DOPAC and HVA levels after dopamine synthesis inhibition was also used as an in vivo index of forebrain dopamine turnover after various pharmacological treatments. Haloperidol pretreatment accelerated this decay, confirming the increase in brain dopamine turnover induced by neuroleptics. After reserpine pretreatment (15 h before), alpha-MPT produced a very sharp decay in levels of DOPAC and HVA. This result indicates that the residual dopamine that cannot be stored after reserpine treatment is very rapidly renewed and metabolized. Nomifensine strongly diminished the slope of DOPAC and HVA level decreases after alpha-MPT, a result which can be explained either by a slower dopamine turnover or by the involvement of storage dopamine pools. These results exemplify the use of monitoring the decay of dopamine metabolites after alpha-MPT administration in the study of the pharmacological action of drugs on the central nervous system of the rat.     

Biotransformation of l-DOPA to Dopamine in the Substantia Nigra of Freely Moving Rats: Effect of Dopamine Receptor Agonists and Antagonists

Abstract: We investigated the effects of continuous intranigral perfusion of dopamine D1 and D2 receptor agonists and antagonists on the biotransformation of locally applied l -DOPA to dopamine in the substantia nigra of freely moving rats by means of in vivo microdialysis. The "dual-probe" mode was used to monitor simultaneously changes in extracellular dopamine levels in the substantia nigra and the ipsilateral striatum. Intranigral perfusion of 10 µ M l -DOPA for 20 min induced a significant 180-fold increase in extracellular nigral dopamine level. No effect of the intranigral l -DOPA administration was observed on dopamine levels in the ipsilateral striatum, suggesting a tight control of extracellular dopamine in the striatum after enhanced nigral dopamine levels. Continuous nigral infusion with the D1 receptor agonist CY 208243 (10 µ M ) and with the D2 receptor agonist quinpirole at 10 µ M (a nonselective concentration) attenuated the l -DOPA-induced increase in dopamine in the substantia nigra by 85 and 75%, respectively. However, perfusion of the substantia nigra with a lower concentration of quinpirole (1 µ M ) and the D1 antagonist SCH 23390 (10 µ M ) did not affect the nigral l -DOPA biotransformation. The D2 antagonist (−)-sulpiride (10 µ M ) also attenuated the l -DOPA-induced dopamine release in the substantia nigra to ∼10% of that of the control experiments. We confirm that there is an important biotransformation of l -DOPA to dopamine in the substantia nigra. The high concentrations of dopamine formed after l -DOPA administration may be the cause of dyskinesias or further oxidative stress in Parkinson's disease. Simultaneous administration of D1 receptor agonists with l -DOPA attenuates the biotransformation of l -DOPA to dopamine in the substantia nigra. The observed effects could occur via changes in nigral GABA release that in turn influence the firing rate of the nigral dopaminergic neurons.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

The Effect of Mesencephalic Lesions on Tyramine and Dopamine in the Caudate Nucleus of the Rat

Abstract: The dopamine (DA)-containing nerve terminals in the caudate nucleus arise from cell bodies located in the substantia nigra (pars compacta), and it is possible that p-tyramine- and m-tyramine-containing neurons may also exist in this nucleus. We have studied the effects of unilateral electrolytic lesions of the pars compacta in rat on levels of DA, p-tyramine, m-tyramine, and homovanillic acid in the caudate nucleus after various survival times. At 12 and 24 h following lesioning the ipsilateral level of p-tyramine was significantly reduced compared with the contralateral side, whereas the concentrations of m-tyramine, DA, and homovanillic acid were significantly increased. Thus, in the short term, the lesion results in an increase in DA turnover, which is accompanied by an increase in m-tyramine levels and a decrease in p-tyramine levels. Similar changes occur following pharmacological treatments (chlorpromazine, d-amphetamine, l-DOPA) that increase DA turnover. At survival times of 2, 11, and 25 days, the ipsilateral concentrations of m-tyramine, DA, and homovanillic acid were reduced along with p-tyramine. These longer-term alterations in amine levels are most likely a consequence of degeneration of nigro-striatal axons. Placement of a lesion 1 mm dorsal to the usual position centering on the pars compacta produced different biochemical changes from those seen after the pars compacta lesion. One day following this lesion the concentration of p-tyramine was slightly reduced; DA was unaffected, but the concentration of m-tyramine was profoundly increased, even more so than after the pars compacta lesion. This could indicate the existence of specific m-tyramine-containing cell bodies located dorsal to the substantia nigra. The results suggest that p- and m-tyramine in the caudate nucleus originate from neurons in or close to the substantia nigra. The results in the short term following the lesion support the observation that there is an inverse relationship between p-tyramine concentration and DA turnover in the caudate nucleus.     

In Vivo Electrochemical Studies of Dopamine Clearance in the Rat Substantia Nigra: Effects of Locally Applied Uptake Inhibitors and Unilateral 6-Hydroxydopamine Lesions

Abstract: High-speed chronoamperometric recordings were used to measure the uptake and clearance of locally applied dopamine (DA) within the substantia nigra (SN) of anesthetized rats. To establish that DA clearance within the SN was mediated primarily by the DA transporter (DAT) rather than the norepinephrine transporter (NET) or the serotonin transporter (SERT), we locally applied uptake inhibitors with different selectivity profiles for the various amine transporters. Nomifensine, a DAT/NET inhibitor, significantly potentiated both the amplitude and the time course of the DA signals. In contrast, neither the selective NET inhibitor desipramine, nor the selective SERT inhibitor citalopram affected the DA signal, suggesting that NET and SERT do not contribute to DA uptake and clearance within the regions of the SN studied over the concentration ranges (1–5 µ M ) used. In unilaterally 6-hydroxydopamine-lesioned rats, the time course of the DA signal was increased in both the lesioned SN and striatum, relative to the unlesioned hemisphere, indicating loss of DAT and decreased DA uptake and clearance. In addition, when identical amounts of DA were injected in the striatum and SN, peak signal amplitudes were larger in the SN, suggesting that the amplitudes are related to the number of DAT sites in a given region of brain tissue. For signals of equivalent amplitudes, clearance rates were lower in the SN than in the striatum, consistent with a lower capacity for DAT-mediated DA uptake within the SN. These results suggest that the DAT is the major transporter responsible for DA clearance within the rat SN.     

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω-conotoxin-GVIA, ω-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.     

Comparison of Serotonin and Dopamine Release in Substantia Nigra and Ventral Tegmental Area: Region and Species Differences

Abstract: In this study, we compare the electrically evoked, somatodendritic release of dopamine (DA) with axonal release of serotonin (5-HT) in the substantia nigra (SN) and ventral tegmental area (VTA) in vitro by using fast-scan cyclic voltammetry with carbon-fibre microelectrodes. Furthermore, we have examined transmitter release in these regions in guinea-pig compared with rat. Somatodendritic DA was released, as shown previously, in guinea-pig VTA, SN pars compacta (SNc), and occasionally in SN pars reticulata (SNr). 5-HT was rarely released, except in SNr, where nonetheless it only contributed to <30% of amine signals. In rat midbrain, somatodendritic DA release was evoked to a similar extent as in guinea-pig. However, a clear species difference was apparent; i.e., 5-HT and DA were detected equally in rat SNc, whereas in rat SNr, 5-HT was the predominant transmitter detected. Nevertheless, electrically evoked extracellular concentrations of 5-HT in SNc and SNr were, respectively, seven- and fourfold less than DA in SNc. 5-HT release was low in all regions in neonatal rat slices before the maturation of 5-HT terminals. Hence, axonal 5-HT transmission in midbrain exhibits both species and site selectivity. Moreover, whereas somatodendritic DA release is conventionally regarded as modest compared with axon terminal release in striatum, somatodendritic DA release can result in significantly greater extracellular levels than a transmitter released from axon terminals in the same locality.     

Phosphorylation of DARPP-32 Is Regulated by GABA in Rat Striatum and Substantia Nigra

Abstract: In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D₁ receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr³⁴, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr³⁴. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABA_A receptor antagonist, bicuculline, but not with the GABA_B receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or l -3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.